# Patent

## Title
Methods and compositions for improving sugar transport, mixed sugar fermentation, and production of biofuels

## Abstract

The present disclosure relates to host cells containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports cellodextrin into the cell. The present disclosure further relates to methods of increasing transport of cellodextrin into a cell, methods of increasing growth of a cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose-derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports cellodextrin into the cell. The present disclosure relates to host cells containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell, methods of increasing transport of a pentose into a cell, methods of increasing growth of a cell on a medium containing pentose sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 13/802,533, filed Mar. 13, 2013, which is a divisional of U.S. application Ser. No. 12/843,844, filed Jul. 26, 2010, now U.S. Pat. No. 8,431,360, which claims the benefit of U.S. Provisional Application No. 61/285,526, filed Dec. 10, 2009, and U.S. Provisional Application No. 61/271,833, filed Jul. 24, 2009, all of which are hereby incorporated by reference in their entirety.


SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 677792000111SeqList.txt, date recorded: May 22, 2014, size: 104 KB).
FIELD OF THE INVENTION
The present disclosure relates to methods and compositions for increasing the transport of sugars into cells, for increasing growth of cells, for increasing synthesis of hydrocarbons and hydrocarbon derivatives, and for co-fermenting cellulose-derived and hemicellulose-derived sugars.
BACKGROUND OF THE INVENTION
Biofuels are under intensive investigation due to the increasing concerns about energy security, sustainability, and global climate change (Lynd et al., 1991). Bioconversion of plant-derived lignocellulosic materials into biofuels has been regarded as an attractive alternative to chemical production of fossil fuels (Lynd et al. 2008; Hahn-Hagerdal et al. 2006). Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin.
The engineering of microorganisms to perform the conversion of lignocellulosic biomass to ethanol efficiently remains a major goal of the biofuels field. Much research has been focused on genetically manipulating microorganisms that naturally ferment simple sugars to alcohol to express cellulases and other enzymes that would allow them to degrade lignocellulosic biomass polymers and generate ethanol within one cell. However, an area that has been less well studied is that of sugar transporters. An understanding of the regulation of sugar transport and the genetic engineering of microorganisms to have improved sugar-uptake ability will greatly improve efficiency (Stephanopoulos 2007). Furthermore, other types of proteins involved in the regulation of cellulase expression and activity remain to be fully explored.
Saccharomyces cerevisiae, also known as baker's yeast, has been used for bioconversion of hexose sugars into ethanol for thousands of years. It is also the most widely used microorganism for large scale industrial fermentation of D-glucose into ethanol. S. cerevisiae is a very suitable candidate for bioconversion of lignocellulosic biomass into biofuels (van Maris et al., 2006). It has a well-studied genetic and physiological background, ample genetic tools, and high tolerance to high ethanol concentration and inhibitors presented in lignocellulosic hydrolysates (Jeffries 2006). The low fermentation pH of S. cerevisiae can also prevent bacterial contamination during fermentation.
Unfortunately, wild type S. cerevisiae cannot utilize pentose sugars (Hector et al., 2008). To overcome this limitation, pentose utilization pathways from pentose-assimilating organisms have been introduced into S. cerevisiae, allowing fermentation of D-xylose and L-arabinose (Hahn-Hagerdal et al., 2007; Brat et al., 2009; Wisselink et al., 2007, 2009; Wiedemann and Boles 2008; Karhumma et al., 2006). However, efficient conversion of pentose sugars into biofuels is limited by multiple issues including cellular redox imbalance, low influx of pentose phosphate pathway, and lack of efficient pentose transport into the cell (Hector et al., 2008).
In addition, both natural and engineered microorganisms show reduced ethanol tolerance during xylose fermentation as compared to glucose fermentation (Jeffries and Jin 2000). Combined with the lower fermentation rate, the reduced ethanol tolerance during xylose fermentation poses a significant problem in fermentation of sugar mixtures containing the high concentrations of glucose ( ̃70-100 g/L) and xylose ( ̃40-60 g/L) present in cellulosic hydrolysates. Since microorganisms utilize glucose preferentially, at the time of glucose depletion (when cells begin to use xylose), the ethanol concentration is already high enough ( ̃35-45 g/L) to further reduce the xylose fermentation rate. As a result, sequential utilization of xylose after glucose depletion because of “glucose repression” is a significant challenge to be overcome in order to successfully utilize mixed sugars in cellulosic hydrolysates.
Thus, a need exists for the identification of additional genes that are critical for the degradation of lignocellulose and for their use in the engineering of microorganisms for improved growth on lignocellulose and uptake of compounds resulting from lignocellulose degradation. A further need exists for improved methods of efficient conversion of pentose sugars into biofuels and of mixed sugar fermentation for the production of biofuels.
BRIEF SUMMARY OF THE INVENTION
In order to meet these needs, the invention described herein provides methods of increasing transport of cellodextrin into a cell, methods of increasing growth of a cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose-derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports cellodextrin into the cell. Further described are host cells containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports cellodextrin into the cell. Further described herein are host cells containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell, methods of increasing transport of a pentose into a cell, methods of increasing growth of a cell on a medium containing pentose sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell.
As used herein, cellodextrin refers to glucose polymers of varying length and includes, without limitation, cellobiose (2 glucose monomers), cellotriose (3 glucose monomers), cellotetraose (4 glucose monomers), cellopentaose (5 glucose monomers), and cellohexaose (6 glucose monomers).
Thus one aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 1 contains SEQ ID NO: 1, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 2 contains SEQ ID NO: 2, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 contains SEQ ID NO: 3, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 5 contains SEQ ID NO: 4, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 6 contains SEQ ID NO: 5, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and sequence between transmembrane α-helix 6 and transmembrane α-helix 7 contains SEQ ID NO: 6, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 7 contains SEQ ID NO: 7, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of cellodextrin into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them contains SEQ ID NO: 8, and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide.
In certain embodiments that may be combined with any of the preceding aspects, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments that may be combined with any of the preceding embodiments, the host cell contains a second recombinant polynucleotide encoding at least a catalytic domain of a β-glucosidase. In certain embodiments that may be combined with the preceding embodiments having a host cell containing a second recombinant polynucleotide encoding at least a catalytic domain of a β-glucosidase, the β-glucosidase is from Neurospora crassa. In certain embodiments that may be combined with the preceding embodiments having a host cell containing a second recombinant polynucleotide encoding at least a catalytic domain of a β-glucosidase from Neurospora crassa, the β-glucosidase is encoded by NCU00130. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. In certain embodiments that may be combined with the preceding embodiments having a host cell further containing one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization, the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains a third recombinant polynucleotide where the third recombinant polynucleotide encodes a pentose transporter. In certain embodiments that may be combined with the preceding embodiments having the host cell further containing a third recombinant polynucleotide where the third recombinant polynucleotide encodes a pentose transporter, the pentose transporter is selected from the group consisting of NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 1 contains SEQ ID NO: 1, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 2 contains SEQ ID NO: 2, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 contains SEQ ID NO: 3, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 5 contains SEQ ID NO: 4, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 6 contains SEQ ID NO: 5, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and sequence between transmembrane α-helix 6 and transmembrane α-helix 7 contains SEQ ID NO: 6, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 7 contains SEQ ID NO: 7, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them contain SEQ ID NO: 8, and the polypeptide is a cellodextrin transporter, and culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
In certain embodiments that may be combined with any of the preceding aspects of increasing growth of cells, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments that may be combined with any of the preceding embodiments, the host cell contains an endogenous or a second recombinant polynucleotide where the polynucleotide encodes at least a catalytic domain of a β-glucosidase. In certain embodiments that may be combined with the preceding embodiments having a host cell containing an endogenous or a second recombinant polynucleotide where the polynucleotide encodes at least a catalytic domain of a β-glucosidase, the β-glucosidase is from Neurospora crassa. In certain embodiments that may be combined with the preceding embodiments having a host cell containing an endogenous or a second recombinant polynucleotide where the polynucleotide encodes at least a catalytic domain of a β-glucosidase from Neurospora crassa, the β-glucosidase is encoded by NCU00130.
Another aspect includes methods of co-fermenting cellulose-derived and hemicellulose-derived sugars, containing providing a host cell, where the host cell contains a first recombinant polynucleotide encoding a cellodextrin transporter and a second recombinant polynucleotide encoding a catalytic domain of a β-glucosidase, and culturing the host cell in a medium containing a cellulose-derived sugar and a hemicellulose-derived sugar, where expression of the recombinant polynucleotides enables co-fermentation of the cellulose-derived sugar and the hemicellulose-derived sugar. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 1 contains SEQ ID NO: 1. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 2 contains SEQ ID NO: 2. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 contains SEQ ID NO: 3. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 5 contains SEQ ID NO: 4. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 6 contains SEQ ID NO: 5. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and sequence between transmembrane α-helix 6 and transmembrane α-helix 7 contains SEQ ID NO: 6. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 7 contains SEQ ID NO: 7. In certain embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them contain SEQ ID NO: 8. In certain embodiments that may be combined with any of the preceding embodiments, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments that may be combined with any of the preceding embodiments, the β-glucosidase is from Neurospora crassa. In certain embodiments that may be combined with the preceding embodiments having a host cell containing a second recombinant polynucleotide encoding a catalytic domain of a β-glucosidase from Neurospora crassa, the β-glucosidase is encoded by NCU00130. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. In certain embodiments that may be combined with the preceding embodiments having a host cell further containing one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization, the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains a third recombinant polynucleotide where the third recombinant polynucleotide encodes a pentose transporter. In certain embodiments that may be combined with the preceding embodiments having the host cell further containing a third recombinant polynucleotide where the third recombinant polynucleotide encodes a pentose transporter, the pentose transporter is selected from the group consisting of NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3. In certain embodiments that may be combined with any of the preceding embodiments, the cellulose-derived sugar is selected from the group consisting of cellobiose, cellotriose, and celltetraose, and the hemicellulose-derived sugar is xylose.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 1 contains SEQ ID NO: 1, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, containing providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 2 contains SEQ ID NO: 2, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 contains SEQ ID NO: 3, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 5 contains SEQ ID NO: 4, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 6 contains SEQ ID NO: 5, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and sequence between transmembrane α-helix 6 and transmembrane α-helix 7 contains SEQ ID NO: 6, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 7 contains SEQ ID NO: 7, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them contain SEQ ID NO: 8, and the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide.
In certain embodiments that may be combined with any of the preceding aspects increasing the synthesis of hydrocarbons or hydrocarbon derivatives, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains a second recombinant polynucleotide where the polynucleotide encodes at least a catalytic domain of a β-glucosidase. In certain embodiments that may be combined with preceding embodiments having the host cell further containing a second recombinant polynucleotide where the polynucleotide encodes at least a catalytic domain of a β-glucosidase, the β-glucosidase is from Neurospora crassa. In certain embodiments that may be combined with preceding embodiments having the host cell further containing a second recombinant polynucleotide where the polynucleotide encodes at least a catalytic domain of a β-glucosidase from Neurospora crassa, the β-glucosidase is encoded by NCU00130. In certain embodiments that may be combined with any of the preceding embodiments, the source of the cellodextrin contains cellulose. In certain embodiments that may be combined with any of the preceding embodiments, the hydrocarbons or hydrocarbon derivatives can be used as fuel. In certain embodiments that may be combined with the preceding embodiments having the hydrocarbons or hydrocarbon derivatives used as fuel, the hydrocarbons or hydrocarbon derivatives contain ethanol. In certain embodiments that may be combined with the preceding embodiments having the hydrocarbons or hydrocarbon derivatives used as fuel, the hydrocarbons or hydrocarbon derivatives contain butanol.
In certain embodiments that may be combined with any of the preceding aspects, the medium contains a cellulase-containing enzyme mixture from an altered organism, where the cellulase-containing mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. In certain embodiments that may be combined with any of the preceding aspects, the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. In certain embodiments that may be combined with any of the preceding aspects, cellodextrin is selected from one or more of the group consisting of cellobiose, cellotriose, and cellotetraose.
Another aspect includes host cells containing a recombinant polynucleotide encoding a polypeptide having transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, an intracellular N-terminus, an intracellular C-terminus, and a sequence selected from the group consisting of SEQ ID NO: 1 in transmembrane α-helix 1, SEQ ID NO: 2 in transmembrane α-helix 2, SEQ ID NO: 3 in a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3, SEQ ID NO: 4 in transmembrane α-helix 5, SEQ ID NO: 5 in transmembrane α-helix 6, SEQ ID NO: 6 in the sequence between transmembrane α-helix 6 and transmembrane α-helix 7, SEQ ID NO: 7 in transmembrane α-helix 7, and SEQ ID NO: 8 in transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them, where the polypeptide is a cellodextrin transporter. In certain embodiments, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments that may be combined with either of the preceding embodiments, the host cell further contains a second recombinant polynucleotide where the second recombinant polynucleotide encodes a catalytic domain of a β-glucosidase. In certain embodiments that may be combined with preceding embodiments having the host cell further containing a second recombinant polynucleotide where the second recombinant polynucleotide encodes a catalytic domain of a β-glucosidase, the β-glucosidase is from Neurospora crassa. In certain embodiments that may be combined with the preceding embodiments having the host cell further containing a second recombinant polynucleotide where the second recombinant polynucleotide encodes a catalytic domain of a β-glucosidase from Neurospora crassa, the β-glucosidase is encoded by NCU00130. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. In certain embodiments that may be combined with the preceding embodiments having the host cell further containing one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization, the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains a third recombinant polynucleotide where the third recombinant polynucleotide encodes a pentose transporter. In certain embodiments that may be combined with the preceding embodiment having the host cell further containing a third recombinant polynucleotide where the third recombinant polynucleotide encodes a pentose transporter, the pentose transporter is selected from the group consisting of NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3.
In certain embodiments that may be combined with any of the preceding aspects, the host cell further contains one or more inducible promoters operably linked to the one or more recombinant polynucleotides.
Another aspect includes a host cell containing a recombinant polynucleotide encoding a polypeptide selected from the group consisting of NCU00821 and STL12/XUT6, where the polypeptide transports xylose into the cell.
Another aspect includes a host cell containing a recombinant polynucleotide encoding a XUT1 polypeptide, where the polypeptide transports arabinose into the cell.
Another aspect includes a host cell containing a recombinant polynucleotide encoding an NCU06138 polypeptide, where the polypeptide transports arabinose and glucose into the cell.
Another aspect includes a host cell containing a recombinant polynucleotide encoding a polypeptide selected from the group consisting of SUT2, SUT3, and XUT3, where the polypeptide transports xylose and glucose into the cell.
Another aspect includes a host cell containing a recombinant polynucleotide encoding an NCU04963 polypeptide, where the polypeptide transports xylose, arabinose, and glucose into the cell.
In certain embodiments that may be combined with any of the preceding aspects having a host cell containing a recombinant polynucleotide encoding a pentose transporter, the host cell further contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. In certain embodiments that may be combined with the preceding embodiment having the host cell further containing one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization, the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase.
Another aspect includes methods of increasing transport of xylose into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide selected from the group consisting of NCU00821 and STL12/XUT6, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of xylose into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of arabinose into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a XUT1 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of arabinose into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of arabinose or glucose into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU06138 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of arabinose or glucose into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of xylose or glucose into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide selected from the group consisting of SUT2, SUT3, and XUT3, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of xylose or glucose into the cell compared with a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing transport of xylose, arabinose, or glucose into a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU04963 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of xylose, arabinose, or glucose into the cell compared with a cell that does not contain the recombinant polynucleotide.
In certain embodiments that may be combined with any of the preceding aspects of increasing transport of xylose, arabinose, or glucose into cells, the method further includes one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. In certain embodiments that may be combined with the preceding embodiments having the method further including one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization, the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a polypeptide selected from the group consisting of NCU00821 and STL12/XUT6, and the polypeptide transports xylose, and culturing the host cell in a medium containing xylose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a XUT1 polypeptide, and the polypeptide transports arabinose, and culturing the host cell in a medium containing arabinose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes method of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes an NCU06138 polypeptide, and the polypeptide transports arabinose and glucose, and culturing the host cell in a medium containing arabinose or glucose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a polypeptide selected from the group consisting of SUT2, SUT3, and XUT3, and the polypeptide transports xylose and glucose, and culturing the host cell in a medium including xylose or glucose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a NCU04963 polypeptide, and the polypeptide transports xylose, arabinose, and glucose, and culturing the host cell in a medium containing xylose, arabinose, or glucose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
In certain embodiments that may be combined with the preceding aspects of increasing growth of cells by culturing a host cell containing a recombinant polynucleotide encoding a polypeptide that transports xylose and/or arabinose and/or glucose, the host cell further contains one or more endogenous or recombinant polynucleotides encoding one or more enzymes involved in pentose utilization. In certain embodiments that may be combined with the preceding embodiments having the host cell further containing one or more endogenous or recombinant polynucleotides encoding one or more enzymes involved in pentose utilization, the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide selected from the group consisting of NCU00821 and STL12/XUT6, where the polypeptide transports xylose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing xylose or a source of xylose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of xylose into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a XUT1 polypeptide, where the polypeptide transports arabinose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing arabinose or a source of arabinose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of arabinose into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding an NCU06138 polypeptide, where the polypeptide transports arabinose or glucose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing arabinose or glucose or a source of arabinose or glucose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of arabinose or glucose into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide selected from the group consisting of SUT2, SUT3, and XUT3, where the polypeptide transports xylose or glucose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing xylose or glucose or a source of xylose or glucose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of xylose or glucose into the cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell, including providing a host cell, where the host cell contains a recombinant polynucleotide encoding an NCU04963 polypeptide, where the polypeptide transports xylose, arabinose, or glucose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing xylose, arabinose, or glucose or a source of xylose, arabinose, or glucose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of xylose, arabinose, or glucose into the cell is increased upon expression of the recombinant polynucleotide.
In certain embodiments that may combine any of the preceding aspects of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by culturing a host cell containing a recombinant polynucleotide encoding a polypeptide that transports glucose, the source of glucose contains cellulose. In certain embodiments that may combine any of the preceding embodiments, the source of xylose or arabinose contains hemicellulose. In certain embodiments that may combine any of the preceding embodiments, the hydrocarbons or hydrocarbon derivatives can be used as fuel. In certain embodiments that may combine the preceding embodiment having the hydrocarbons or hydrocarbon derivatives used as fuel, the hydrocarbons or hydrocarbon derivatives contain ethanol. In certain embodiments that may combine the preceding embodiment having the hydrocarbons or hydrocarbon derivatives used as fuel, the hydrocarbons or hydrocarbon derivatives contain butanol.
In certain embodiments that may combine any of the preceding embodiments, the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. 
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a NCU07705 polypeptide, and culturing the cell in a medium containing cellulose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. In certain embodiments, the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. In certain embodiments, the host cell further contains an inducible promoter operably linked to the recombinant polynucleotide. In certain embodiments, expression of cellulases is increased in the host cell upon expression of the recombinant polynucleotide.
Another aspect includes methods of increasing growth of a cell on a biomass polymer, including providing a host cell, where the host cell contains an endogenous polynucleotide where the polynucleotide encodes an NCU05137 polypeptide, inhibiting expression of the endogenous polynucleotide, and culturing the cell in a medium containing the biomass polymer, where the host cell grows at a faster rate in the medium than a cell in which expression of the endogenous polynucleotide is not inhibited. In certain embodiments, the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. In certain embodiments, cellulase activity of the host cell is increased upon inhibiting expression of the endogenous polynucleotide. In certain embodiments, hemicellulase activity of the host cell is increased upon inhibiting expression of the endogenous polynucleotide. In certain embodiments, inhibiting expression of the endogenous polynucleotide contains mutating or deleting a gene containing the endogenous polynucleotide. In certain embodiments, the biomass polymer is cellulose. In certain embodiments, the biomass polymer is hemicellulose.
Another aspect includes methods of increasing growth of a cell, including providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a polypeptide selected from the group consisting of NCU01517, NCU09133, and NCU10040, and culturing the cell in a medium containing hemicellulose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. In certain embodiments, the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, and Bacillus subtilis. In certain embodiments, the host cell further contains an inducible promoter operably linked to the recombinant polynucleotide. In certain embodiments, hemicellulase activity of the host cell is increased upon expression of the recombinant polynucleotide.
Another aspect includes methods of degrading cellulose, including providing a composition containing cellulose, and contacting the composition with a cellulase-containing enzyme mixture from an altered organism, where the cellulase-containing mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism, and where the cellulose is degraded by the cellulase-containing mixture. In certain embodiments, the organism is altered by mutation of a gene encoding a β-glucosidase. In certain embodiments, the organism is altered by reducing the expression of a β-glucosidase. In certain embodiments that may be combined with any of the preceding embodiments, the organism is selected from the group consisting of a fungus and a bacterium. In certain embodiments that may be combined with any of the preceding embodiments having the organism selected from the group consisting of a fungus and a bacterium, the organism is a filamentous fungus. In certain embodiments that may be combined with any of the preceding embodiments, the cellulose is from plant material. In certain embodiments that may be combined with the preceding embodiments having the cellulose from plant material, the plant material is selected from the group consisting of switchgrass, Miscanthus, rice hulls, bagasse, flax, bamboo, sisal, abaca, straw, leaves, grass clippings, corn stover, corn cobs, distillers grains, legume plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust, and biomass crops.
Yet another aspect includes methods of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell comprising providing a host cell, wherein the host cell comprises a recombinant polynucleotide wherein the polynucleotide encodes a polypeptide encoded by a sequence selected from the group consisting of NCU00801, NCU00988, NCU01231, NCU04963, NCU05519, NCU05853, NCU05897, NCU06138, NCU00809, NCU08114, NCU10021, and any of the genes listed in Table 15 and culturing the host cell in a medium comprising a source of a compound to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, wherein the compound is a substrate for the synthesis of the hydrocarbons or hydrocarbon derivatives, and wherein transport of the compound into the cell is increased upon expression of the recombinant polynucleotide. In certain embodiments, the host cell is selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, Zymomonas mobilis, Neurospora crassa, Candida shehatae, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Moorella thermocetica, Thermoanaerobacterium saccharolyticum, Klebsiella oxytoca, and Pichia stipitis. In certain embodiments, the host cell further comprises an inducible promoter operably linked to the recombinant polynucleotide. In certain embodiments, the recombinant polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by a sequence selected from the group consisting of NCU00801, NCU00988, NCU01231, NCU04963, NCU05519, NCU05853, NCU05897, NCU06138, NCU00809, NCU08114, NCU10021, and any of the genes listed in Table 15. In some embodiments, the hydrocarbons or hydrocarbon derivatives can be used as fuel. In certain embodiments, the medium comprises cellulose. In other embodiments, the medium comprises hemicellulose. In certain embodiments, the compound is a sugar. In certain embodiments that may be combined with the preceding embodiments, the sugar is a pentose. In certain embodiments that may be combined with the preceding embodiments, the sugar is a hexose. In certain embodiments that may be combined with the preceding embodiments, the sugar is a disaccharide. In certain embodiments that may be combined with the preceding embodiments, the sugar is an oligosaccharide. In other embodiments, the compound is a plant phenol. In certain embodiments that may be combined with the preceding embodiments, the plant phenol is quinic acid. In certain embodiments that may be combined with the preceding embodiments, the plant phenol is nicotinamide. In other embodiments, the compound is pyruvate or lactate.
Another aspect includes methods of increasing growth of a cell on a biomass polymer comprising providing a host cell, wherein the host cell comprises a recombinant polynucleotide wherein the polynucleotide encodes a polypeptide encoded by any of the Neurospora or Pichia stipitis genes listed in Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al., PNAS, 2009, vol. 106, no. 52, 22157-22162, the disclosure of which is hereby incorporated by reference, in Table 15, or NCU01517, NCU09133, or NCU10040 and culturing the cell in a medium comprising the biomass polymer, wherein the host cell grows at a faster rate in the medium than a cell that does not comprise the recombinant polynucleotide. In certain embodiments, the polynucleotide encodes a polypeptide encoded by any of the sequences NCU00130.2, NCU00248.2, NCU00326.2, NCU00762.2, NCU00810.2, NCU00890.2, NCU03328.2, NCU03415.2, NCU03731.2, NCU03753.2, NCU04197.2, NCU04249.2, NCU04287.2, NCU04349.2, NCU04475.2, NCU04997.2, NCU05057.2, NCU05159.2, NCU05493.2, NCU05751.2, NCU05770.2, NCU05932.2, NCU06009.2, NCU06490.2, NCU07340.2, NCU07853.2, NCU07997.2, NCU08744.2, NCU08746.2, NCU08760.2, NCU09108.2, NCU09495.2, NCU09680.2, or NCU10045.2. In certain embodiments, the polynucleotide encodes a polypeptide encoded by NCU07705. In certain embodiments, the recombinant polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by any of the Neurospora or Pichia stipitis genes listed in Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al., 2009, or in Table 15. In certain embodiments, the polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by any of the sequences NCU00130.2, NCU00248.2, NCU00326.2, NCU00762.2, NCU00810.2, NCU00890.2, NCU03328.2, NCU03415.2, NCU03731.2, NCU03753.2, NCU04197.2, NCU04249.2, NCU04287.2, NCU04349.2, NCU04475.2, NCU04997.2, NCU05057.2, NCU05159.2, NCU05493.2, NCU05751.2, NCU05770.2, NCU05932.2, NCU06009.2, NCU06490.2, NCU07340.2, NCU07853.2, NCU07997.2, NCU08744.2, NCU08746.2, NCU08760.2, NCU09108.2, NCU09495.2, NCU09680.2, or NCU10045.2. In certain embodiments, the recombinant polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by NCU07705. In certain embodiments, the biomass polymer is cellulose. In other embodiments, the biomass polymer is hemicellulose. In certain embodiments, the host cell is selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, Zymomonas mobilis, Neurospora crassa, Candida shehatae, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Moorella thermocetica, Thermoanaerobacterium saccharolyticum, Klebsiella oxytoca, and Pichia stipitis. In certain embodiments, the host cell further comprises an inducible promoter operably linked to the recombinant polynucleotide. In certain embodiments, expression of cellulases is increased in the host cell upon expression of the recombinant polynucleotide. In other embodiments, expression of hemicellulases is increased in the host cell upon expression of the recombinant polynucleotide.
Yet another aspect includes methods of increasing growth of a cell on a biomass polymer comprising providing a host cell, wherein the host cell comprises an endogenous polynucleotide wherein the polynucleotide encodes a polypeptide encoded by any of the Neurospora or Pichia stipitis genes listed in Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al., 2009, or in Table 15, or, inhibiting expression of the endogenous polynucleotide, and culturing the cell in a medium comprising the biomass polymer, wherein the host cell grows at a faster rate in the medium than a cell in which expression of the endogenous polynucleotide is not inhibited. In certain embodiments, the endogenous polynucleotide encodes a polypeptide encoded by any of the sequences NCU00130.2, NCU00248.2, NCU00326.2, NCU00762.2, NCU00810.2, NCU00890.2, NCU03328.2, NCU03415.2, NCU03731.2, NCU03753.2, NCU04197.2, NCU04249.2, NCU04287.2, NCU04349.2, NCU04475.2, NCU04997.2, NCU05057.2, NCU05159.2, NCU05493.2, NCU05751.2, NCU05770.2, NCU05932.2, NCU06009.2, NCU06490.2, NCU07340.2, NCU07853.2, NCU07997.2, NCU08744.2, NCU08746.2, NCU08760.2, NCU09108.2, NCU09495.2, NCU09680.2, or NCU10045.2. In certain embodiments, the endogenous polynucleotide encodes a polypeptide encoded by NCU05137. In certain embodiments, the endogenous polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by any of the Neurospora or Pichia stipitis genes listed in Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al., 2009, or in Table 15. In certain embodiments, the endogenous polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by any of the sequences NCU00130.2, NCU00248.2, NCU00326.2, NCU00762.2, NCU00810.2, NCU00890.2, NCU03328.2, NCU03415.2, NCU03731.2, NCU03753.2, NCU04197.2, NCU04249.2, NCU04287.2, NCU04349.2, NCU04475.2, NCU04997.2, NCU05057.2, NCU05159.2, NCU05493.2, NCU05751.2, NCU05770.2, NCU05932.2, NCU06009.2, NCU06490.2, NCU07340.2, NCU07853.2, NCU07997.2, NCU08744.2, NCU08746.2, NCU08760.2, NCU09108.2, NCU09495.2, NCU09680.2, or NCU10045.2. In certain embodiments, the endogenous polynucleotide encodes a polypeptide having at least 50% amino acid identity to the polypeptide encoded by NCU05137. In certain embodiments, the host cell is selected from the group consisting of Saccharomyces cerevisiae, Escherichia coli, Zymomonas mobilis, Neurospora crassa, Candida shehatae, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Moorella thermocetica, Thermoanaerobacterium saccharolyticum, Klebsiella oxytoca, and Pichia stipitis. In certain embodiments, the biomass polymer is cellulose. In other embodiments, the biomass polymer is hemicellulose. In certain embodiments, cellulase activity of the host cell is increased upon inhibiting expression of the endogenous polynucleotide. In other embodiments, hemicellulase activity of the host cell is increased upon inhibiting expression of the endogenous polynucleotide. In certain embodiments, inhibiting expression of the endogenous polynucleotide comprises mutating or deleting a gene comprising the endogenous polynucleotide.



BRIEF DESCRIPTION OF THE DRAWINGS
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FIG. 1 shows the domain structure of the polypeptide encoded by NCU07705.
FIG. 2 shows the phylogenetic analysis of NCU05137. The predicted orthologs of N. crassa NCU05137 were retrieved from NCBI and JGI based on amino acid sequences showing significant similarity by BLAST. All identified filamentous fungal orthologs are shown; NCBI E values were 0.0 except for B. fuckeliana, which was 9e-175. Homologs of NCU05137 were also identified in a number of bacteria (E value ̃e-30). YP 981875 from Polaromonas naphthalenivorans (a beta-proteobacterium) was used as an outgroup. A.=Aspergillus; N.=Neosartorya; P. chyrosogenum=Penicillium; S.=Sclerotinia; B.=Botryotinia; P.=Pyrenophora; C.=Cochliobolus; N. haematococca=Nectria; P. anserina=Podospora; N.=Neurospora. The tree was made by MEGA3, NJ. Bar=0.2 substitutions per amino acid site.
FIG. 3 shows an analysis of N. crassa FGSC2489 and T. reesei QM9414 endoglucanase activity when grown on Miscanthus and Avicel as a sole carbon source. Endoglucanase activity in culture filtrates of N. crassa WT strain FGSC2489 and T. reesei QM9414. N. crassa was grown on Vogel's minimal medium containing 2% of either Avicel or Miscanthus powder as a sole carbon source at 25° C. T. reesei strain was inoculated in MA medium with either 1% Avicel or Miscanthus powder as sole carbon source at 25° C. Both strains were inoculated with the same amount of conidia (1×106/mL in 100 mL culture). The endoglucanase activity in the cultures at different time points were measured at pH 4.5 using Azo-CM-cellulose as a substrate according to the manufacturer's instructions (Megazyme, Ireland).
FIGS. 4A-C show transcriptional profiling of N. crassa grown on Miscanthus and Avicel. FIG. 4A shows hierarchical clustering analysis of 769 genes showing expression differences in Miscanthus culture. Dark shading indicates higher relative expression and light shading indicates lower relative expression. Lane 1: Expression profile of a 16 hr Vogel's minimal medium N. crassa culture (Vogel 1956). Lane 2: Expression profile of a culture grown on Miscanthus as a sole carbon source for 16 hrs. Lanes 3, 4, 5: Expression profiles from cultures grown on Miscanthus for 40 hrs, 5 days, and 10 days. The three clusters are shown as C1, C2, and C3. The cluster that showed increased expression levels of most of the cellulase and hemicellulase genes is boxed (C3 cluster). FIG. 4B shows analysis of the overlap in expression profiles between the N. crassa Miscanthus versus Avicel grown cultures (Top). Analysis and overlap of proteins detected in the culture filtrates of N. crassa grown on Miscanthus and Avicel by tandem mass spectrometry (Bottom). FIG. 4C shows functional category (FunCat) enrichment analysis (Ruepp 2004) of the 231 genes that showed an increase in relative expression levels in Miscanthus cultures. Functional categories that showed significant enrichment (p<0.001), including the unclassified group are shown.
FIGS. 5A-B show the relative expression levels of N. crassa genes encoding cellulases (A) and hemicellulases (B) during growth on minimal medium (MM) and during growth on Miscanthus for 16 hr, 40 hr, 5 days and 10 days. FIG. 5A shows cellulases. FIG. 5B shows hemicellulases.
FIGS. 6A-C show the protein profile and enzymatic activity of culture supernatants from strains containing deletions of genes encoding secreted proteins identified by MS. FIG. 6A shows SDS-PAGE of proteins present in the culture filtrates of 16 deletion strains as compared to wild type when grown on Avicel for 7 days. Deletion strains were chosen based on identification of the protein by mass spectrometry in both Miscanthus and Avicel culture filtrates. Strains are ordered based on gene NCU number, the wild-type strain is FGSC 2489. Missing protein bands that correspond to the deleted genes are marked with boxes. FIG. 6B shows the total secreted protein, azo-CMCase, and β-glucosidase activity assays (see Example 5) performed on 16 deletion strains and the wild-type parental strain (FGSC 2489) using the same sample from FIG. 6A. Activities and protein concentrations were normalized compared to wild type levels and represent the average of triplicate biological measurements. FIG. 6C show cellulase activity of the culture filtrates from the 16 deletion strains using the same samples as in FIG. 6A. Culture filtrates were diluted 10 fold and mixed with 5 mg/mL Avicel (see Example 5) to assess Avicelase activity. Glucose (black) and cellobiose (white) were measured after 8 hours of incubation at 40° C.
FIG. 7 shows the identity of N. crassa secreted proteins based on mutant analysis from a culture grown on Avicel as a sole carbon source. SDS-PAGE of secreted proteins from WT N. crassa (FGSC 2489) grown on 2% Avicel in 100 mL shake flasks for 7 days at 25° C. 15 μL of unconcentrated culture filtrate was loaded onto Criterion 4-15% 26-well gel. Proto Blue Safe (Coomassie) from National Diagnostics was used to stain the gel. The protein bands were identified in this study as shown in FIG. 6A based on analysis of secreted proteins in deletion strains.
FIG. 8 shows the profile of secreted proteins and expression of cbh-1 (NCU07340) and gh6-2 (NCU09680; CBHII) in ΔNCU04952 and ΔNCU05137. FIG. 8A shows SDS-PAGE of total secreted proteins in WT, ΔNCU04952, and ΔNCU05137. Cultures were grown on Avicel from conidia, and harvested at 30 hrs, two days (48 hrs) and three days (72 hrs) (see Example 5). Lanes 1-3, 20× concentrated culture filtrates after 30 hrs of growth on Avicel from WT, ΔNCU04952, and ΔNCU05137 strains, respectively. Lanes 4-6, unconcentrated culture filtrates after two days of growth from WT, ΔNCU04952, and ΔNCU05137 strains, respectively. Lane 7-9, unconcentrated culture filtrates after three days of growth from WT and ΔNCU04952 and ΔNCU05137 strains, respectively. FIG. 8B shows RT-PCR of cbh-1 (NCU07340; CBHI) and gh6-2 (NCU09680; CBHII) in the WT, ΔNCU04952, and ΔNCU05137 strains during growth on Avicel. The WT and deletion strains were grown on Avicel from conidia, and harvested at 48 hrs and 72 hrs (see Example 5). The minimal medium (MM) culture, with sucrose as a sole carbon source (Vogel 1956), was grown for 16 hrs (similar developmental time point). The fold induction of cbh-1 and gh6-2 were relative to the expression of these genes under MM conditions, with actin gene expression used as the control in all samples.
FIG. 9 shows a model of plant cell wall deconstruction in N. crassa. Induction: Extracellular enzymes expressed at low levels generate secondary metabolites that signal N. crassa to dramatically increase the expression level of genes encoding plant cell wall degrading enzymes, most of which are secreted. Utilization: Extracellular enzymes and transporters specific for translocation of cell wall degradation products enable N. crassa to utilize plant cell material for growth. Some extracellular proteins (NCU05137, NCU05057, and NCU04952) may generate metabolites that modulate gene expression of cellulases and hemicellulase during the utilization phase; double hexagon (cellobiose), double pentagon (xylobiose), hexagon (glucose), and pentagon (xylose). The depicted plant cell wall-degrading enzymes include CBH(I), CBH(II), EG2, EG1, EG6, and xylanase. Additional cellulolytic enzymes are not shown. Thickness of arrows indicates relative strength of response.
FIG. 10-1, FIG. 10-2, FIG. 10-3, FIG. 10-4, FIG. 10-5, FIG. 10-6, FIG. 10-7, FIG. 10-8, FIG. 10-9, FIG. 10-10, FIG. 10-11, and FIG. 10-12 show BLAST results from searching the sequences of N. crassa putative transporters against a database of S. thermophile protein sequences or from searching the sequences of S. thermophile putative transporters against a database of N. crassa protein sequences.
FIGS. 11A-B show the growth phenotype of a N. crassa strain lacking NCU08114. FIG. 11A shows shaker flasks of WT (left) and ΔNCU08114 (right) N. crassa strains after 3 days of growth with crystalline cellulose as a carbon source. FIG. 11B shows the mean Alamar Blue© fluorescence from N. crassa cultures grown with either sucrose or crystalline cellulose as a carbon source for 16 or 28 hours, respectively. Fluorescence was normalized by setting WT to 100%. Error bars were the standard deviation between measurements from three biological replicates. N. crassa lacking NCU00801 did not have an obvious phenotype. N. crassa secreted β-glucosidases (Tian et al., 2009) that hydrolyzed cellodextrins to glucose, which was subsequently taken up by monosaccharide transporters (Scarborough 1973). This alternate route of consumption led to an underestimate of the cellodextrin transport defect in these deletion lines.
FIGS. 12A-D show (A) cellobiose consumption for S. cerevisiae strains expressing NCU00801, NCU05853, or NCU08114 along with NCU00130; (B) cellotriose consumption for S. cerevisiae strains expressing NCU00801, NCU05853, or NCU08114 along with NCU00130; (C) cellotetraose consumption for S. cerevisiae strains expressing NCU00801, NCU05853, or NCU08114 along with NCU00130; and (D) cellohexaose consumption for S. cerevisiae strains expressing NCU00801, NCU05853, or NCU08114 along with NCU00130. FIG. 12A shows cellobiose consumption. FIG. 12B shows cellotriose consumption. FIG. 12C shows cellotetraose consumption. FIG. 12D shows cellohexaose consumption.
FIG. 13 shows cellodextrin consumption by N. crassa strains lacking NCU008114 or NCU00801. The indicated N. crassa strains were incubated with 90 μm of the respective sugars for 15 minutes. Bars represent the mean concentration of sugars remaining in the supernatant following the incubation from two independent experiments. Error bars were the standard deviation between these experiments.
FIG. 14 shows cellobiose transport by a S. cerevisiae strain expressing NCU00801/cbt1. Shown is cellobiose transport by yeast with (◯) or without (●) CBT1. Both strains expressed the intracellular β-glucosidase, NCU00130. The initial concentration of cellobiose was 50 μM. All values were the mean between two measurements, with error bars representing the standard deviation between these measurements.
FIGS. 15A-B show localization and quantification of GFP fused to CBT1 and CBT2. FIG. 15A shows images of S. cerevisiae strains expressing cbt1 (left), or cbt2 (right), fused to GFP at their C-terminus. FIG. 15B shows GFP fluorescence of yeast strains without a cellobiose transporter, or expressing cbt1 or cbt2 fused to GFP at their C-terminus. Values were the mean from three biological replicates, and error bars represent the standard deviation between these replicates.
FIGS. 16A-C show cellodextrin transport by N. crassa transport systems expressed in S. cerevisiae. FIG. 16A shows cellobiose-mediated growth of yeast strains expressing the gene NCU00801 (named cbt1, ◯), NCU08114 (named cbt2, ▾), or no transporter (●). All strains also expressed the intracellular β-glucosidase, NCU00130. A representative experiment is shown. Growth rates from three independent experiments were as follows: cbt1, 0.0341±0.0010 hr−1; cbt2, 0.0131±0.0008 hr−1; no transporter, 0.0026±0.0001 hr−1. FIG. 16B shows growth of yeast strains on cellotriose and cellotetraose. Strains expressing the intracellular β-glucosidase, NCU00130, as well as the transporters listed in the legend, were grown with 0.5% (w/v) of cellotriose (G3) or cellotetraose (G4) serving as the sole carbon source. A representative experiment is shown. Growth rates from three independent experiments were as follows: cbt1 cellotriose, 0.0332±0.0004 hr−1; cbt1 cellotetraose 0.0263±0.0020 hr−1; no transporter cellotriose, 0.0043±0.0015 hr−1; cbt2 cellotriose, 0.0178±0.0005 hr−1; cbt2 cellotetraose 0.0041±0.0003 hr−1; no transporter cellotetraose, 0.0031±0.0008 hr−1. FIG. 16C shows glucose produced from cellobiose (G2), cellotriose (G3), and cellotetraose (G4) hydrolysis by purified NCU00130. The mean and standard deviation of three independent measurements are shown. Residual glucose in incubations without enzyme (2 nmol) was subtracted from the values shown.
FIG. 17 shows growth of S. cerevisiae strains expressing cbt1 (◯), cbt2 (▾), or no transporter (●) on glucose. All strains expressed the β-glucosidase, NCU00130. A representative experiment is shown.
FIG. 18 shows cellobiose-mediated growth of S. cerevisiae strains in 250 mL flasks. Values represent the mean OD between two replicate cultures of yeast strains expressing the β-glucosidase, NCU00130, cbt1 or cbt2, or a strain expression NCU00130, but lacking any transporters. Error bars represent the standard deviation between replicates.
FIG. 19 shows kinetics of cellobiose transport by CBT1 and CBT2. The rate of cellobiose transport was determined as a function of cellobiose concentration by yeast strains expressing either cbt1 or cbt2. The transport rate was normalized for transporter abundance.
FIG. 20 shows the ability of S. cerevisiae expressing the combinations of Neurospora genes shown on the x-axis to grow on cellobiose, cellotriose, or cellotetraose.
FIG. 21 shows competition by cellodextrins for cellobiose transport in strains carrying cbt1 or cbt2. A 5-fold excess of the respective unlabeled sugar was included during assays of [3H]-cellobiose transport. Substrates of CBT1 or CBT2 would decrease the [3H]-cellobiose transport rate by competing for binding. Bars represent the mean from three replicates. Error bars represent the standard deviation between these replicates. Values were normalized by setting the rate of [3H]-cellobiose transport without a competing sugar to 100.
FIG. 22 shows the SDS-PAGE gel of purified NCU00130. Lane 1, Protein molecular weight standards, in kDa. Lane 2, NCU00130 after purification over nickel-NTA resin. Molecular weights in kDa are shown to the left.
FIG. 23 shows maximum likelihood phylogenetic analysis of the cellobiose transporters NCU08114 and NCU00801. With the exception of S. cerevisiae HXT1 and K. lactis LACP, all genes encoding proteins shown are reported to increases in expression level when the fungus comes into contact with plant cell wall material or cellobiose (Tian et al., 2009; Noguchi et al., 2009; Wymelenberg et al., 2010; Martin et al., 2010). S. cerevisiae HXT1, a low affinity glucose transporter (Reifenberger et al., 1997), was used as an outgroup.
FIGS. 24A-C show cellobiose fermentation, and simultaneous saccharification and fermentation of cellulose, by S. cerevisiae expressing the cellobiose transport system from N. crassa. FIG. 24A shows cellobiose fermentation to ethanol. Ethanol produced by yeast strains with CBT1 (●), or without CBT1 (◯). Cellobiose concentration during the fermentation reaction using yeast strains with CBT1 (▾), or without CBT1 (Δ). FIG. 24B shows SSF using yeast strains with and without CBT1. Cellobiose (●) and glucose (▾) concentrations in the presence of a strain with CBT1, and cellobiose (◯) and glucose (A) concentrations in the presence of a strain lacking CBT1. Note, 0.1 mg/mL cellobiose=292 μM. FIG. 24C shows ethanol produced during SSF using a strain with CBT1 (●), or without CBT1 (◯). In all panels, values are the mean of 3 biological replicates. Error bars were the standard deviation between these replicates. All strains also expressed the intracellular β-glucosidase, NCU00130.
FIG. 25 shows use of cellodextrin transport pathways from filamentous fungi during simultaneous saccharification and fermentation of cellulose by yeast. The cellodextrin (Cdex) transport pathway (black) includes a cellodextrin transporter (CBT) and intracellular β-glucosidase (βG). The sugar catabolism pathway presented in standard yeast includes hexose transporters (HXT). In SSF, both cellulases (GH) and extracellular β-glucosidase (βG) could be used.
FIGS. 26A-C show residues in NCU00801 and NCU08114 that are critical for function. FIG. 26A shows Ala-scan of cbt1/NCU00801. FIG. 26B shows polypeptide sequence (important residues marked) of cbt1/NCU00801. FIG. 26C shows polypeptide sequence (important residues marked) of cbt2/NCU08114.
FIGS. 27A-C show a comparison of S. cerevisiae strains expressing cellobiose transporters from P. stipitis. FIG. 27A shows cell growth of S. cerevisiae strains expressing β-glucosidase and orthologs of cellobiose transporters NCU00801, NCU08114, and NCU05853. FIG. 27B shows a comparison of cellobiose transporters from P. stipitis: cell growth of S. cerevisiae strains expressing β-glucosidase and cellobiose transporters. FIG. 27C shows a comparison of cellobiose transporters from P. stipitis: xylose consumption and ethanol production by S. cerevisiae strains expressing β-glucosidase and cellobiose transporters.
FIGS. 28A-B show alignments of cellobiose transporter orthologs. FIG. 28A shows alignment of cellobiose transporter orthologs including ones that did not appear to have transporter function under the conditions tested. FIG. 28B shows alignment of cellobiose transporter orthologs that had transport function. FIG. 28C shows alignment of NCU00801 and NCU08114.
FIGS. 29A-B show functionally important motifs marked in homology models of NCU00801 and NCU08114. FIG. 29A shows location of cellobiose transporters motifs on NCU00801 homology model. Motif [LIVM]-Y-[FL]-x(13)-[YF]-D (SEQ ID NO: 1) is shown in red. Motif [YF]-x(2)-G-x(5)-[PVF]-x(6)-[DQ] (SEQ ID NO: 2) is shown in light green. Motif G-R-[RK] (SEQ ID NO: 3) is shown in dark blue. Motif R-x(6)-[YF]-N(SEQ ID NO: 4) is shown in yellow. Motif WR-[IVLA]-P-x(3)-Q (SEQ ID NO: 5) is shown in magenta. Motif P-E-S-P-R-x-L-x(8)-A-x(3)-L-x(2)-Y-H (SEQ ID NO: 6) is shown in cyan. Motif F-[GST]-Q-x-S-G-N-x-[LIV] (SEQ ID NO: 7) is shown in orange. Motif L-x(3)-[YIV]-x(2)-E-x-L-x(4)-R-[GA]-K-G (SEQ ID NO: 8) is shown in dark green. I. View of NCU00801 from the cytoplasmic side looking into the putative cellobiose binding pore. Note that in this image, some of the residues connecting transmembrane helices 6 and 7 have been removed for clarity as they occlude the pore. II. View of one side of NCU00801. III. View of the side opposite to that shown in II. FIG. 29B shows location of cellobiose transporters motifs on NCU08114 homology model. Motif [LIVM]-Y-[FL]-x(13)-[YF]-D (SEQ ID NO: 1) is shown in red. Motif [YF]-x(2)-G-x(5)-[PVF]-x(6)-[DQ] (SEQ ID NO: 2) is shown in light green. Motif G-R-[RK] (SEQ ID NO: 3) is shown in dark blue. Motif R-x(6)-[YF]-N (SEQ ID NO: 4) is shown in yellow. Motif WR-[IVLA]-P-x(3)-Q (SEQ ID NO: 5) is shown in magenta. Motif P-E-S-P-R-x-L-x(8)-A-x(3)-L-x(2)-Y-H (SEQ ID NO: 6) is shown in cyan. Motif F-[GST]-Q-x-S-G-N-x-[LIV] (SEQ ID NO: 7) is shown in oranges. Motif L-x(3)-[YIV]-x(2)-E-x-L-x(4)-R-[GA]-K-G (SEQ ID NO: 8) is shown in dark green. I. View of NCU08114 from the cytoplasmic side looking into the putative cellobiose binding pore. Note that in this image, some of the residues connecting transmembrane helices 6 and 7 have been removed for clarity as they occlude the pore. II. View of one side of NCU08114. III. View of the side opposite to that shown in II.
FIGS. 30A-B show the cloning process used in the construction of plasmid expressing: (A) putative transporters and (B) transporter-GFP fusion proteins. FIG. 30A shows putative transporters. FIG. 30B shows transporter-GFP fusion proteins.
FIG. 31 shows pentose transport activity of putative transporters identified to have glucose-uptake activity.
FIG. 32 shows pentose transport activity of putative transporters identified to not have glucose-uptake activity.
FIGS. 33A-B show pentose uptake of NCU00821 (AN25), STL12/XUT6 (Xyp29), and XUT1 (Xyp32). FIG. 33A shows xylose uptake. FIG. 33B shows arabinose uptake.
FIG. 34 shows 14C-labeled sugar uptake by S. cerevisiae expressing STL12/XUT6 (Xyp29).
FIG. 35 shows localizations of transporters expressed in S. cerevisiae cells as monitored by GFP fluorescence. First row from left to right: NCU00821-GFP fluorescence, NCU00821 nuclei; second row from left to right: STL12/XUT6-GFP fluorescence, STL12/XUT6 nuclei.
FIGS. 36A-C show the effect on pH upon addition of maltose to un-buffered cell suspension expressing: (a) NCU00821 (AN25), (b) STL12/XUT6 (Xyp29), and (c) XUT1 (Xyp32). The black arrows indicate the time points when maltose was added. FIG. 36A shows NCU00821 (AN25). FIG. 36B shows STL12/XUT6 (Xyp29). FIG. 36C shows XUT1 (Xyp32).
FIGS. 37A-F show results of a symporter assay of NCU00821, STL12/XUT6, and XUT1. FIG. 37A shows NCU00821 for xylose. FIG. 37B shows NCU00821 for arabinose. FIG. 37C shows XUT1 for arabinose. FIG. 37D shows XUT1 for xylose. FIG. 37E shows STL12/XUT6 for arabinose. FIG. 37F shows STL12/XUT6 for xylose. The black arrows the time points when maltose was added.
FIGS. 38A-G show phenotypic analyses of transporter overexpression. FIG. 38A shows OD. FIG. 38B shows xylose concentration. FIG. 38C shows xylose consumption in 0.5% xylose-containing media. FIG. 38D shows OD. FIG. 38E shows xylose concentration. FIG. 38F shows xylose consumption in 5% xylose-containing media. FIG. 38G shows the growth curve of S. cerevisiae containing pentose transporters introduced on pRS424, a multicopy plasmid.
FIG. 39 shows maps of the plasmids used for cloning of heterologous transporters.
FIG. 40 shows results of the sugar-uptake assay by S. cerevisiae strains expressing pentose transporter orthologs.
FIGS. 41A-C show sequence alignments of the pentose transporter orthologs by Clustal W (1.81). FIG. 41A shows alignment of the xylose transporter orthologs. FIG. 41B shows alignment of the arabinose transporters. FIG. 41C shows alignment of xylose and arabinose transporters. Consensus key: *—single, fully conserved residue; :—conservation of strong groups; .—conservation of weak groups.
FIG. 42 describes the different S. cerevisiae strains engineered to express xylose-utilizing enzymes.
FIG. 43 shows xylose metabolism (as monitored by xylose consumption, ethanol production, etc.) of three S. cerevisiae strains of different backgrounds expressing identical cassettes containing xylose utilization pathway enzymes.
FIG. 44 shows xylose-uptake rates and metabolite yields of three S. cerevisiae strains of different backgrounds expressing identical cassettes containing xylose utilization pathway enzymes.
FIGS. 45A-C show xylose fermentation by the S. cerevisiae strain DA24 under various conditions. FIG. 45A shows 40 g/L xylose in a shaker flask. FIG. 45B shows 80 g/L xylose in a shaker flask. FIG. 45C shows 80 g/L xylose in a bioreactor. Symbols: xylose (▪), ethanol (♦), and OD600 (●).
FIGS. 46A-B show a comparison of xylose consumption and ethanol production between (a) S. cerevisiae DA24 and (b) P. stipitis. Symbols: xylose (▪), ethanol (♦), and OD600 (●). FIG. 46A shows S. cerevisiae DA24. FIG. 46B shows P. stipitis. 
FIG. 47 describes the experimental design used to test the effect of XYL2 over-expression levels on xylose metabolism in engineered S. cerevisiae. 
FIG. 48 shows the effect of additional XYL2 integration (i.e. increased XYL2 expression level) into the genome of engineered xylose-fermenting S. cerevisiae. 
FIG. 49 shows the effect of additional simultaneous over-expression of XYL2 and XYL3 on xylose fermentation by engineered S. cerevisiae. 
FIG. 50 describes S. cerevisiae strains expressing different levels of xylose-fermenting enzymes.
FIG. 51 shows the effect of differential XYL1 expression of fermentation by engineered S. cerevisiae. 
FIG. 52 describes S. cerevisiae strains engineered to over-express identical XYL2 and XYL3 but different reductases (XYL1 vs. GRE3).
FIG. 53 shows the effect of over-expressing XYL1 versus GRE3 on xylose fermentation by engineered S. cerevisiae grown in 40 g/L xylose.
FIG. 54 shows the effect of over-expressing XYL1 versus GRE3 on xylose fermentation by engineered S. cerevisiae grown in 80 g/L xylose.
FIGS. 55A-C show the thermal and pH-dependent properties of different wild-type LAD enzymes: anLAD (▪), tlLAD (♦), and pcLAD (●). FIG. 55A shows temperature-dependent catalytic activities. FIG. 55B shows thermal inactivation at 50° C. over time. FIG. 55C shows pH-dependent catalytic activities. Error bars indicate standard error of the mean (n=3).
FIG. 56 shows an alignment of XDH from N. crassa (ncXDH) and P. stipitis (psXDH).
FIG. 57 show a comparison of pH rate profiles of N. crassa LAD and XDH. Data taken from the characterization of LAD was performed in universal buffer MES/Tris/glycine, and overlapped with data for ncXDH (closed triangles) and ncLAD (closed circles) performed in universal buffer acetic acid/MES/Tris for lower pH values.
FIG. 58 shows ethanol production by S. cerevisiae strain L2612 transformed with xylose isomerase enzyme from Bacteroids stercoris (BtXI), Bifidobacterium longum (BfXI), and BtXIO coding for codon-optimized BtXI. The XI gene was cloned into the pRs424TEF vector.
FIG. 59 shows xylose consumption and ethanol production by S. cerevisiae strain D452-2, which had BtXI integrated into its genome by the vector pRS403TEF. Comparison is also made to xylose-fermentation by S. cerevisiae strain L2612, which expresses BtXI from a plasmid
FIG. 60 shows xylose fermentation by S. cerevisiae strain, containing integrated BtXI and expressing XYL2 or XYL3 or XYL2 and XYL3.
FIG. 61 shows the necessity of XYL3 expression in S. cerevisiae engineered to over-express enzymes, such as GND1, involved in the pentose phosphate pathway in order to efficiently metabolize xylose.
FIGS. 62A-B show the effect of over-expression of NCU09705 homologs in E. coli, S. cerevisiae, and P. stipitis on fermentation parameters. Over-expression of galM, GAL10-Sc, GAL10-Ps, YHR210C, and YNR071C on (A) cellobiose consumption, growth, and ethanol production; and on (B) ethanol yield and productivity. FIG. 62A shows cellobiose consumption, growth, and ethanol production. FIG. 62B shows ethanol yield and productivity.
FIGS. 63A-C show the experimental design enabling simultaneous co-fermentation of cellobiose and xylose without glucose repression through integration of a cellodextrin assimilation pathway from filamentous fungi (N. crassa) and modified xylose metabolic pathway from the xylose-fermenting yeast P. stipitis into S. cerevisiae. FIG. 63A shows a strain improvement strategy to engineer yeast strain capable of fermenting two non-metabolizable sugars (cellobiose and xylose). The cellodextrin assimilation pathway consists of a cellodextrin transporter (NCU00801) and an intracellular β-glucosidase (NCU00130) from N. crassa. The modified xylose metabolic pathway utilizes xylose reductase isozymes (wild-type XR and mutant XRR276H), xylitol dehydrogenase (XYL2), and xylulokinase (XKS1). FIG. 63B shows fermentation profile of a sugar mixture containing glucose and xylose by the engineered S. cerevisiae developed in this study. Glucose fermentation repressed xylose fermentation completely so that xylose fermentation begins only after glucose depletion. FIG. 63C shows fermentation profile of a sugar mixture containing cellobiose and xylose by the engineered S. cerevisiae developed in this study. Cellobiose and xylose are simultaneously utilized, as neither carbon source repressed consumption of the other.
FIG. 64 shows the scheme for plasmid construction. The pRS425 shuttle vector was linearized followed by assembly of the cellobiose transporter and β-glucosidase genes using the DNA assembler method (Shao et al., 2009).
FIGS. 65A-G show the change in concentrations of cellobiose (▪), glucose (●), D-xylose (▴), ethanol (▾), and biomass (□) during co-fermentation of 4% cellobiose and 5% D-xylose by S. cerevisiae strains (a) SL01, (b) SL04, (c) SL02, (d) SL05, (e) SL03, (f) SL06, and (g) SL00 as a function of time. FIG. 65A shows SL01. FIG. 65B shows SL04. FIG. 65C shows SL02. FIG. 65D shows SL05. FIG. 65E shows SL03. FIG. 65F shows SL06. FIG. 65G shows SL00.
FIGS. 66A-D show the change in concentrations of cellobiose (▪), glucose (●), D-xylose (▴), ethanol (▾), and biomass (□) in S. cerevisiae strains SL01 (a, c) and SL00 (b, d) grown in cellobiose-xylose mixtures in shake-flasks (a, b) or bioreactors (c, d) plotted as a function of time. FIG. 66A shows S. cerevisiae strain SL01 grown in cellobiose-xylose mixtures in shake-flasks. FIG. 66B shows S. cerevisiae strain SL00 grown in cellobiose-xylose mixtures in shake-flasks. FIG. 66C shows S. cerevisiae strain SL01 grown in cellobiose-xylose mixtures in bioreactors. FIG. 66D shows S. cerevisiae strain SL00 grown in cellobiose-xylose mixtures in bioreactors.
FIGS. 67A-D show the change in concentrations of cellobiose (▪), glucose (●), D-xylose (▴), ethanol (▾), and biomass (□) in S. cerevisiae strains SL01 (a, c) and SL00 (b, d) grown in media containing 5 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture (a, b) or 10 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture (c, d) in bioreactors, plotted as a function of time. FIG. 67A shows S. cerevisiae strain SL01 grown in media containing 5 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture. FIG. 67B shows S. cerevisiae strains SL00 grown in media containing 5 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture. FIG. 67C shows S. cerevisiae strain SL01 grown in media containing 10 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture. FIG. 67D shows S. cerevisiae strains SL00 grown in media containing 10 g/L glucose-40 g/L cellobiose-50 g/L xylose mixture.
FIGS. 68A-C show a comparison of cellobiose utilizations by β-glucosidase (NCU00130)-containing S. cerevisiae strain expressing (a) NCU00801, (b) NCU00809, and (c) NCU08114. Symbols: cellobiose (▪), ethanol (♦), and OD600 (●). FIG. 68A shows NCU00801. FIG. 68B shows NCU00809. FIG. 68C shows NCU08114.
FIGS. 69A-C show co-fermentation of cellobiose and xylose by the S. cerevisiae strain DA24-16BT3 grown in mixtures containing various concentrations of the two sugars: (a) 20 g/L (each) of cellobiose and xylose, (b) 30 g/L (each) of cellobiose and xylose, and (c) 40 g/L (each) of cellobiose and xylose. Symbols: cellobiose (▴), xylose (▪), ethanol (♦), and OD600 (●). FIG. 69A shows 20 g/L (each) of cellobiose and xylose. FIG. 69B shows 30 g/L (each) of cellobiose and xylose. FIG. 69C shows 40 g/L (each) of cellobiose and xylose.
FIGS. 70A-C show the synergistic effects of co-fermentation of cellobiose and xylose by the S. cerevisiae strain DA24-16BT3. Symbols: cellobiose (▴), xylose (▪), ethanol (♦), and OD600 (●). FIG. 70A shows 40 g/L cellobiose. FIG. 70B shows 40 g/L (each) of cellobiose and xylose. FIG. 70C shows 40 g/L xylose.
FIGS. 71A-B show co-fermentation of glucose, cellobiose, and xylose by the S. cerevisiae strain DA24-16BT3 and the wild-type P. stipitis strain. Symbols: cellobiose (▴), xylose (▪), ethanol (♦), OD600 (●), and glucose (▾). FIG. 71A shows DA24-16BT3. FIG. 71B shows P. stipitis. 
FIG. 72 shows HPLC chromatograms from each time point, suggesting cellotriose and cellotetraose accumulation during c-fermentation of cellobiose and xylose by the S. cerevisiae strain DA24-16BT3.
FIG. 73 shows HPAEC analysis demonstrating cellodextrin accumulation in fermentation medium after 22 hours fermentation by the S. cerevisiae strain DA24-16BT3 during co-fermentation of cellobiose and xylose. (G1: glucose, G2: cellobiose, G3: cellotriose, G4: cellotetraose, and G5: cellopentaose).
FIGS. 74A-B show a comparison of sugar utilization by S. cerevisiae transformants expressing (a) an integrated copy of NCU00801 and (b) NCU00801 on a multi-copy plasmid, during co-fermentation of 40 g/L (each) of cellobiose and xylose. Symbols: cellobiose (▴), xylose (▪), ethanol (♦), and OD600 (●). FIG. 74A shows S. cerevisiae transformants expressing an integrated copy of NCU00801. FIG. 74B shows S. cerevisiae transformants expressing NCU00801 on a multi-copy plasmid.
FIGS. 75A-B show ethanol production by cultivation of two different yeast strains. FIG. 75A shows the two different S. cerevisiae strains used in study: DA24-16 and D452BT. A xylose molecule is shown as a pentagon and a cellobiose molecule is shown as two hexagons. FIG. 75B shows mixed cultures of xylose-fermenting strain and cellobiose-fermenting strain.
FIG. 76 shows a listing of 354 xylan-induced genes in N. crassa. 
FIG. 77 shows secreted protein levels, reducing sugar, and azo-xylanase activity for various N. crassa knock-out strains. Secreted protein levels were relatively constant for all strains.
FIG. 78A shows total secreted protein and CMC-activity for wild type, ΔNCU05137, and ΔNCU05137/ΔNCU05137-GFP Neurospora strains. FIG. 78B shows a Coomassie stain of total protein in supernatants from cultures of the three different strains.
FIG. 79 shows localization of NCU05137-GFP in conidia.
FIG. 80 shows localization of NCU05137-GFP in the hypha tip.



DETAILED DESCRIPTION OF THE INVENTION
The present disclosure relates to host cells containing a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, and α-helix 12, where one or more of the following is true: transmembrane α-helix 1 comprises SEQ ID NO: 1, transmembrane α-helix 2 comprises SEQ ID NO: 2, the loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 5 comprises SEQ ID NO: 4, transmembrane α-helix 6 comprises SEQ ID NO: 5, sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6, transmembrane α-helix 7 comprises SEQ ID NO: 7, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8, and where the polypeptide transports cellodextrin into the cell. Further described herein are methods of increasing transport of cellodextrin into a cell, methods of increasing growth of a cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose-derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives using the host cells. Further described herein are host cells containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell, methods of increasing transport of a pentose into a cell, methods of increasing growth of a cell on a medium containing pentose sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell.
As used herein, cellodextrin refers to glucose polymers of varying length and includes, without limitation, cellobiose (2 glucose monomers), cellotriose (3 glucose monomers), cellotetraose (4 glucose monomers), cellopentaose (5 glucose monomers), and cellohexaose (6 glucose monomers).
As used herein, sugar refers to monosaccharides (e.g., glucose, fructose, galactose, xylose, arabinose), disaccharides (e.g., cellobiose, sucrose, lactose, maltose), and oligosaccharides (typically containing 3 to 10 component monosaccharides).
Polynucleotides of the Invention
The invention herein relates to host cells and methods of using such host cells where the host cells comprise recombinant polynucleotides encoding polypeptides capable of transporting various sugars.
As used herein, the terms “polynucleotide,” “nucleic acid sequence,” “sequence of nucleic acids,” and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; inter-nucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970).
As used herein, a “polypeptide” is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues (e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues). In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transporter, a transcription factor, a predicted protein of unknown function, or a domain or portion or fragment thereof. A transporter is involved in the movement of ions, small molecules, or macromolecules, such as a carbohydrate, across a biological membrane. A transcription factor can regulate gene expression and may increase or decrease gene expression in a host cell. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues.
As used herein, “protein” refers to an amino acid sequence, oligopeptide, peptide, polypeptide, or portions thereof whether naturally occurring or synthetic.
Recombinant polynucleotides of the invention include any polynucleotides that encode a polypeptide encoded by any of the genes listed in Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al., 2009; in Tables 14, 15, 16, 29; or in FIG. 76. In preferred embodiments, polynucleotides of the invention include any polynucleotides that encode a polypeptide encoded by any of the sequences NCU00801, NCU00809, NCU08114, NCU00130, NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, XUT3, NCU07705, NCU05137, NCU01517, NCU09133, or NCU10040.







TABLE 1







shows polynucleotides of the invention including sequences listed 


below or sequences encoding polypeptides listed below.












NCBI Reference



Gene
Alternate
Sequence/GenBank



Name/Locus
Name
Accession Number
Organism





NCU00801
cbt1
XP_963801.1/EAA34565
N. crassa


NCU00809

XP_964302.1/EAA35116.1
N. crassa


NCU00821
AN25
XP_964364.2/EAA35128.2
N. crassa


NCU00988
Xy33
XP_963898.1/EAA34662.1
N. crassa


NCU01231

XP_961597.2/EAA32361.2
N. crassa


NCU01494
AN49
XP_955927.2/EAA26691.2
N. crassa


NCU02188
AN28-3
XP_959582.2/EAA30346.2
N. crassa


NCU04537
Xy50
XP_955977.1/EAA26741.1
N. crassa


NCU04963
AN29-2
XP_959411.2/EAA30175.2
N. crassa


NCU05519

XP_960481.1/EAA31245.1
N. crassa


NCU05853

XP_959844.1/EAA30608.1
N. crassa


NCU05897

XP_959888.1/EAA30652.1 
N. crassa


NCU06138
Xy31
XP_960000.1/EAA30764.1
N. crassa


NCU08114
cbt2
XP_963873.1/EAA34637.1
N. crassa


NCU09287
AN41
XP_958139.1/EAA28903.1
N. crassa


NCU10021

XP_958069.2/EAA28833.2 
N. crassa


XP_001387242
Ap26
XP_001387242
P. stipitis


HGT3
Xyp30-1
XP_001386715.1/ABN68686.1
P. stipitis


STL1
Xyp30
XP_001383774.1/ABN65745.1
P. stipitis


STL12/XUT6
Xyp29
XP_001386589.1/ABN68560.1
P. stipitis


SUT2
Ap31
XP_001384295.2/ABN66266.2
P. stipitis


SUT3
Xyp37
XP_001386019.2/ABN67990.2
P. stipitis


XUT1
Xyp32
XP_001385583.1/ABN67554.1
P. stipitis


XUT2
Xyp31
XP_001387242.1/EAZ63219.2
P. stipitis


XUT3
Xyp33
XP_001387138.1/EAZ63115.1
P. stipitis


XUT7
Xyp28
XP_001387067.1/EAZ63044.1
P. stipitis


NCU07705
cdr-1
XP_962291.1/EAA33055
N. crassa


NCU05137

XP_956635.1/EAA27399
N. crassa


NCU01517

XP_956966.1/EAA27730
N. crassa


NCU09133

XP_958905.1/EAA29669
N. crassa


NCU10040


N. crassa









In certain embodiments, the recombinant polynucleotides of the invention encode polypeptides having at least about 20%, or at least about 29%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 92%, or at least about 94%, or at least about 96%, or at least about 98%, or at least about 99%, or at least about 100% amino acid residue sequence identity to a polypeptide encoded by any of the genes listed in genes listed in Table 10, in Supplemental Data, Dataset S1, page 3 in Tian et al., 2009; in Tables 14, 15, 16, 29; or in FIG. 76. In preferred embodiments, the polynucleotides of the invention encode polypeptides having at least about 20%, or at least about 29%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 92%, or at least about 94%, or at least about 96%, or at least about 98%, or at least about 99%, or at least about 100% amino acid residue sequence identity to a polypeptide encoded by any of the sequences NCU00801, NCU00809, NCU08114, NCU00130, NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, XUT3, NCU07705, NCU05137, NCU01517, NCU09133, or NCU10040.
Polynucleotides of the invention further include polynucleotides that encode conservatively modified variants of polypeptides encoded by the genes listed above. “Conservatively modified variants” as used herein include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
Polynucleotides of the invention further include polynucleotides that encode homologs or orthologs of polypeptides encoded by any of the genes listed in Table 10, in Supplemental Data, Dataset 51, page 3 in Tian et al., 2009; in Tables 14, 15, 16, 29; or in FIG. 76. “Homology” as used herein refers to sequence similarity between a reference sequence and at least a fragment of a second sequence. Homologs may be identified by any method known in the art, preferably, by using the BLAST tool to compare a reference sequence to a single second sequence or fragment of a sequence or to a database of sequences. As described below, BLAST will compare sequences based upon percent identity and similarity. “Orthology” as used herein refers to genes in different species that derive from a common ancestor gene.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity. For blastn, the default parameters are Gap opening penalty=5 and Gap extension penalty=2. For blastp, the default parameters are Gap opening penalty=11 and Gap extension penalty=1.
A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions including, but not limited to from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the search for similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA 85(8):2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection [see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].
Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919] alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
As described herein, polynucleotides of the invention include members of the Major Facilitator Superfamily sugar transporter family, including NCU00988, NCU10021, NCU04963, NCU06138, NCU00801, NCU08114, and NCU05853. Members of the Major Facilitator Superfamily (MFS) (Transporter Classification #2.A.1) of transporters almost always consist of 12 transmembrane α-helices, with an intracellular N- and C-terminus (S. S. Pao, I. T. Paulsen, M. H. Saier, Jr., Microbiol Mol Biol Rev 62, 1 (March, 1998)). While the primary sequence of MFS transporters varies widely, all are thought to share the tertiary structure of the E. coli lactose permease (LacY) (J. Abramson et al., Science 301, 610 (Aug. 1, 2003)), and the E. coli Pi/glycerol-3-phospate (GlpT) (Y. Huang, M. J. Lemieux, J. Song, M. Auer, D. N. Wang, Science 301, 616 (Aug. 1, 2003)). In these examples the six N- and C-terminal helices form two distinct domains connected by a long cytoplasmic loop between helices 6 and 7. This symmetry corresponds to a duplication event thought to have given rise to the MFS. Substrate binds within a hydrophilic cavity formed by helices 1, 2, 4, and 5 of the N-terminal domain, and helices 7, 8, 10, and 11 of the C-terminal domain. This cavity is stabilized by helices 3, 6, 9, and 12.
The Sugar Transporter family of the MFS (Transporter Classification #2.A.1.1) is defined by motifs found in transmembrane helices 6 and 12 (PESPR (SEQ ID NO: 9)/PETK (SEQ ID NO: 10)), and loops 2 and 8 (GRR/GRK) (M. C. Maiden, E. O. Davis, S. A. Baldwin, D. C. Moore, P. J. Henderson, Nature 325, 641 (Feb. 12-18, 1987)). The entire Hidden Markov Model (HMM) for this family can be viewed at pfam.janelia.org/family/PF00083#tabview=tab3. PROSITE (N. Hulo et al., Nucleic Acids Res 34, D227 (Jan. 1, 2006)) uses two motifs to identify members of this family. The first is [LIVMSTAG]-[LIVMFSAG]-{SH}-{RDE}-[LIVMSA]-[DE]-{TD}-[LIVMFYWA]-G-R-[RK]-x(4,6)-[GSTA] (SEQ ID NO: 11). The second is [LIVMF]-x-G-[LIVMFA]-{V}-x-G-{KP}-x(7)-[LIFY]-x(2)-[EQ]-x(6)-[RK] (SEQ ID NO: 12). As an example of how to read a PROSITE motif, the following motif, [AC]-x-V-x(4)-{ED}, is translated as: [Ala or Cys]-any-Val-any-any-any-any-{any but Glu or Asp} (SEQ ID NO: 13).
As described herein, NCU00801, NCU00809, NCU08114, XP—001268541.1, and LAC2 were discovered to encode polypeptides that transport cellodextrins. Further, alanine scanning experiments and sequence analyses were used to determine that a recombinant polypeptide containing 12 transmembrane α-helices, and one or more of the sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 encodes a polypeptide that transports cellodextrin.
Thus, in one aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 1 comprises SEQ ID NO: 1. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 2 comprises SEQ ID NO: 2. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 5 comprises SEQ ID NO: 4. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 6 comprises SEQ ID NO: 5. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 7 comprises SEQ ID NO: 7. In another aspect, the invention provides a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8.
Each of the above described aspects may be combined in any number of combinations. A polynucleotide according to any of these aspects may encode a polypeptide containing 1, 2, 3, 4, 5, 6, or 7 of any of SEQ ID NOs: 1-8, or the polypeptide may contain all of SEQ ID NOs: 1-8. For example, a polynucleotide may encode a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 1 comprises SEQ ID NO: 1, a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, and transmembrane α-helix 7 comprises SEQ ID NO: 7. Or, in another example, a polynucleotide may encode a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 2 comprises SEQ ID NO: 2, transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 6 comprises SEQ ID NO: 5, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8.
In certain embodiments of the above described aspects, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114.
As further described herein, NCU08221 and STL12/XUT6 were discovered to encode polypeptides that transport xylose. XUT1 was discovered to encode a polypeptide that transports arabinose. NCU06138 was discovered to encode a polypeptide that transports arabinose or glucose. SUT2, SUT3, and XUT3 were discovered to encode polypeptides that transport xylose or glucose. NCU04963 was discovered to encode a polypeptide that transports xylose, arabinose, or glucose. In preferred embodiments, polynucleotides of the invention include recombinant polynucleotides encoding a NCU08221 or STL12/XUT6 polypeptide, where the polypeptide transports xylose. In other preferred embodiments, polynucleotides of the invention include recombinant polynucleotides encoding a XUT1 polypeptide, where the polypeptide transports arabinose. In other preferred embodiments, polynucleotides of the invention include recombinant polynucleotides encoding a NCU06138 polypeptide, where the polypeptide transports arabinose or glucose. In other preferred embodiments, polynucleotides of the invention include recombinant polynucleotides encoding a SUT2, SUT3, or XUT3 polypeptide, where the polypeptide transports xylose or glucose. In other preferred embodiments, polynucleotides of the invention include recombinant polynucleotides encoding a NCU04963 polypeptide, where the polypeptide transports xylose, arabinose, or glucose.
The polynucleotides of the invention that encode polypeptides encoded by NCU07705 are predicted by FunCat (Ruepp, 2004; webpage broad.mit.edu/annotation/genome/neurospora/Home.html) to encode an unclassified protein. However, BLAST analysis of the polypeptide encoded by NCU07705 revealed that the polypeptide has high similarity to many C6 zinc finger domain containing transcription factors (see FIG. 1; a list of exemplary homologs can be found in FIG. 23 of related U.S. Appl. No. 61/271,833). Polynucleotides of the invention include polynucleotides that encode these homologs of the polypeptide encoded by NCU07705 or any other homologs identified with any methods known in the art.
In another aspect of the invention, polynucleotides of the invention include those polynucleotides that encode polypeptides encoded by NCU05137. FunCat classifies the polypeptide encoded by NCU05137 to be an unclassified protein. However, NCU05137 is highly conserved in the genomes of a number of filamentous ascomycete fungi (see FIG. 2). Polynucleotides of the invention include polynucleotides that encode these homologs of the polypeptide encoded by NCU05137 or any other homologs identified with any methods known in the art.
In another aspect of the invention, polynucleotides of the invention include those polynucleotides that encode polypeptides encoded by NCU01517, NCU09133, or NCU10040. FunCat classifies the polypeptide encoded by NCU01517 to be a glucoamylase precursor. FunCat classifies the polypeptides encoded by NCU09133 and NCU10040 to be unclassified proteins. Polynucleotides of the invention include polynucleotides that encode these homologs of the polypeptide encoded by NCU01517, NCU09133, or NCU10040 or any other homologs identified with any methods known in the art.
Predicted functions of these polypeptides can be confirmed by performing functional analyses of the polynucleotide and its encoded protein. These analyses may include, for example, phenotypic analysis of strains containing deletions of the polynucleotide, genetic complementation experiments, phenotypic analysis of strains over expressing a wild-type copy of the polynucleotide, expression and purification of a recombinant form of the polypeptide, and subsequent characterization of the biochemical properties and activity of the recombinant polypeptide.
Sequences of the polynucleotides of the invention are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a polymer of nucleic acids typically involves sequential addition of 3′-blocked and 5′-blocked nucleotide monomers to the terminal 5′-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5′-hydroxyl group of the growing chain on the 3′-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. Such methodology is known to those of ordinary skill in the art and is described in the pertinent texts and literature [e.g., in Matteucci et al., (1980) Tetrahedron Lett 21:719-722; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637]. In addition, the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired nucleic acid sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No. 4,683,195).
Each polynucleotide of the invention can be incorporated into an expression vector. “Expression vector” or “vector” refers to a compound and/or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell. An “expression vector” contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Preferred expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.
Incorporation of the individual polynucleotides may be accomplished through known methods that include, for example, the use of restriction enzymes (such as BamHI, EcoRI, HhaI, XhoI, XmaI, and so forth) to cleave specific sites in the expression vector, e.g., plasmid. The restriction enzyme produces single stranded ends that may be annealed to a polynucleotide having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. Annealing is performed using an appropriate enzyme, e.g., DNA ligase. As will be appreciated by those of ordinary skill in the art, both the expression vector and the desired polynucleotide are often cleaved with the same restriction enzyme, thereby assuring that the ends of the expression vector and the ends of the polynucleotide are complementary to each other. In addition, DNA linkers maybe used to facilitate linking of nucleic acids sequences into an expression vector.
A series of individual polynucleotides can also be combined by utilizing methods that are known to those having ordinary skill in the art (e.g., U.S. Pat. No. 4,683,195).
For example, each of the desired polynucleotides can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3′ ends overlap and can act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are “spliced” together. In this way, a series of individual polynucleotides may be “spliced” together and subsequently transduced into a host cell simultaneously. Thus, expression of each of the plurality of polynucleotides is affected.
Individual polynucleotides, or “spliced” polynucleotides, are then incorporated into an expression vector. The invention is not limited with respect to the process by which the polynucleotide is incorporated into the expression vector. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a polynucleotide into an expression vector. A typical expression vector contains the desired polynucleotide preceded by one or more regulatory regions, along with a ribosome binding site, e.g., a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initiation codon in E. coli. See Shine and Dalgarno (1975) Nature 254(5495):34-38 and Steitz (1979) Biological Regulation and Development (ed. Goldberger, R. F.), 1:349-399 (Plenum, New York).
The term “operably linked” as used herein refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the DNA sequence or polynucleotide such that the control sequence directs the expression of a polypeptide.
Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired polynucleotide, thereby initiating transcription of the polynucleotide via an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. Examples include lactose promoters (Lad repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator). Another example is the tac promoter (see de Boer et al., (1983) Proc Natl Acad Sci USA 80(1):21-25). As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present invention, and the invention is not limited in this respect.
Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSClOl, pBR322, pBBRlMCS-3, pUR, pEX, pMRlOO, pCR4, pBAD24, pUC19; bacteriophages, such as Ml 3 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.
Host Cells of the Invention
The invention herein relates to host cells containing recombinant polynucleotides encoding polypeptides where the polypeptides transport cellodextrin or a pentose into the cell. Further described herein are methods of increasing transport of cellodextrin into a host cell, methods of increasing growth of a host cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose-derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports cellodextrin into the cell. Further described herein are methods of increasing transport of a pentose into a host cell, methods of increasing growth of a host cell on a medium containing pentose sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell.
“Host cell” and “host microorganism” are used interchangeably herein to refer to a living biological cell that can be transformed via insertion of recombinant DNA or RNA. Such recombinant DNA or RNA can be in an expression vector. Thus, a host organism or cell as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.
Any prokaryotic or eukaryotic host cell may be used in the present invention so long as it remains viable after being transformed with a sequence of nucleic acids. Preferably, the host cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., transporters), or the resulting intermediates. Suitable eukaryotic cells include, but are not limited to, fungal, plant, insect or mammalian cells.
In preferred embodiments, the host is a fungal strain. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
In particular embodiments, the fungal host is a yeast strain. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
In a more preferred embodiment, the yeast host is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain.
In certain embodiments, the yeast host is a Saccharomyces carlsbergensis (Todkar, 2010), Saccharomyces cerevisiae (Duarte et al., 2009), Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces monacensis (GB-Analysts Reports, 2008), Saccharomyces bayanus (Kristen Publicover, 2010), Saccharomyces pastorianus (Nakao et al., 2007), Saccharomyces pombe (Mousdale, 2008), or Saccharomyces oviformis strain. In other preferred embodiments, the yeast host is Kluyveromyces lactis (O. W. Merten, 2001), Kluyveromyces fragilis (Pestal et al., 2006; Siso, 1996), Kluyveromyces marxiamus (K. Kourkoutas et al., 2008), Pichia stipitis (Almeida et al., 2008), Candida shehatae (Ayhan Demirbas, 2003), or Candida tropicalis (Jamai et al., 2006). In other embodiments, the yeast host may be Yarrowia lipolytica (Biryukova E. N., 2009), Brettanomyces custersii (Spindler D. D. et al., 1992), or Zygosaccharomyces roux (Chaabane et al., 2006).
In another particular embodiment, the fungal host is a filamentous fungal strain. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
In preferred embodiments, the filamentous fungal host is, but not limited to, an Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma strain.
In certain embodiments, the filamentous fungal host is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae strain. In other embodiments, the filamentous fungal host is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum strain. In yet other preferred embodiments, the filamentous fungal host is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Scytalidium thermophilum, Sporotrichum thermophile (Topakas et al., 2003), or Thielavia terrestris strain. In a further embodiment, the filamentous fungal host is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain.
In other preferred embodiments, the host cell is prokaryotic, and in certain embodiments, the prokaryotes are E. coli (Dien, B. S. et al., 2003; Yomano, L. P. et al., 1998; Moniruzzaman et al., 1996), Bacillus subtilis (Susana Romero et al., 2007), Zymomonas mobilis (B. S. Dien et al, 2003; Weuster Botz, 1993; Alterthum and Ingram, 1989), Clostridium sp. (Zeikus, 1980; Lynd et al., 2002; Demain et al., 2005), Clostridium phytofermentans (Leschine S., 2010), Clostridium thermocellum (Lynd et al., 2002), Clostridium beijerinckii (Giles Clark, 2008), Clostridium acetobutylicum (Moorella thermoacetica) (Huang W. C. et al., 2004; Dominik et al., 2007), Thermoanaerobacterium saccharolyticum (Marietta Smith, 2009), or Klebsiella oxytoca (Dien, B. S. et al., 2003; Zhou et al., 2001; Brooks and Ingram, 1995). In other embodiments, the prokaryotic host cells are Carboxydocella sp. (Dominik et al., 2007), Corynebacterium glutamicum (Masayuki Inui, et al., 2004), Enterobacteriaceae (Ingram et al., 1995), Erwinia chrysanthemi (Zhou and Ingram, 2000; Zhou et al., 2001), Lactobacillus sp. (McCaskey, T. A., et al., 1994), Pediococcus acidilactici (Zhou, S. et al., 2003), Rhodopseudomonas capsulata (X. Y. Shi et al., 2004), Streptococcus lactis (J.C. Tang et al., 1988), Vibrio furnissii (L. P. Wackett, 2010), Vibrio furnissii M1 (Park et al, 2001), Caldicellulosiruptor saccharolyticus (Z. Kadar et al., 2004), or Xanthomonas campestris (S.T. Yang et al., 1987). In other embodiments, the host cells are cyanobacteria. Additional examples of bacterial host cells include, without limitation, those species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, and Paracoccus taxonomical classes.
In especially preferred embodiments of the invention, the host cell is Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, or Bacillus subtilis. Saccharomyces sp. may include Industrial Saccharomyces strains. Argueso et al. discuss the genome structure of an Industrial Saccharomyces strain commonly used in bioethanol production as well as specific gene polymorphisms that are important for bioethanol production (Genome Research, 19: 2258-2270, 2009).
The host cells of the present invention may be genetically modified in that recombinant nucleic acids have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing one or more nucleic acid constructs encoding one or more proteins for different functions.
“Recombinant nucleic acid” or “heterologous nucleic acid” or “recombinant polynucleotide” as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present invention describes the introduction of an expression vector into a host cell, wherein the expression vector contains a nucleic acid sequence coding for a protein that is not normally found in a host cell or contains a nucleic acid coding for a protein that is normally found in a cell but is under the control of different regulatory sequences. With reference to the host cell's genome, then, the nucleic acid sequence that codes for the protein is recombinant.
In some embodiments, the host cell naturally produces any of the proteins encoded by the polynucleotides of the invention. The genes encoding the desired proteins may be heterologous to the host cell or these genes may be endogenous to the host cell but are operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the host cell. In other embodiments, the host cell does not naturally produce the desired proteins, and comprises heterologous nucleic acid constructs capable of expressing one or more genes necessary for producing those molecules.
“Endogenous” as used herein with reference to a nucleic acid molecule or polypeptide and a particular cell or microorganism refers to a nucleic acid sequence or peptide that is in the cell and was not introduced into the cell using recombinant engineering techniques; for example, a gene that was present in the cell when the cell was originally isolated from nature.
“Genetically engineered” or “genetically modified” refer to any recombinant DNA or RNA method used to create a prokaryotic or eukaryotic host cell that expresses a protein at elevated levels, at lowered levels, or in a mutated form. In other words, the host cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby been altered so as to cause the cell to alter expression of a desired protein. Methods and vectors for genetically engineering host cells are well known in the art; for example various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Genetically engineering techniques include but are not limited to expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Pat. No. 5,272,071 to Chappel) and trans-activation by engineered transcription factors (see, for example, Segal et al., (1999) Proc Natl Acad Sci USA 96(6):2758-2763).
Genetic modifications that result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. More specifically, reference to increasing the action (or activity) of enzymes or other proteins discussed herein generally refers to any genetic modification of the host cell in question which results in increased expression and/or functionality (biological activity) of the enzymes or proteins and includes higher activity or action of the proteins (e.g., specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the proteins, and overexpression of the proteins. For example, gene copy number can be increased, expression levels can be increased by use of a promoter that gives higher levels of expression than that of the native promoter, or a gene can be altered by genetic engineering or classical mutagenesis to increase the biological activity of an enzyme or action of a protein. Combinations of some of these modifications are also possible.
Genetic modifications which result in a decrease in gene expression, in the function of the gene, or in the function of the gene product (i.e., the protein encoded by the gene) can be referred to as inactivation (complete or partial), deletion, interruption, blockage, silencing, or down-regulation, or attenuation of expression of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the protein encoded by such gene, can be the result of a complete deletion of the gene (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene which results in incomplete or no translation of the protein (e.g., the protein is not expressed), or a mutation in the gene which decreases or abolishes the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity or action). More specifically, reference to decreasing the action of proteins discussed herein generally refers to any genetic modification in the host cell in question, which results in decreased expression and/or functionality (biological activity) of the proteins and includes decreased activity of the proteins (e.g., decreased transport), increased inhibition or degradation of the proteins as well as a reduction or elimination of expression of the proteins. For example, the action or activity of a protein of the present invention can be decreased by blocking or reducing the production of the protein, reducing protein action, or inhibiting the action of the protein. Combinations of some of these modifications are also possible. Blocking or reducing the production of a protein can include placing the gene encoding the protein under the control of a promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the protein (and therefore, of protein synthesis) could be turned off. Blocking or reducing the action of a protein could also include using an excision technology approach similar to that described in U.S. Pat. No. 4,743,546, incorporated herein by reference. To use this approach, the gene encoding the protein of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Pat. No. 4,743,546, or by some other physical or nutritional signal.
In general, according to the present invention, an increase or a decrease in a given characteristic of a mutant or modified protein (e.g., enzyme activity, ability to transport compounds) is made with reference to the same characteristic of a wild-type (i.e., normal, not modified) protein that is derived from the same organism (from the same source or parent sequence), which is measured or established under the same or equivalent conditions. Similarly, an increase or decrease in a characteristic of a genetically modified host cell (e.g., expression and/or biological activity of a protein, or production of a product) is made with reference to the same characteristic of a wild-type host cell of the same species, and preferably the same strain, under the same or equivalent conditions. Such conditions include the assay or culture conditions (e.g., medium components, temperature, pH, etc.) under which the activity of the protein (e.g., expression or biological activity) or other characteristic of the host cell is measured, as well as the type of assay used, the host cell that is evaluated, etc. As discussed above, equivalent conditions are conditions (e.g., culture conditions) which are similar, but not necessarily identical (e.g., some conservative changes in conditions can be tolerated), and which do not substantially change the effect on cell growth or enzyme expression or biological activity as compared to a comparison made under the same conditions.
Preferably, a genetically modified host cell that has a genetic modification that increases or decreases the activity of a given protein (e.g., a transporter, an enzyme) has an increase or decrease, respectively, in the activity or action (e.g., expression, production and/or biological activity) of the protein, as compared to the activity of the wild-type protein in a wild-type host cell, of at least about 5%, and more preferably at least about 10%, and more preferably at least about 15%, and more preferably at least about 20%, and more preferably at least about 25%, and more preferably at least about 30%, and more preferably at least about 35%, and more preferably at least about 40%, and more preferably at least about 45%, and more preferably at least about 50%, and more preferably at least about 55%, and more preferably at least about 60%, and more preferably at least about 65%, and more preferably at least about 70%, and more preferably at least about 75%, and more preferably at least about 80%, and more preferably at least about 85%, and more preferably at least about 90%, and more preferably at least about 95%, or any percentage, in whole integers between 5% and 100% (e.g., 6%, 7%, 8%, etc.). The same differences are preferred when comparing an isolated modified nucleic acid molecule or protein directly to the isolated wild-type nucleic acid molecule or protein (e.g., if the comparison is done in vitro as compared to in vivo).
In another aspect of the invention, a genetically modified host cell that has a genetic modification that increases or decreases the activity of a given protein (e.g., a transporter, an enzyme) has an increase or decrease, respectively, in the activity or action (e.g., expression, production and/or biological activity) of the protein, as compared to the activity of the wild-type protein in a wild-type host cell, of at least about 2-fold, and more preferably at least about 5-fold, and more preferably at least about 10-fold, and more preferably about 20-fold, and more preferably at least about 30-fold, and more preferably at least about 40-fold, and more preferably at least about 50-fold, and more preferably at least about 75-fold, and more preferably at least about 100-fold, and more preferably at least about 125-fold, and more preferably at least about 150-fold, or any whole integer increment starting from at least about 2-fold (e.g., 3-fold, 4-fold, 5-fold, 6-fold, etc.).
Host Cell Components
In one aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 1 comprises SEQ ID NO: 1. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 2 comprises SEQ ID NO: 2. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where the loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 5 comprises SEQ ID NO: 4. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 6 comprises SEQ ID NO: 5. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 7 comprises SEQ ID NO: 7. In another aspect, host cells of the invention contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8.
Each of the above described aspects may be combined in any number of combinations. A host cell may contain a polynucleotide encoding a polypeptide containing 1, 2, 3, 4, 5, 6, or 7 of any of SEQ ID NOs: 1-8, or the polypeptide may contain all of SEQ ID NOs: 1-8. For example, a host cell may contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 1 comprises SEQ ID NO: 1, a loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, and transmembrane α-helix 7 comprises SEQ ID NO: 7. Or, in another example, a host cell may contain a polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, where transmembrane α-helix 2 comprises SEQ ID NO: 2, transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 6 comprises SEQ ID NO: 5, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8.
In certain embodiments of the above described aspects, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114.
In preferred embodiments, the host cells further contain a polynucleotide, where the polynucleotide encodes a catalytic domain of a β-glucosidase. As used herein, β-glucosidase refers to a β-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues with the release of β-D-glucose. A catalytic domain of β-glucosidase has β-glucosidase activity as determined, for example, according to the basic procedure described by Venturi et al., 2002. A catalytic domain of a β-glucosidase is any domain that catalyzes the hydrolysis of terminal non-reducing residues in β-D-glucosides with release of glucose. In preferred embodiments, the β-glucosidase is a glycosyl hydrolase family 1 member. Members of this group can be identified by the motif, [LIVMFSTC]-[LIVFYS]-[LIV]-[LIVMST]-E-N-G-[LIVMFAR]-[CSAGN] (SEQ ID NO: 14). Here, E is the catalytic glutamate (webpage expasy.org/cgi-bin/prosite-search-ac?PD0000495). In certain embodiments, the polynucleotide encoding a catalytic domain of β-glucosidase is heterologous to the host cell. In preferred embodiments, the catalytic domain of β-glucosidase is located intracellularly in the host cell. In preferred embodiments, the β-glucosidase is from N. crassa, and in particularly preferred embodiments, the β-glucosidase is NCU00130. In certain embodiments, the β-glucosidase may be an ortholog of NCU00130. Examples of orthologs of NCU00130 include, without limitation, T. melanosporum, CAZ82985.1; A. oryzae, BAE57671.1; P. placenta, EED81359.1; P. chrysosporium, BAE87009.1; Kluyveromyces lactis, CAG99696.1; Laccaria bicolor, EDR09330; Clavispora lusitaniae, EEQ37997.1; and Pichia stipitis, ABN67130.1. Other β-glucosidases could be used include those from the glycosyl hydrolase family 3. These β-glucosidases can be identified by the following motif according to PROSITE: [LIVM](2)-[KR]-x-[EQKRD]-x(4)-G-[LIVMFTC]-[LIVT]-[LIVMF]-[ST]-D-x(2)-[SGADNIT] (SEQ ID NO: 15). Here D is the catalytic aspartate. Typically, any β-glucosidase may be used that contains the conserved domain of β-glucosidase/6-phospho-β-glucosidase/β-galactosidase found in NCBI sequence COG2723. Catalytic domains from specific β-glucosidases may be preferred depending on the cellodextrin transporter contained in the host cell.
In certain embodiments, the host cell contains one or more polynucleotides, where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. The one or more polynucleotides may be endogenous or heterologous to the host cell. Pentose, as used herein, refers to any monosaccharide with five carbon atoms. Examples of pentoses include, without limitation, xylose, arabinose, mannose, galactose, and rhamnose. The one or more enzymes involved in pentose utilization may include, for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabitinol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase in any combination. These enzymes may come from any organism that naturally metabolizes pentose sugars. Examples of such organisms include, for example, Kluyveromyces sp., Zymomonas sp., E. coli, Clostridium sp., and Pichia sp.
Examples 12-15 describe ways in which the pentose utilization pathway in the host cell may be improved or made to be more efficient. Strain background of a host cell can affect the efficiency of its pentose utilization pathway. In embodiments of the invention where the host cell is a Saccharomyces sp., preferred pentose utilizing strains include DA24-16 (see Example 13) and L2612 (see Example 16). Other host cells containing polynucleotides encoding enzymes involved in pentose utilization include a DuPont Zymomonas strain (WO 2009/058927) and a Saccharomyces strain (U.S. Pat. No. 5,789,210).
In certain embodiments of the invention, the host cell contains a recombinant polynucleotide encoding a pentose transporter. In certain embodiments, pentose transporters include those transporters discovered and described herein, including NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3 (see Example 11). In other embodiments, pentose transporters may include Gxs1 from C. intermedia, Aut1 from P. stipitis, Xylhp from D. hansenii (Nobre et al., 1999), xylose transporter from K. marxianus (Stambuk et al., 2003), LAT1 and LAT2 from Ambrosiozyma monospora (EMBL AY923868 and AY923869, respectively, R. Verho et al.), ART1 from C. arabinofermentans (Fonseca et al., 2007), KmLAT1 from K. marxiamus (Knoshaug et al., 2007), PgLAT2 from P. guilliermondii (Knoshaug et al., 2007), and araT from P. stipitis (Boles & Keller, 2008).
Methods of Producing and Culturing Host Cells of the Invention
The invention herein relates to host cells containing recombinant polynucleotides encoding polypeptides where the polypeptide transports cellodextrin or a pentose into the cell. Further described herein are methods of increasing transport of cellodextrin into a host cell, methods of increasing growth of a host cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose-derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports cellodextrin into the cell. Further described herein are methods of increasing transport of a pentose into a host cell, methods of increasing growth of a host cell on a medium containing pentose sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a polypeptide where the polypeptide transports a pentose into the cell.
Methods of producing and culturing host cells of the invention may include the introduction or transfer of expression vectors containing the recombinant polynucleotides of the invention into the host cell. Such methods for transferring expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host cell. Also, microinjection of the nucleic acid sequences provides the ability to transfect host cells. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.
The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host, or a transposon may be used.
The vectors preferably contain one or more selectable markers which permit easy selection of transformed hosts. A selectable marker is a gene the product of which provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selection of bacterial cells may be based upon antimicrobial resistance that has been conferred by genes such as the amp, gpt, neo, and hyg genes.
Suitable markers for yeast hosts are, for example, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in Aspergillus are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. Preferred for use in Trichoderma are bar and amdS.
The vectors preferably contain an element(s) that permits integration of the vector into the host's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host genome, the vector may rely on the gene's sequence or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host. The additional nucleotide sequences enable the vector to be integrated into the host genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a sequence that enables a plasmid or vector to replicate in vivo. Examples of origins of replication for use in a yeast host are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991; Cullen et al., 1987; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
For other hosts, transformation procedures may be found, for example, in Jeremiah D. Read, et al., Applied and Environmental Microbiology, August 2007, p. 5088-5096, for Kluyveromyces, in Osvaldo Delgado, et al., FEMS Microbiology Letters 132, 1995, 23-26, for Zymomonas, in U.S. Pat. No. 7,501,275 for Pichia stipitis, and in WO 2008/040387 for Clostridium. 
More than one copy of a gene may be inserted into the host to increase production of the gene product. An increase in the copy number of the gene can be obtained by integrating at least one additional copy of the gene into the host genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the gene, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
The host cell is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the nucleic acid sequences necessary.
Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. Methods of the invention may include culturing the host cell such that recombinant nucleic acids in the cell are expressed. For microbial hosts, this process entails culturing the cells in a suitable medium. Typically cells are grown at 35° C. in appropriate media. Preferred growth media in the present invention include, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular host cell will be known by someone skilled in the art of microbiology or fermentation science. Temperature ranges and other conditions suitable for growth are known in the art (see, e.g., Bailey and Ollis 1986).
According to some aspects of the invention, the culture media contains a carbon source for the host cell. Such a “carbon source” generally refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, for example, various monosaccharides such as glucose, oligosaccharides, polysaccharides, a biomass polymer such as cellulose or hemicellulose, xylose, arabinose, disaccharides, such as sucrose, saturated or unsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., or mixtures thereof. The carbon source can additionally be a product of photosynthesis, including, but not limited to glucose.
In preferred embodiments, the carbon source is a biomass polymer such as cellulose or hemicellulose. “A biomass polymer” as described herein is any polymer contained in biological material. The biological material may be living or dead. A biomass polymer includes, for example, cellulose, xylan, xylose, hemicellulose, lignin, mannan, and other materials commonly found in biomass. Non-limiting examples of sources of a biomass polymer include grasses (e.g., switchgrass, Miscanthus), rice hulls, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, leaves, grass clippings, corn stover, corn cobs, distillers grains, legume plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust, and biomass crops (e.g., Crambe).
In addition to an appropriate carbon source, media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathways necessary for the fermentation of various sugars and the production of hydrocarbons and hydrocarbon derivatives. Reactions may be performed under aerobic or anaerobic conditions where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism. As the host cell grows and/or multiplies, expression of the enzymes, transporters, or other proteins necessary for growth on various sugars or biomass polymers, sugar fermentation, or synthesis of hydrocarbons or hydrocarbon derivatives is affected.
Methods of Increasing Transport of a Sugar into a Cell
The present invention provides methods of increasing transport of a sugar into a cell. In one aspect, the invention provides a method of transporting cellodextrin into a cell, including a first step of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, and α-helix 12, where one or more of the following is true: transmembrane α-helix 1 comprises SEQ ID NO: 1, transmembrane α-helix 2 comprises SEQ ID NO: 2, the loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 5 comprises SEQ ID NO: 4, transmembrane α-helix 6 comprises SEQ ID NO: 5, sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6, transmembrane α-helix 7 comprises SEQ ID NO: 7, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8. The method includes a second step of culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not contain the recombinant polynucleotide. Transport of cellodextrin into a cell may be measured by any method known to one of skill in the art, including those methods described in Example 9 such as measuring uptake of [3H]-cellobiose into cells or measuring the ability of an S. cerevisiae host cell to grow when cellobiose is the sole carbon source. Typically, the host cell containing the recombinant polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background.
In certain embodiments, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments, the host cell also contains a recombinant polynucleotide encoding a catalytic domain of a β-glucosidase. Such embodiments are useful for host cells lacking the endogenous ability to utilize cellodextrins. Preferably, the catalytic domain of the β-glucosidase is intracellular. In preferred embodiments, the β-glucosidase is from Neurospora crassa. In particularly preferred embodiments, the β-glucosidase is encoded by NCU00130.
In methods of increasing transport of cellodextrin into a cell, the cell may be cultured in a medium containing a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.
In another aspect, the invention provides a method of increasing transport of xylose into a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU00821 or STL12/XUT6 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of xylose into the cell compared with a cell that does not contain the recombinant polynucleotide. In another aspect, the invention provides a method of increasing transport of arabinose into a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a XUT1 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of arabinose into the cell compared with a cell that does not contain the recombinant polynucleotide. In yet another aspect, the invention provides a method of increasing transport of arabinose or glucose into a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU06138 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of arabinose or glucose into the cell compared with a cell that does not contain the recombinant polynucleotide. In yet another aspect the invention provides a method of increasing transport of xylose or glucose into a cell, including the steps of providing a host cell, where the host cell comprises a recombinant polynucleotide encoding a SUT2, SUT3, or XUT3 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of xylose or glucose into the cell compared with a cell that does not contain the recombinant polynucleotide. In another aspect, the invention provides a method of increasing transport of xylose, arabinose, or glucose into a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU04963 polypeptide, and culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of xylose, arabinose, or glucose into the cell compared with a cell that does not contain the recombinant polynucleotide.
Transport of xylose, arabinose, or glucose into a cell may be measured by any method known to one of skill in the art, including those methods described in Example 11. These methods include, for example, measuring D-xylose or L-arabinose transport by extracting accumulated D-xylose and xylitol or L-arabinose and arabinitol from the host cell using osmosis and analyzing it using high performance liquid chromatography and measuring glucose transport by using host cells lacking the ability to grow on glucose as a sole carbon source. Typically, the host cell containing the recombinant polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background.
In certain embodiments, the host cell also contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. The one or more enzymes may be, for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or any other pentose utilization enzymes known in the art.
Methods of Increasing Growth of a Cell
The present invention further provides methods of increasing the growth of a cell. In one aspect the invention provides methods of increasing growth of a cell, including a first step of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, and α-helix 12, where one or more of the following is true: transmembrane α-helix 1 comprises SEQ ID NO: 1, transmembrane α-helix 2 comprises SEQ ID NO: 2, the loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 5 comprises SEQ ID NO: 4, transmembrane α-helix 6 comprises SEQ ID NO: 5, sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6, transmembrane α-helix 7 comprises SEQ ID NO: 7, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8, and the polypeptide transports cellodextrin. The method includes a second step of culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. The growth rate of a host cell may be measured by any method known to one of skill in the art. Typically, growth rate of a cell will be measured by evaluating cell concentration in suspension by optical density. Preferably, the host cell containing the recombinant polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background. Media containing cellodextrins may have resulted from enzymatic treatment of biomass polymers such as cellulose.
In certain embodiments, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments, the host cell also contains a recombinant polynucleotide encoding a catalytic domain of a β-glucosidase. Such embodiments are useful for host cells lacking the endogenous ability to utilize cellodextrins. Preferably, the catalytic domain of the β-glucosidase is intracellular. In preferred embodiments, the β-glucosidase is from Neurospora crassa. In particularly preferred embodiments, the β-glucosidase is encoded by NCU00130.
In methods of increasing growth of a cell, the culturing medium may contain a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.
In another aspect, the invention provides a method of increasing growth of a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a NCU00821 or STL12/XUT6 polypeptide, and the polypeptide transports xylose, and culturing the host cell in a medium containing xylose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. In another aspect the invention provides a method of increasing growth of a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a XUT1 polypeptide, and the polypeptide transports arabinose, and culturing the host cell in a medium containing arabinose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. In yet another aspect, the invention provides a method of increasing growth of a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a NCU06138 polypeptide, and the polypeptide transports arabinose and glucose, and culturing the host cell in a medium containing arabinose or glucose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. In another aspect, the invention provides a method of increasing growth of a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a SUT2, SUT3, or XUT3 polypeptide, and the polypeptide transports xylose and glucose, and culturing the host cell in a medium containing xylose or glucose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide. In yet another aspect, the invention provides a method of increasing growth of a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a NCU04963 polypeptide, and the polypeptide transports xylose, arabinose, and glucose, and culturing the host cell in a medium containing xylose, arabinose, or glucose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
The growth rate of a host cell may be measured by any method known to one of skill in the art. Typically, growth rate of a cell will be measured by evaluating cell concentration in suspension by optical density. Preferably, the host cell containing the recombinant polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background. Media containing xylose or arabinose may have resulted from acid treatment of biomass polymers such as hemicellulose. Media containing glucose may have resulted from enzymatic treatment of biomass polymers such as cellulose.
In certain embodiments, the host cell also contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. The one or more enzymes may be, for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or any other pentose utilization enzymes known in the art.
In one aspect, the invention provides methods of increasing growth of a cell on a biomass polymer. In preferred embodiments, the biomass polymer is cellulose. In other preferred embodiments, the biomass polymer is hemicellulose. According to one aspect of the invention, the method includes providing a host cell comprising a recombinant polynucleotide that encodes a NCU07705 polypeptide. According to another aspect of the invention, the method includes culturing the cell in a medium comprising the biomass polymer wherein the host cell grows at a faster rate in the medium than a cell that does not comprise the recombinant polynucleotide.
In another aspect of the invention, the invention provides a method of increasing growth of a cell, including the steps of providing a host cell, where the host cell contains a recombinant polynucleotide where the polynucleotide encodes a NCU01517, NCU09133, or NCU10040 polypeptide, and culturing the cell in a medium containing hemicellulose, where the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.
According to another aspect of the invention, the method includes providing a host cell comprising an endogenous polynucleotide that encodes a NCU05137 polypeptide. According to another aspect of the invention, the method includes inhibiting expression of the endogenous polynucleotide and culturing the cell in a medium comprising a biomass polymer wherein the host cell grows at a faster rate in the medium than a cell in which expression of the endogenous polynucleotide is not inhibited.
Methods of the invention may include culturing the host cell such that recombinant nucleic acids in the cell are expressed. For microbial hosts, this process entails culturing the cells in a suitable medium. Typically cells are grown at 35° C. in appropriate media. Preferred growth media in the present invention include, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular host cell will be known by someone skilled in the art of microbiology or fermentation science. Temperature ranges and other conditions suitable for growth are known in the art (see, e.g., Bailey and Ollis 1986).
The source of the biomass polymer in the medium may include, for example, grasses (e.g., switchgrass, Miscanthus), rice hulls, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, leaves, grass clippings, corn stover, corn cobs, distillers grains, legume plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust, and biomass crops (e.g., Crambe). In addition to a biomass polymer, the medium must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures. The rate of growth of the host cell may be measured by any methods known to one of skill in the art.
In certain embodiments of the invention, the expression of cellulases is increased in the host cell upon expression of a recombinant polynucleotide. “Cellulase” as used herein refers to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cello-oligosaccharide oligomers, cellobiose, and/or glucose. Cellulases include, without limitation, exoglucanases, exocellobiohydrolases, endoglucanases, and glucosidases. Expression of cellulases may be measured by RT-PCR or other methods known in the art.
In certain embodiments of the invention, the expression of hemicellulases is increased in the host cell upon expression of a recombinant polynucleotide. “Hemicellulase” as used herein refers to a category of enzymes capable of hydrolyzing hemicellulose polymers. Hemicellulases include, without limitation, xylanases, mannanases, arabinases (both endo and exo kinds) and their corresponding glycosidases. Expression of hemicellulases may be measured by RT-PCR or other methods known in the art.
Inhibition of expression of the endogenous polynucleotide may be achieved, for example, by genetic modifications which result in a decrease in gene expression, in the function of the gene, or in the function of the gene product (i.e., the protein encoded by the gene) and can be referred to as inactivation (complete or partial), deletion, interruption, blockage, silencing, or down-regulation, or attenuation of expression of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the protein encoded by such a gene can be the result of a complete deletion of the gene (i.e., the gene does not exist, and therefore the protein does not exist), a mutation in the gene which results in incomplete or no translation of the protein (e.g., the protein is not expressed), or a mutation in the gene which decreases or abolishes the natural function of the protein (e.g., a protein is expressed which has decreased or no enzymatic activity or action). More specifically, reference to decreasing the action of proteins discussed herein generally refers to any genetic modification in the host cell in question which results in decreased expression and/or functionality (biological activity) of the proteins and includes decreased activity of the proteins (e.g., decreased transport), increased inhibition or degradation of the proteins, as well as a reduction or elimination of expression of the proteins. For example, the action or activity of a protein of the present invention can be decreased by blocking or reducing the production of the protein, reducing protein action, or inhibiting the action of the protein. Combinations of some of these modifications are also possible. Blocking or reducing the production of a protein can include placing the gene encoding the protein under the control of a promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the protein (and therefore, of protein synthesis) could be turned off. Blocking or reducing the action of a protein could also include using an excision technology approach similar to that described in U.S. Pat. No. 4,743,546. To use this approach, the gene encoding the protein of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Pat. No. 4,743,546, or by some other physical or nutritional signal.
In certain embodiments of the invention, cellulase activity of the host cell is increased upon inhibiting expression of an endogenous polynucleotide. Cellulase activity may be measured as described in Example 5 and by any other methods known in the art.
In certain embodiments of the invention, hemicellulase activity of the host cell is increased upon inhibiting expression of an endogenous polynucleotide. Hemicellulase activity may be measured as described in Example 17 and by any other methods known in the art.
Methods of Co-Fermentation
One aspect of the present invention provides methods of co-fermenting cellulose-derived and hemicellulose-derived sugars. As used herein, co-fermentation refers to simultaneous utilization by a host cell of more than one sugar in the same vessel. The method includes the steps of providing a host cell, where the host cell contains a first recombinant polynucleotide encoding a cellodextrin transporter and a second recombinant polynucleotide encoding a catalytic domain of a β-glucosidase, and culturing the host cell in a medium containing a cellulose-derived sugar and a hemicellulose-derived sugar, where expression of the recombinant polynucleotides enables co-fermentation of the cellulose-derived sugar and the hemicellulose-derived sugar.
The first recombinant polynucleotide may encode any polypeptide that is capable of transporting cellodextrin into a cell. Cellodextrin transport may be measured by any method known to one of skill in the art, including the methods discussed in Example 9. In preferred embodiments, the first recombinant polynucleotide encodes a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, and α-helix 12, where one or more of the following is true: transmembrane α-helix 1 comprises SEQ ID NO: 1, transmembrane α-helix 2 comprises SEQ ID NO: 2, the loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 5 comprises SEQ ID NO: 4, transmembrane α-helix 6 comprises SEQ ID NO: 5, sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6, transmembrane α-helix 7 comprises SEQ ID NO: 7, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8. Examples of such polypeptides include NCU00801, NCU00809, NCU08114, XP—001268541.1, and LAC2. In preferred embodiments, the first recombinant polypeptide encodes NCU00801.
The second recombinant polynucleotide may encode any catalytic domain capable of catalyzing the hydrolysis of terminal non-reducing residues in β-D-glucosides with release of glucose. Preferably, the β-glucosidase catalytic domain is located intracellularly in the host cell. In certain embodiments the source of the β-glucosidase domain is a N. crassa β-glucosidase. In preferred embodiments the source of the β-glucosidase domain is NCU00130. Catalytic domains from different sources may work best with different cellodextrin transporters.
In certain embodiments, the host cell also contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. Alternatively, one or more polynucleotides encoding one or more enzymes involved in pentose utilization may be endogenous to the host cell. The one or more enzymes may include, for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or any other pentose-utilizing enzymes known to one of skill in the art.
In certain embodiments, the host cell contains a third recombinant polynucleotide where the polynucleotide encodes a pentose transporter. Alternatively, the host cell may contain an endogenous polynucleotide encoding a pentose transporter. In preferred embodiments, the pentose transporter transports xylose and/or arabinose into the cell. In certain embodiments, the third recombinant polynucleotide encodes a polypeptide such as NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, or XUT3. The expression of a pentose transporter in the host cell may enhance the efficiency of co-fermentation if glucose is present along with a pentose sugar is the growth medium.
In methods of co-fermentation as described herein, cellulose-derived sugars preferably include cellobiose, cellotriose, and celltetraose, and hemicellulose-derived sugars preferably include xylose and arabinose. Typically, in order to prepare the cellulose-derived sugars and hemicellulose-derived sugars for co-fermentation by a host cell, lignocellulosic biomass is first pretreated to alter its structure and allow for better enzymatic hydrolysis of cellulose. Pretreatment may include physical or chemical methods, including, for example, ammonia fiber/freeze explosion, the lime method based on calcium or sodium hydroxide, and steam explosion with or without an acid catalyst. Acid treatment will release xylose and arabinose from the hemicellulose component of the lignocellulosic biomass. Next, preferably, the cellulose component of the pretreated biomass is hydrolyzed by a mixture of cellulases. Examples of commercially available cellulase mixtures include Celluclast 1.5L® (Novozymes), Spezyme CP® (Genencor) (Scott W. Pryor, 2010, Appl Biochem Biotechnol), and Cellulyve 50L (Lyven).
Cellulase mixtures typically contain endoglucanases, exoglucanases, and β-glucosidases. In methods of co-fermentation as described herein, the amount of β-glucosidase activity in the cellulase mixture should be minimized as much as possible. For example, the culturing medium may contain a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.
Surprisingly, as described in Example 17, co-fermentation of cellobiose and xylose by the methods of the invention resulted in a synergistic effect on sugar consumption and ethanol production by the host cell.
Methods of Synthesis of Hydrocarbons or Hydrocarbon Derivatives
One aspect of the present invention provides methods for increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell.
“Hydrocarbons” as used herein are organic compounds consisting entirely of hydrogen and carbon. Hydrocarbons include, without limitation, methane, ethane, ethene, ethyne, propane, propene, propyne, cyclopropane, allene, butane, isobutene, butene, butyne, cyclobutane, methylcyclopropane, butadiene, pentane, isopentane, neopentane, pentene, pentyne, cyclopentane, methylcyclobutane, ethylcyclopropane, pentadiene, isoprene, hexane, hexene, hexyne, cyclohexane, methylcyclopentane, ethylcyclobutane, propylcyclopropane, hexadiene, heptane, heptene, heptyne, cycloheptane, methylcyclohexane. heptadiene, octane, octene, octyne, cyclooctane, octadiene, nonane, nonene, nonyne, cyclononane, nonadiene, decane, decene, decyne, cyclodecane, and decadiene.
“Hydrocarbon derivatives” as used herein are organic compounds of carbon and at least one other element that is not hydrogen. Hydrocarbon derivatives include, without limitation, alcohols (e.g., arabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); organic acids (e.g., acetic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid); esters; ketones (e.g., acetone); aldehydes (e.g., furfural); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); and gases (e.g., carbon dioxide and carbon monoxide).
In preferred embodiments, the hydrocarbon or hydrocarbon derivative can be used as fuel. In particularly preferred embodiments, the hydrocarbon or hydrocarbon derivative is ethanol or butanol.
According to one aspect of the invention, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes a first step of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a polypeptide containing transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, and α-helix 12, where one or more of the following is true: transmembrane α-helix 1 comprises SEQ ID NO: 1, transmembrane α-helix 2 comprises SEQ ID NO: 2, the loop connecting transmembrane α-helix 2 and transmembrane α-helix 3 comprises SEQ ID NO: 3, transmembrane α-helix 5 comprises SEQ ID NO: 4, transmembrane α-helix 6 comprises SEQ ID NO: 5, sequence between transmembrane α-helix 6 and transmembrane α-helix 7 comprises SEQ ID NO: 6, transmembrane α-helix 7 comprises SEQ ID NO: 7, and transmembrane α-helix 10 and transmembrane α-helix 11 and the sequence between them comprise SEQ ID NO: 8, and where the polypeptide transports cellodextrin into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives. The method includes a second step of culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide. In certain embodiments, the polypeptide has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114. In certain embodiments, the host cell also contains a recombinant polynucleotide encoding a catalytic domain of a β-glucosidase. Such embodiments are useful for host cells lacking the endogenous ability to utilize cellodextrins. Preferably, the catalytic domain of the β-glucosidase is intracellular. In preferred embodiments, the β-glucosidase is from Neurospora crassa. In particularly preferred embodiments, the β-glucosidase is encoded by NCU00130. Transport of cellodextrin into the cell may be measured by any methods known to one of skill in the art, including the methods described in Example 9. Typically, the source of the cellodextrin is cellulose.
The culturing medium may contain a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.
According to another aspect of the invention, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU00821 or STL12/XUT6 polypeptide, where the polypeptide transports xylose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing xylose or a source of xylose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of xylose into the cell is increased upon expression of the recombinant polynucleotide.
According to another aspect, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a XUT1 polypeptide, where the polypeptide transports arabinose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing arabinose or a source of arabinose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of arabinose into the cell is increased upon expression of the recombinant polynucleotide.
According to yet another aspect, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU06138 polypeptide, where the polypeptide transports arabinose or glucose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium comprising arabinose or glucose or a source of arabinose or glucose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of arabinose or glucose into the cell is increased upon expression of the recombinant polynucleotide.
According to yet another aspect, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a SUT2, SUT3, or XUT3 polypeptide, where the polypeptide transports xylose or glucose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium containing xylose or glucose or a source of xylose or glucose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of xylose or glucose into the cell is increased upon expression of the recombinant polynucleotide.
According to another aspect, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes the steps of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a NCU04963 polypeptide, where the polypeptide transports xylose, arabinose, or glucose into the host cell for the synthesis of hydrocarbons or hydrocarbon derivatives, and culturing the host cell in a medium comprising xylose, arabinose, or glucose or a source of xylose, arabinose, or glucose to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of xylose, arabinose, or glucose into the cell is increased upon expression of the recombinant polynucleotide.
Transport of xylose, arabinose, or glucose into the cell may by measured by any methods known to one of skill in the art, including the methods described in Example 11. Typically, the source of glucose is cellulose, and the source of xylose and arabinose is hemicellulose.
Methods of Degrading Cellulose
One aspect of the present invention provides methods of degrading cellulose. The methods include a first step of providing a composition comprising cellulose. The cellulose is preferably from plant material, such as switchgrass, Miscanthus, rice hulls, bagasse, flax, bamboo, sisal, abaca, straw, leaves, grass clippings, corn stover, corn cobs, distillers grains, legume plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust, and biomass crops.
The methods include a second step of contacting the composition with a cellulase-containing enzyme mixture from an altered organism, where the cellulase-containing mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The cellulose is degraded by the cellulase-containing mixture. The organism may be altered by mutation of a gene encoding a β-glucosidase or by reducing the expression of a β-glucosidase with a technique such as RNA interference. The organism may be a fungus or a bacterium. In preferred embodiments, the organism is a filamentous fungus such as T. reesei. 
Alternatively, the methods include a second step of contacting the composition with a cellulase-containing enzyme mixture that has been altered to reduce its β-glucosidase activity. For example, the cellulase-containing enzyme mixture may be altered by affinity chromatography where β-glucosidase enzymes are captured during the chromatography, and thus removed from the mixture. In another example, the cellulase-containing enzyme mixture is altered by inactivation of β-glucosidase enzymes in the mixtures with an inhibitor. Examples of commercially available cellulase mixtures include Celluclast 1.5L® (Novozymes), Spezyme CP® (Genencor) (Scott W. Pryor, 2010, Appl Biochem Biotechnol), and Cellulite 50L (Lyven).
It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
EXAMPLES
Example 1
Transcriptome Analysis of N. crassa Grown on Miscanthus and Avicel
In this example, the expression profile of the N. crassa genome was examined during growth on Miscanthus or Avicel. Growth and cellulase activity of N. crassa (FGSC 2489) cultured on Vogel's minimal media with crystalline cellulose (Avicel) as the sole carbon source was similar to that of T. reesei (QM9414) (FIG. 3); N. crassa completely degraded Avicel in approximately 4 days. N. crassa also grew rapidly on ground Miscanthus stems, suggesting functional cellulase and hemicellulase degradative capacity. To determine the transcriptome associated with plant cell wall deconstruction in N. crassa, we used full genome microarrays (Kasuga and Glass 2008; Tian et al., 2007; Kasuga et al., 2005) to monitor gene expression profiles during growth of N. crassa on ground Miscanthus stems. RNA was sampled after 16 hrs of growth on sucrose and compared to RNA isolated from N. crassa grown on Miscanthus medium at 16 and 40 hrs, 5 days and 10 days (FIGS. 4A-B; also see Supplemental Data, Dataset S1, page 1 in Tian et al., 2009; data can also be found at bioinfo.townsend.yale.edu/browse.jsp, Experiment IDs 52 and 53).
A total of 769 N. crassa genes showed a statistically significant difference in relative expression level among the four Miscanthus samples as compared to the sucrose sample (see Supplemental Data, Dataset S1, page 3 in Tian et al., 2009). Hierarchical clustering showed that these genes fell into three main clusters (FIG. 4A). The first cluster of genes (C1; 300 genes) showed the highest expression levels in minimal medium with sucrose as a carbon source. Functional category (FunCat) analysis (Ruepp 2004) of these genes showed an enrichment for ribosomal proteins and other functional categories associated with primary metabolism, such as respiration, electron transport and amino acid metabolism (see Supplemental Data, Dataset S1, page 4 in Tian et al., 2009). The second cluster (C2) included 327 genes that showed the highest expression levels in Miscanthus cultures at later time points (40 hrs to 10 days; FIG. 4A). Within this group were 89 genes that showed a high relative expression level in Miscanthus cultures at all time points. For further analyses, these 89 genes were added to the cluster of genes that showed the highest expression levels from the 16 hr Miscanthus cultures (C3 cluster, see below). FunCat analysis (Ruepp 2004) of the remaining 238 genes showed one functional category (C-compound and carbohydrate metabolism) was slightly enriched (see Supplemental Data, Dataset S1, page 5 in Tian et al., 2009).
A third cluster of 142 genes showed the highest relative expression level after 16 hrs of growth of N. crassa on Miscanthus (C3, FIG. 4A). FunCat analysis (Ruepp 2004) of these 142 genes plus the 89 genes that showed high expression levels in Miscanthus cultures at all time points (C3+ cluster; total 231 genes) showed an enrichment for proteins involved with carbon metabolism, including predicted cellulases and hemicellulases (FIG. 4C; also see Supplemental Data, Dataset S1, page 6 in Tian et al., 2009). Of the 23 predicted cellulase genes in the N. crassa genome, 18 showed significant increases in expression levels during growth on Miscanthus (see Table 1 in Tian et al., 2009), particularly at the 16 hr time point (FIG. 5A). Five genes showed an increase in expression level over 200-fold (cbh-1 (CBH(I); NCU07340, gh6-2 (CBH(II)-like gene; NCU09680), gh6-3 (NCU07190) and two GH61 genes (gh61-4; NCU01050 and NCU07898))).
Plant cell walls are complex structures composed of cellulose microfibrils, hemicellulose, lignin, pectin, cutin, and protein. Thus, we compared expression profiles of N. crassa grown on Miscanthus to expression profiles of N. crassa grown on Avicel, a pure form of crystalline cellulose (see Tian et al., 2009, Supplemental Data, Dataset S1, page 2; data can also be found at bioinfo.townsend.yale.edu/browse.jsp, Experiment IDs 52 and 53). Over 187 genes showed a significant increase in relative expression level during growth of N. crassa on Avicel. Of these genes, 114 overlapped with the 231 genes in the C3+ cluster (FIG. 4B). FunCat analysis of the 114-overlap gene set showed a clear enrichment for genes predicted to be involved in carbon metabolism (see Supplemental Data, Dataset S1, page 6 in Tian et al., PNAS, 2009). Within this gene set, there was a further enrichment for secreted proteins; 53 of the 114 gene products were predicted to be secreted. Of these 53 genes, 32 encode predicted proteins that have annotation suggesting a role in plant cell wall degradation, while 16 encode putative or hypothetical proteins that lack any functional prediction. The remaining 61 genes encode predicted intracellular proteins, including ten predicted major facilitator superfamily transporters (NCU00801, NCU00988, NCU01231, NCU04963, NCU05519, NCU05853, NCU05897, NCU06138, NCU08114 and NCU10021) and 23 putative or hypothetical proteins.
Of the 117 genes within the Miscanthus-specific cluster (FIG. 4B), 37 encode proteins predicted to be secreted. Nine predicted hemicellulases or enzymes related to the degradation of hemicellulose were identified (NCU00710, NCU04265, NCU04870, NCU05751, NCU05965, NCU09170, NCU09775, NCU09923 and NCU09976) (Tian et al., 2009-Table 2). The remaining 80 Miscanthus-specific genes encode predicted intracellular proteins, including genes involved in the metabolism of pentose sugars (for example, NCU00891, xylitol dehydrogenase and NCU00643, a predicted arabinitol dehydrogenase), a predicted sugar transporter (NCU01132), and 48 proteins of unknown function.
Example 2
Secretome Analysis of N. crassa Grown on Miscanthus and Avicel
Lignocellulose degradation by fungi takes place extracellularly and requires the secretion of proteins associated with depolymerization of cell wall constituents (Lynd et al., 2002). To compare with transcriptional profiling data, which showed that genes encoding predicted cellulases, hemicellulases, and other secreted proteins increased in expression levels when N. crassa was grown on Miscanthus or Avicel, we analyzed the secretome of N. crassa using a shotgun proteomics approach (FIG. 4B). Supernatants from seven day old Miscanthus and Avicel cultures were digested with trypsin and analyzed by liquid chromatography nano-electrospray ionization tandem mass spectrometry (MS; see Example 5). Secreted proteins that bound to phosphoric acid swollen cellulose (PASC) were enriched and also analyzed by MS.
A total of 50 proteins were identified with confidence by tandem MS (Tables 2 and 3). There were 34 proteins detected in the Miscanthus grown N. crassa cultures, while 38 proteins were identified from Avicel grown cultures; twenty-two proteins were detected in both samples. Of these 22 proteins, 21 were predicted to be secreted based on computational analyses and 19 showed increased expression levels in both the Miscanthus and Avicel grown cultures (Table 2). The overlap dataset included eight of the 23 predicted cellulases in N. crassa (Table 3). There were also five predicted hemicellulases, a predicted β-glucosidase (gh3-4; NCU04952), five proteins with predicted activity on carbohydrates, and two proteins with unknown function (NCU07143 and NCU05137) (Table 4-5).
For Table 2, the annotation was generated by the Broad Institute at webpage broad.mit.edu/annotation/genome/neurospora/Home.html. The “sample detected” was the sample in which peptides were detected for a particular protein. Peptides were validated by manual inspection. A protein was determined to be present if at least 1 peptide was detected in each biological repeat. “TOTAL” refers to peptides detected from a tryptic digest of all extracellular proteins. “PASC BOUND” refers to peptides detected after enrichment for proteins that bind to phosphoric acid swollen cellulose. “UNBOUND” refers to proteins remaining in solution after removal of PASC bound proteins.







TABLE 2







Proteins identified by LC-MS/MS In the culture filtrates of


Avicel grown Neurospora crassa









GENE ID
ANNOTATION
SAMPLE DETECTED





NCU00206
Neurospora crassa hypothetical protein similar to cellobiose dehydrogenase 830 nt
TOTAL


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
TOTAL


NCU01050
Neurospora crassa hypothetical protein similar to endoglucanase II 239 nt
TOTAL


NCU02343
Neurospora crassa hypothetical protein similar to alpha L arabinofuranosidase A
TOTAL



668 nt



NCU04870
Neurospora crassa hypothetical protein similar to acetyl xylan esterase 313 nt
TOTAL


NCU04952
Neurospora crassa hypothetical protein similar to beta D glucoside glucohydrolase
TOTAL



736 nt



NCU05137
Neurospora crassa conserved hypothetical protein 692 nt
TOTAL


NCU05159
Neurospora crassa acetylxylan esterase precursor 301 nt
TOTAL


NCU05924
Neurospora crassa endo 1 4 beta xylanase 330 nt
TOTAL


NCU07143
Neurospora crassa predicted protein 391 nt
TOTAL


NCU07190
Neurospora crassa conserved hypothetical protein 384 nt
TOTAL


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
TOTAL


NCU07326
Neurospora crassa predicted protein 327 nt
TOTAL


NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt
TOTAL


NCU07898
Neurospora crassa predicted protein 242 nt
TOTAL


NCU08189
Neurospora crassa hypothetical protein similar to endo 1 4 beta xylanase 385 nt
TOTAL


NCU08398
Neurospora crassa conserved hypothetical protein 391 nt
TOTAL


NCU08760
Neurospora crassa predicted protein 343 nt
TOTAL


NCU08785
Neurospora crassa conserved hypothetical protein 291 nt
TOTAL


NCU09491
Neurospora crassa feruloyl esterase B precursor 293 nt
TOTAL


NCU09680
Neurospora crassa exoglucanase 2 precursor 485 nt
TOTAL


NCU09923
Neurospora crassa hypothetical protein similar to beta xylosidase 775 nt
TOTAL


NCU00206
Neurospora crassa hypothetical protein similar to cellobiose dehydrogenase 830 nt 
PASC BOUND


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
PASC BOUND


NCU05159
Neurospora crassa acetylxylan esterase precursor 301 nt
PASC BOUND


NCU05955
Neurospora crassa hypothetical protein similar to Ce174a 862 nt
PASC BOUND


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
PASC BOUND


NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt
PASC BOUND


NCU08760
Neurospora crassa predicted protein 343 nt
PASC BOUND


NCU09680
Neurospora crassa exoglucanase 2 precursor 485 nt
PASC BOUND


NCU09708
Neurospora crassa conserved hypothetical protein 465 nt
PASC BOUND


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
UNBOUND


NCU01651
Neurospora crassa conserved hypothetical protein 783 nt
UNBOUND


NCU02343
Neurospora crassa hypothetical protein similar to alpha L arabinofuranosidase A
UNBOUND



668 nt



NCU04202
Neurospora crassa nucleoside diphosphate kinase 1 153 nt
UNBOUND


NCU04870
Neurospora crassa hypothetical protein similar to acetyl xylan esterase 313 nt
UNBOUND


NCU04952
Neurospora crassa hypothetical protein similar to beta D glucoside glucohydrolase
UNBOUND



736 nt



NCU05057
Neurospora crassa endoglucanase EG 1 precursor 439 nt
UNBOUND


NCU05137
Neurospora crassa conserved hypothetical protein 692 nt
UNBOUND


NCU05751
Neurospora crassa conserved hypothetical protein 242 nt
UNBOUND


NCU05924
Neurospora crassa endo 1 4 beta xylanase 330 nt
UNBOUND


NCU06239
Neurospora crassa conserved hypothetical protein 514 nt
UNBOUND


NCU07143
Neurospora crassa predicted protein 391 nt
UNBOUND


NCU07190
Neurospora crassa conserved hypothetical protein 384 nt
UNBOUND


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
UNBOUND


NCU07326
Neurospora crassa predicted protein 327 nt
UNBOUND


NCU07898
Neurospora crassa predicted protein 242 nt
UNBOUND


NCU08189
Neurospora crassa hypothetical protein similar to endo 1 4 beta xylanase 385 nt
UNBOUND


NCU08398
Neurospora crassa conserved hypothetical protein 391 nt
UNBOUND


NCU08412
Neurospora crassa conserved hypothetical protein 401 nt
UNBOUND


NCU08760
Neurospora crassa predicted protein 343 nt
UNBOUND


NCU08785
Neurospora crassa conserved hypothetical protein 291 nt
UNBOUND


NCU09024
Neurospora crassa conserved hypothetical protein 625 nt
UNBOUND


NCU09175
Neurospora crassa conserved hypothetical protein 411 nt
UNBOUND


NCU09267
Neurospora crassa conserved hypothetical protein 1048 nt
UNBOUND


NCU09491
Neurospora crassa feruloyl esterase B precursor 293 nt
UNBOUND


NCU09775
Neurospora crassa hypothetical protein similar to alpha L arabinofuranosidase 343
UNBOUND



nt



NCU09923
Neurospora crassa hypothetical protein similar to beta xylosidase 775 nt
UNBOUND
















TABLE 3







22 secreted proteins detected in both Miscanthus and Avicel cultures












Gene name
Gene annotation
Profiling
kos
CBM1
Signal P





NCU00206.2
CBDH
both
heter
yes
yes


NCU00762.2
probable cellulase precursor
both
16747
yes
yes


NCU01050.2
related to cell protein precursor
both
16543
no
yes


NCU04952.2
probable beta-D-glucoside glucohydrolase
both
13732
no
yes


NCU05057.2
probable endo-1,4-beta-glucanase
both
13343
no
yes


NCU05137.2
conserved hypothetical protein
both
11682
no
yes


NCU05924.2
probable endo-beta-1,4-D-xylanase
both
15626
no
yes


NCU05955.2
probable endoglucanase C
both
13535
yes
yes


NCU07143.2
hypothetical
both
No
no
yes


NCU07190.2
CBHII homolog
both
19315
no
yes


NCU07225.2
probable endo-1,4-beta-xylanase B
both
heter
yes
yes



precursor






NCU07326.2
related to putative arabinase
both
19534
no
yes


NCU07340.2
CBHI
both
15630
yes
yes


NCU07898.2
related to cell protein precursor
both
19600
no
yes


NCU08189.2
similar to endo-1,4-beta xylanase 
both
19861
no
yes


NCU08398.2
related to aldose 1-epimerase
both
20310
no
yes


NCU08412.2
hypothetical protein 401 nt
none
No
no
no


NCU08760.2
related to family 61 endoglucanase
both
15664
yes
yes


NCU09024.2
hypothetical protein 625 nt
none
No
no
yes


NCU09175.2
glucan 1,3-beta-glucosidase precursor
both
11750
no
yes


NCU09491.2
feruloyl esterase B precursor
mis
No
no
yes


NCU09680.2
CBHII
both
15633
yes
yes
















TABLE 4







shows predicted cellulase genes in Neurospora crassa














GH1



EL5 
EL5


Gene
Family
CBM12
SP3
MS4
Miscanthus
Avicel
















NCU00762
5
yes
yes
both
29.6
31.5


NCU03996
6
no
no
ND6
ND
ND


NCU07190
6
no
yes
both
526.0
119


NCU09680
6
yes
yes
both
230.9
251.3


NCU04854
7
no
yes
ND
32.9
10.8


NCU05057
7
no
yes
both
8.7
7.4


NCU05104
7
no
yes
ND
11.6
NC7


NCU07340
7
yes
yes
both
426.4
382.2


NCU05121
45
yes
yes
avi
8.6
17.2


NCU00836
61
yes
yes
ND
91.2
31


NCU01050
61
no
yes
both
206.7
382.1


NCU01867
61
yes
yes
ND
2.2
NC


NCU02240
61
yes
yes
avi
193.5
84


NCU02344
61
no
yes
ND
8.1
4.1


NCU02916
61
yes
yes
ND
85.2
17.7


NCU03000
61
no
yes
ND
NC
ND


NCU03328
61
no
yes
ND
26.4
23.8


NCU05969
61
no
yes
ND
ND
12.7


NCU07520
61
no
yes
ND
ND
ND


NCU07760
61
yes
yes
ND
3.7
NC


NCU07898
61
no
yes
both
376.3
230


NCU07974
61
no
yes
ND
NC
NC


NCU08760
61
yes
yes
both
107.5
44.7





1GlucosideHydrolase;


2CBM1, carbohydrate binding module;


3Signal peptide prediction (signalP = webpage cbs.dtu.dk/services/SignalP/);


4MS, mass spectrometry analysis;


5EL, relative expression level;


6ND, not detected;


7NC, no change in expression level versus minimal media.













TABLE 5







Cellulases and Hemicellulases identified by LC-MS/MS













GH
AV
MIS 
AV 
MIS


Gene ID 
Family
MS
MS
ARRAY
ARRAY










Predicted cellulases in the genome of Neurospora crassa












NCU00762 
5
+
+
31.5
29.6


NCU00836
61
−
−
31
91.2


NCU01050
61
+
+
382.1
206.7


NCU01867 
61
−
−
1
1


NCU02240 
61
+
−
84
193.5


NCU02344 
61
−
−
4.1
8.1


NCU02916 
61
−
−
17.7
85.2


NCU03000 
61
−
−
1
1


NCU03328 
61
−
−
23.8
26.4


NCU03996 
6
−
−
2.5
6.3


NCU04854
7
−
−
10.8
32.9


NCU05057 
7
+
+
7.4
8.7


NCU05104 
7
−
−
1
1


NCU05121 
45
+
−
17.2
8.6


NCU05969 
61
−
−
12.7
12.3


NCU07190
6
+
+
119
526


NCU07340
7
+
+
382.2
426.4


NCU07520 
61
−
−
1
1


NCU07760
61
−
−
1
3.4


NCU07898 
61
+
+
230.5
376.3


NCU07974 
61
−
−
1
1


NCU08760 
61
+
+
44.7
107.5


NCU09680 
6
+
+
251.3
230.9







Predicted cellulases in the genome of Neurospora crassa












NCU00852
43
−
−
1
1


NCU00972
53
−
−
9.03
15.6


NCU01900
43
−
−
10.03
26


NCU02343
51
−
+
6.63
174.6


NCU02855
11
+
−
10.2
364


NCU04997
10
−
−
1
25.6


NCU05924
10
+
+
55.9
149.3


NCU05955
74
+
+
19.9
50.5


NCU05965
43
−
−
1
5.4


NCU06861
43
−
−
1
1


NCU07130
10
−
−
1
1


NCU07225
11
+
+
11.43
33.5


NCU07326
43
+
+
104.5
426.6


NCU07351
67
−
−
1
1


NCU08087
26
−
−
1
1


NCU08189
10
+
+
39.8
94.4


NCU09170
43
−
−
1
16.7


NCU09652
43
−
−
12.2
95.4


NCU09775
54
−
+
1
48.3





GH Family—Glycosyl Hydrolase Family;


AV MS - Protein detected by LC-MS/MS in the culture filtrates of Avicel grown Neurospora crassa . (+) detected, (−) not detected;


MIS MS - Protein detected by LC-MS/MS in the culture filtrates of Miscanthus grown Neurospora crassa . (+) detected, (−) not detected;


AV ARRAY - Fold upregulation after 30 hours of growth on Avicel relative to 16 hours of growth on sucrose from profiling data;


MIS ARRAY - Fold upregulation after 16 hours of growth on Miscanthus relative to 16 hours of growth on sucrose from profiling data, peptides detected only in Miscanthus culture filtrates.






There were 16 proteins identified with confidence only in the Avicel culture and 14 of these were predicted to be secreted (Table 6) including two predicted cellulases (gh61-1; NCU02240 and gh45-1; NCU05121), one xylanase (gh11-1; NCU02855), one predicted protease (NCU04205), three other proteins with predicted activity on carbohydrates (NCU08909, NCU05974 and gh30-1 (NCU04395)), three Neurospora-specific proteins of unknown function, and four conserved hypothetical proteins, including one protein with a cellulose binding domain (NCU09764). Twelve proteins were specific for culture filtrates of Miscanthus cultures and seven of these were predicted to be secreted (Table 3). Three of the five predicted intracellular proteins were conserved hypothetical proteins. The remaining two included a predicted glyoxal oxidase (NCU09267, identified from the N. crassa Miscanthus transcriptome) and a nucleoside diphosphate kinase (ndk-1; NCU04202, not identified in the N. crassa transcriptome). The seven proteins predicted to be secreted included three predicted esterases (NCU04870, NCU05159, and NCU08785), two predicted xylanases (GH51; NCU02343 and GH54; NCU09775), a predicted β-xylosidase (gh3-7; NCU09923) and a conserved hypothetical protein (NCU05751).







TABLE 6







Proteins identified by LC-MS/MS In the culture filtrates of Avicel grown Neurospora crassa









GENE ID
ANNOTATION
SAMPLE DETECTED





NCU00206
Neurospora crassa hypothetical protein similar to cellobiose dehydrogenase 830 nt
TOTAL


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
TOTAL


NCU00798
Neurospora crassa predicted protein 313 nt
TOTAL


NCU01050
Neurospora crassa hypothetical protein similar to endoglucanase II 239 nt
TOTAL


NCU01595
Neurospora crassa protein SOF1 446 nt
TOTAL


NCU02240
Neurospora crassa hypothetical protein similar to endoglucanase II 323 nt
TOTAL


NCU02696
Neurospora crassa hypothetical protein similar to DEAD DEAH box RNA helicase 
TOTAL



1195 nt



NCU02855
Neurospora crassa endo 1 4 beta xylanase A precursor 221 nt
TOTAL


NCU04952
Neurospora crassa hypothetical protein similarnt to beta D glucoside glucohydrolase 
TOTAL



736 nt



NCU05057
Neurospora crassa endoglucanase EG 1 precursor 439 nt
TOTAL


NCU05137
Neurospora crassa conserved hypothetical protein 692 nt
TOTAL


NCU05924
Neurospora crassa endo 1 4 beta xylanase 330 nt
TOTAL


NCU05955
Neurospora crassa hypothetical protein similar to Cel74a 862 nt
TOTAL


NCU07143
Neurospora crassa predicted protein 391 nt
TOTAL


NCU07190
Neurospora crassa conserved hypothetical protein 384 nt
TOTAL


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
TOTAL


NCU07326
Neurospora crassa predicted protein 327 nt
TOTAL


NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt
TOTAL


NCU07898
Neurospora crassa predicted protein 242 nt
TOTAL


NCU08171
Neurospora crassa predicted protein 382 nt
TOTAL


NCU08412
Neurospora crassa conserved hypothetical protein 401 nt
TOTAL


NCU08760
Neurospora crassa predicted protein 343 nt
TOTAL


NCU09491
Neurospora crassa feruloyl esterase B precursor 293 nt
TOTAL


NCU09680
Neurospora crassa exoglucanase 2 precursor 485 nt
TOTAL


NCU09764
Neurospora crassa conserved hypothetical protein 406 nt
TOTAL


NCU00206
Neurospora crassa hypothetical protein similar to cellobiose dehydrogenase 830 nt
PASC BOUND


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
PASC BOUND


NCU05121
Neurospora crassa endoglucanase V 294 nt
PASC BOUND


NCU05955
Neurospora crassa hypothetical protein similar to Ce174a 862 nt
PASC BOUND


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
PASC BOUND


NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt
PASC BOUND


NCU08760
Neurospora crassa predicted protein 343 nt
PASC BOUND


NCU09680
Neurospora crassa exoglucanase 2 precursor 485 nt
PASC BOUND


NCU00206
Neurospora crassa hypothetical protein similar to cellobiose dehydrogenase 830 nt
UNBOUND


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
UNBOUND


NCU00798
Neurospora crassa predicted protein 313 nt
UNBOUND


NCU01050
Neurospora crassa hypothetical protein similar to endoglucanase II 239 nt
UNBOUND


NCU04205
Neurospora crassa predicted protein 346 nt
UNBOUND


NCU04395
Neurospora crassa endo 1 6 beta D glucanase precursor 481 nt
UNBOUND


NCU04952
Neurospora crassa hypothetical protein similarnt to beta D glucoside glucohydrolase 736
UNBOUND



nt



NCU05057
Neurospora crassa endoglucanase EG 1 precursor 439 nt
UNBOUND


NCU05134
Neurospora crassa hypothetical protein 124 nt
UNBOUND


NCU05137
Neurospora crassa conserved hypothetical protein 692 nt
UNBOUND


NCU05852
Neurospora crassa conserved hypothetical protein 254 nt
UNBOUND


NCU05924
Neurospora crassa endo 1 4 beta xylanase 330 nt
UNBOUND


NCU05974
Neurospora crassa hypothetical protein similar to cell wall glucanosyltransferase Mwg1
UNBOUND



365 nt



NCU07143
Neurospora crassa predicted protein 391 nt
UNBOUND


NCU07190
Neurospora crassa conserved hypothetical protein 384 nt
UNBOUND


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
UNBOUND


NCU07326
Neurospora crassa predicted protein 327 nt
UNBOUND


NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt
UNBOUND


NCU07898
Neurospora crassa predicted protein 242 nt
UNBOUND


NCU08171
Neurospora crassa predicted protein 382 nt
UNBOUND


NCU08189
Neurospora crassa hypothetical protein similar to endo 1 4 beta xylanase 385 nt
UNBOUND


NCU08398
Neurospora crassa conserved hypothetical protein 391 nt
UNBOUND


NCU08412
Neurospora crassa conserved hypothetical protein 401 nt
UNBOUND


NCU08760
Neurospora crassa predicted protein 343 nt
UNBOUND


NCU08909
Neurospora crassa hypothetical protein similar to beta 1 3 glucanosyltransferase 543 nt
UNBOUND


NCU08936
Neurospora crassa clock controlled gene 15 412 nt
UNBOUND


NCU09024
Neurospora crassa conserved hypothetical protein 625 nt
UNBOUND


NCU09046
Neurospora crassa predicted protein 187 nt
UNBOUND


NCU09175
Neurospora crassa conserved hypothetical protein 411 nt
UNBOUND


NCU09491
Neurospora crassa feruloyl esterase B precursor 293 nt
UNBOUND





ANNOTATION - Generated by the Broad Institute (webpage at broad.mit.edu/annotation/genome/neurospora/Home.html);


SAMPLE DETECTED - Sample in which peptides were detected for a particular protein. Peptides were validated by manual inspection. A protein was determined to be present if at least 1 peptide was detected in each biological repeat.


TOTAL, peptides detected from a tryptic digest of all extracellular proteins;


PASC BOUND, peptides detected after enrichment for proteins that bind to phosphoric acid swollen cellulose;


UNBOUND, proteins remaining in solution after removal of PASC bound proteins.






Many plant cell wall degrading enzymes contain a cellulose-binding module (CBM), which aids in attachment of the enzyme to the substrate (Linder and Teeri 1996). Within the N. crassa genome, proteins encoded by 19 genes are predicted to contain a CBM1 domain (Cantarel et al., 2009). Of these 19 genes, 16 showed an increase in relative gene expression in Miscanthus-grown cultures (Table 7).







TABLE 7







Effect of Miscanthus and Avicel on N. crassa gene expression












Gene
CBM

Mis 
Avicel



name
prediction
Annotation
Array
array
MS















NCU00206
cazy and mips
probable 
164
12
both




cellobiose







dehydrogenase





NCU00710
cazy and mips
acetylxylan 
30
no 
none




esterase

detect



NCU00762
cazy and mips
EG2
29
31
both


NCU00836
cazy and mips
EG, GH61
91
30
none


NCU01867
cazy and mips
EG, GH61
2.2-d10
no
none






difference



NCU02240
cazy and mips
EG, GH61
193
84
avi


NCU02916
cazy and mips
EG, GH61
85
17
none


NCU04500
cazy and mips
similar to 
no 
no 
none




chitinase 4
detect
detect



NCU04997
cazy and mips
similar to 
no 
no 
none




xylanase
detect
detect



NCU05121
cazy and mips
EG, GH45
8.5
17
avi


NCU05159
cazy and mips
acetylxylan 
34
10
mis




esterase







precursor





NCU05955
cazy and mips
GH74
50
19
both


NCU07225
cazy and mips
xylanase
33
11
both


NCU07340
cazy and mips
CBH1
426
382
both


NCU07760
cazy and mips
EG, GH61
3.7
no
none






difference



NCU08760
cazy and mips
EG, GH61
107
44
both


NCU09416
cazy and mips
hypothetical
no 
27
none





detect




NCU09680
cazy and mips
CBH2
230
251
both


NCU09764
cazy and mips
hypothetical
18
16.6
avi









From the 50 proteins identified by MS, 11 contained a CBM1 domain. PASC was used to enrich for proteins that bind to cellulose (see Example 4 for methods). Nine proteins were identified by MS that bound to PASC from the supernatant of Miscanthus-grown N. crassa cultures, while eight proteins from the Avicel supernatants were identified; seven cellulose binding proteins were identified in both (Tables 2, 3, 8). These included NCU00206, a predicted cellobiose dehydrogenase; gh5-1 (NCU00762), a predicted endoglucanase; NCU05955, a predicted GH74 xyloglucanase; gh11-2 (NCU07225), a predicted endoxylanase; cbh-1 (NCU07340); gh61-5 (NCU08760), a predicted endoglucanase; and gh6-2 (NCU09680), a predicted cellobiohydrolase 2 precursor.







TABLE 8







Proteins identified by LC-MS/MS in the culture filtrates of Avicel-grown Neurospora crassa









GENE ID
ANNOTATION
CULTURE





NCU00206
Neurospora crassa hypothetical protein similar to cellobiose dehydrogenase 830 nt
BOTH


NCU00762
Neurospora crassa endoglucanase 3 precursor 391 nt
BOTH


NCU01050
Neurospora crassa hypothetical protein similar to endoglucanase II 239 nt
BOTH


NCU04952
Neurospora crassa hypothetical protein similar to beta D glucoside glucohydrolase
BOTH



736 nt



NCU05057
Neurospora crassa endoglucanase EG 1 precursor 439 nt
BOTH


NCU05137
Neurospora crassa conserved hypothetical protein 692 nt
BOTH


NCU05924
Neurospora crassa endo 1 4 beta xylanase 330 nt
BOTH


NCU05955
Neurospora crassa hypothetical protein similar to Cel74a 862 nt
BOTH


NCU07143
Neurospora crassa predicted protein 391 nt
BOTH


NCU07190
Neurospora crassa conserved hypothetical protein 384 nt
BOTH


NCU07225
Neurospora crassa endo 1 4 beta xylanase 2 precursor 255 nt
BOTH


NCU07326
Neurospora crassa predicted protein 327 nt
BOTH


NCU07340
Neurospora crassa exoglucanase 1 precursor 522 nt
BOTH


NCU07898
Neurospora crassa predicted protein 242 nt
BOTH


NCU08189
Neurospora crassa hypothetical protein similar to endo 1 4 beta xylanase 385 nt
BOTH


NCU08398
Neurospora crassa conserved hypothetical protein 391 nt
BOTH


NCU08412
Neurospora crassa conserved hypothetical protein 401 nt
BOTH


NCU08760
Neurospora crassa predicted protein 343 nt
BOTH


NCU09024
Neurospora crassa conserved hypothetical protein 625 nt
BOTH


NCU09175
Neurospora crassa conserved hypothetical protein 411 nt
BOTH


NCU09491
Neurospora crassa feruloyl esterase B precursor 293 nt
BOTH


NCU09680
Neurospora crassa exoglucanase 2 precursor 485 nt
BOTH


NCU00798
Neurospora crassa predicted protein 313 nt
AV


NCU01595
Neurospora crassa protein SOF1 446 nt
AV


NCU02240
Neurospora crassa hypothetical protein similar to endoglucanase II 323 nt
AV


NCU02696
Neurospora crassa hypothetical protein similar to DEAD DEAH box RNA helicase
AV



1195 nt



NCU02855
Neurospora crassa endo 1 4 beta xylanase A precursor 221 nt
AV


NCU04205
Neurospora crassa predicted protein 346 nt
AV


NCU04395
Neurospora crassa endo 1 6 beta D glucanase precursor 481 nt
AV


NCU05121
Neurospora crassa endoglucanase V 294 nt
AV


NCU05134
Neurospora crassa hypothetical protein 124 nt
AV


NCU05852
Neurospora crassa conserved hypothetical protein 254 nt
AV


NCU05974
Neurospora crassa hypothetical protein similar to cell wall glucanosyltransferase
AV



Mwg1 365 nt



NCU08171
Neurospora crassa predicted protein 382 nt
AV


NCU08909
Neurospora crassa hypothetical protein similar to beta 1 3 glucanosyltransferase 
AV



543 nt



NCU08936
Neurospora crassa clock controlled gene 15 412 nt
AV


NCU09046
Neurospora crassa predicted protein 187 nt
AV


NCU09764
Neurospora crassa conserved hypothetical protein 406 nt
AV


NCU01651
Neurospora crassa conserved hypothetical protein 783 nt
MIS


NCU02343
Neurospora crassa hypothetical protein similar to alpha L arabinofuranosidase 
MIS



A 668 nt



NCU04202
Neurospora crassa nucleoside diphosphate kinase 1 153 nt
MIS


NCU04870
Neurospora crassa hypothetical protein similar to acetyl xylan esterase 313 nt
MIS


NCU05159
Neurospora crassa acetylxylan esterase precursor 301 nt
MIS


NCU05751
Neurospora crassa conserved hypothetical protein 242 nt
MIS


NCU06239
Neurospora crassa conserved hypothetical protein 514 nt
MIS


NCU08785
Neurospora crassa conserved hypothetical protein 291 nt
MIS


NCU09267
Neurospora crassa conserved hypothetical protein 1048 nt
MIS


NCU09708
Neurospora crassa conserved hypothetical protein 465 nt
MIS


NCU09775
Neurospora crassa hypothetical protein similar to alpha L arabinofuranosidase 
MIS



343 nt



NCU09923
Neurospora crassa hypothetical protein similar to beta xylosidase 775 nt
MIS





ANNOTATION - Generated by the Broad Institute (webpage broad.mit.edu/annotation/genome/neurospora/Home.html);


CULTURE- Culture in which peptides were detected for a particular protein.


BOTH, peptides detected in both Avicel and Miscanthus culture filtrates;


AV, peptides detected only in Avicel culture filtrates;


MIS, peptides detected only in Miscanthus culture filtrates.






Example 3
Characterization of Extracellular Proteins and Cellulase Activity in Strains Containing Deletions in Genes Identified in the Overlap of the Transcriptome/Secretome Datasets
Of the 22 extracellular proteins detected in both the Miscanthus and Avicel grown cultures, homokaryotic strains containing deletions in genes encoding 16 of these extracellular proteins were available to the public (Dunlap et al., 2007). None of these 16 deletion strains had been previously characterized with respect to their influence on plant cell wall or cellulose degradation in N. crassa. The 16 deletion strains were grown both on media containing sucrose or Avicel as a preferred carbon source. All strains showed a wild type growth phenotype on sucrose. On medium containing Avicel, the bulk growth of the 16 deletion strains was monitored for a 7-day period. After seven days, the total secreted protein, endoglucanase activity, β-glucosidase activity, and aggregate Avicelase activity of the culture filtrates was measured and compared with the wild-type strain from which all the mutants were derived (FIGS. 6A-C). SDS-PAGE was also done on unconcentrated culture supernatants to investigate the relative abundance of secreted proteins.
There were growth deficiencies on Avicel for strains containing deletions of two predicted exoglucanases (cbh-1; NCU07340 and gh6-2; NCU09680) and a predicted β-glucosidase (gh3-4; NCU04952). The cbh-1 mutant was the most severe; after seven days much of the Avicel remained, while in the wild-type strain all of the Avicel was degraded by this time. For 10 of the 16 deletion strains, SDS-PAGE analysis of the secreted proteins showed an altered extracellular protein profile where a single band disappeared, thus allowing assignment of a particular protein band to a predicted gene (FIG. 6A, boxes; FIG. 7). These included NCU00762 (gh5-1), NCU04952 (gh3-4), NCU05057 (gh7-1), NCU05137, NCU05924 (gh10-1), NCU05955, NCU07190 (gh6-3), NCU07326, NCU07340 (cbh-1), and NCU09680 (gh6-2).
For the majority of the deletion strains, the total secreted protein, endoglucanase, β-glucosidase, and Avicelase activities of the culture supernatants were similar to wild type (FIG. 6B, C and Table 9).







TABLE 9







Enzyme Activity of Deletion Strains
















Azo-






Growth
[Secreted Protein]
CMCase
Bgl
[CB]
[GLC]


Gene Name
on Avicel
(% of WT)
(% of WT)
(% of WT)
(mM)
(mM)





NCU00762
***
113 ± 8  
33 ± 2 
102 ± 2  
0.9 ± 0.0
2.6 ± 0.1


NCU01050
***
98 ± 12
92 ± 8 
88 ± 5 
0.8 ± 0.2
2.9 ± 0.3


NCU04952
***
146 ± 6  
124 ± 5  
  1 ± 0.3
2.24 ± 0.2 
0.6 ± 0.0


NCU05057
***
143 ± 10 
98 ± 3 
100 ± 10 
1.7 ± 0.1
3.6 ± 0.1


NCU05137
***
154 ± 12 
156 ± 10 
178 ± 3  
1.0 ± 0.0
3.8 ± 0.1


NCU05924
***
108 ± 3  
108 ± 5  
101 ± 4  
1.1 ± 0.1
2.6 ± 0.2


NCU05955
***
92 ± 10
94 ± 8 
98 ± 7 
0.9 ± 0.1
2.3 ± 0.1


NCU07190
***
111 ± 7  
136 ± 6  
92 ± 1 
1.1 ± 0.0
2.6 ± 0.0


NCU07326
***
105 ± 4  
114 ± 17 
85 ± 11
1.0 ± 0.0
2.3 ± 0.0


NCU07340
*
 41 ± 2.2
43 ± 9 
56 ± 9 
0.1 ± 0.0
0.5 ± 0.1


NCU07898
***
84 ± 7 
 86 ± 1.5
59 ± 15
0.5 ± 0.3
2.3 ± 0.5


NCU08189
***
83 ± 12
80 ± 8 
69 ± 15
0.5 ± 0.1
2.3 ± 0.4


NCU08398
***
95 ± 11
107 ± 7  
97 ± 3 
0.6 ± 0.1
1.8 ± 0.0


NCU08760
***
115 ± 3  
126 ± 6  
115 ± 8  
0.9 ± 0.1
2.6 ± 0.1


NCU09175
***
96 ± 7 
115 ± 0  
101 ± 8  
0.7 ± 0.0
1.9 ± 0.1


NCU09680
**
118 ± 7  
165 ± 7  
150 ± 1  
0.23 ± 0.1 
1.7 ± 0.1


WT
***
100 ± 7  
100 ± 12 
100 ± 6  
0.97 ± 0.0 
2.4 ± 0.1









Deviations from this trend were seen with the Δgh5-1 (NCU00762), Δgh3-4 (NCU04952), ΔNCU05137, Δcbh-1 (NCU07340), and Δgh6-2 (NCU09680) mutants. In Δgh5-1 (NCU00762), Δgh3-4 (NCU04952), and Δcbh-1 (NCU07340), Avicelase, endoglucanase or Δ-glucosidase activities were lower than the corresponding wild-type activities. In particular, the deletion of NCU04952 eliminated all β-glucosidase activity from the culture supernatant, as evidenced by PNPGase activity and by higher levels of cellobiose and lower levels of glucose in the Avicelase enzyme assays (FIG. 6B, C). Despite lowering endoglucanase activity, the culture filtrate from Δgh5-1 (NCU00762) showed no significant deficiency in Avicelase activity relative to the wild-type strain (FIG. 6C). As expected, mutations in cbh-1 (NCU07340) resulted in lower endoglucanase and Avicelase activity, due to poor growth. A strain containing a deletion of NCU09680, encoding a CBH(II)-like protein (gh6-2), also showed reduced cellobiose accumulation, as observed with Δcbh-1 mutant (FIG. 6C).
Mutations in three strains resulted in an increased level of secreted proteins, especially CBH(I) (FIG. 6A); gh3-4 (NCU04952), gh7-1 (NCU05057) and a hypothetical protein gene (NCU05137). In addition to increased levels of secreted proteins, the ΔNCU05137 mutant showed increased endoglucanase, β-glucosidase, and Avicelase activity (FIG. 6B, C). NCU05137 is highly conserved in the genomes of a number of filamentous ascomycete fungi, including other cellulolytic fungi, but notably does not have an ortholog in T. reesei (FIG. 2). It is possible that the increase in CBH(I) levels observed in Δgh3-4, Δgh7-1, and ΔNCU05137 could be due to either increased secretion, protein stability or, alternatively, feedback that results in an increase in expression of cbh-1. To differentiate these possibilities, the profile of extracellular proteins produced by ΔNCU05137 and Δgh3-4 (NCU04952) was compared with gene expression levels of cbh-1 (NCU07340) and gh6-2 (CBH(II); NCU09680) as assayed by quantitative RT-PCR (FIG. 8). The strains ΔNCU05137 and Δgh3-4 showed a higher level of CBH(I) protein as early as two days in an Avicel-grown culture. Quantitative RT-PCR of cbh-1 and gh6-2 from Avicel-grown cultures showed that both genes exhibited high expression levels in wild type and the ΔNCU05137 and Δgh3-4 mutants after two days of growth. However, although expression of both of these genes decreased significantly on day three in the wild-type strain, both cbh-1 and gh6-2 expression levels increased in the ΔNCU05137 mutant, and decreased less than wild type in Δgh3-4 (FIG. 8). Sustained expression of cbh-1 and gh6-2 genes in the ΔNCU05137 and Δgh3-4 mutants could be responsible for the observed increase in CBH(I) and CBH(II) protein levels.
Example 4
Materials and Methods for Transcriptome and Secretome Studies
Strains
All Neurospora crassa strains were obtained from the Fungal Genetics Stock Center (FGSC; webpage fgsc.net) (Supplemental Data, Dataset S1, page 1 in Tian et al., 2009). Gene deletion strains were from the N. crassa functional genomics project (Dunlap et al., 2007). Trichoderma reesei QM9414 was a gift from Dr. Monika Schmoll (Vienna University of Technology). Strains were grown on Vogel's salts (Vogel 1956) with 2% (w/v) carbon source (Miscanthus, sucrose or Avicel (Sigma)). Miscanthus x giganteus (milled stem to  ̃0.1 mm) was a gift from the University of Illinois.
Enzyme Activity Measurements
Total extracellular protein content was determined using a Bio-Rad DC Protein Assay kit (Bio-Rad). Endoglucanase activity in culture supernatants was measured with an azo-CMC kit (Megazyme SCMCL). β-glucosidase activity was measured by mixing 10-fold diluted culture supernatant with 500 μM 4-nitrophenyl β-D-glucopyranoside in 50 mM sodium acetate buffer, pH 5.0, for 10 minutes at 40° C. The reaction was quenched with 5% w/v sodium carbonate, and the absorbance at 400 nm was measured. Avicelase activity was measured by mixing 2-fold diluted culture supernatant with 50 mM sodium acetate, pH 5.0, and 5 mg/mL Avicel at 40° C. Supernatants were analyzed for glucose content using a coupled enzyme assay with glucose oxidase/peroxidase. Fifty μL of the avicelase reaction was transferred to 150 μL of glucose detection reagent containing 100 mM sodium acetate pH 5.0, 10 U/mL horseradish peroxidase, 10 U/mL glucose oxidase, and 1 mM o-dianisidine. After 30 minutes absorption was measured at 540 nm. Cellobiose concentrations were determined using a coupled enzyme assay with cellobiose dehydrogenase (CDH) from Sporotrichum thermophile. CDH was isolated from S. thermophile similar to previous reports (Canevascini 1988). Fifty μL of the avicelase reaction was transferred to 250 μL of cellobiose detection reagent containing 125 mM sodium acetate pH 5.0, 250 μM dichlorophenol indophenol, and 0.03 mg/mL CDH. After 10 minutes absorption was measured at 530 nm.
RNA Isolation, Microarray Analysis, and Signal Peptide Predictions
Mycelia were harvested by filtration and flash frozen in liquid nitrogen. Total RNA was isolated using trizol (Tian et al., 2007; Kasuga et al., 2005). Microarray hybridization and data analysis were as previously described (Tian et al., 2007). Normalized expression values were analyzed using BAGEL (Bayesian analysis of gene expression levels) (Townsend 2004; Townsend and Hartl 2002), which infers relative gene expression levels and credible intervals for each gene at each experimental time point. Signal peptides were predicted using the N-terminal 70 amino acid region of each predicted protein with the signalP3 program (webpage cbs.dtu.dk/services/SignalP-3.0/). Original profiling data is obtainable at (webpage yale.edu/townsend/Links/ffdatabase/).
Protein Gel Electrophoresis
Except where otherwise noted, unconcentrated culture supernatants were treated with 5×SDS loading dye and boiled for 5 minutes before loading onto Criterion 4-15% Tris-HCl polyacrylamide gels. Coomassie dye was used for staining.
Preparation of Tryptic Peptides for Secretome Analysis
Culture supernatants were concentrated with 10 kDa MWCO PES spin concentrators. Cellulose binding proteins were isolated from the culture supernatant by addition of phosphoric acid swollen cellulose (PASC). Five mL of a suspension of 10 mg/mL PASC was added to 10 mL of culture supernatant. After incubation at 4° C. for 5 minutes, the mixture was centrifuged and the pelleted PASC was then washed with 20 pellet volumes of 100 mM sodium acetate pH 5.0. The supernatant after treatment with PASC was saved as the unbound fraction and concentrated. 36 mg of urea, 5 μL of 1M Tris PH 8.5, and 5 μL of 100 mM DTT were then added to 100 μL of concentrated culture supernatant or protein-bound PASC and the mixture was heated at 60° C. for 1 hour. After heating 700 μL of 25 mM ammonium bicarbonate and 140 μL of methanol were added to the solution followed by treatment with 50 μL of 100 μg/mL trypsin in 50 mM sodium acetate pH 5.0. For the PASC bound proteins, the PASC was removed by centrifugation after heating, and the supernatant was then treated with trypsin. The trypsin was left to react overnight at 37° C. After digestion the volume was reduced by speedvac and washed with MilliQ water three times. Residual salts in the sample were removed by using OMIX microextraction pipette tips according to the manufacturer's instructions.
Liquid Chromatography of Tryptic Peptides
Trypsin-digested proteins were analyzed using a tandem mass spectrometer that was connected in-line with ultraperformance liquid chromatography (UPLC). Peptides were separated using a nanoAcquity UPLC (Waters, Milford, Mass.) equipped with C18 trapping (180 μm×20 mm) and analytical (100 μm×100 mm) columns and a 10 μL sample loop. Solvent A was 0.1% formic acid/99.9% water and solvent B was 0.1% formic acid/99.9% acetonitrile (v/v). Sample solutions contained in 0.3 mL polypropylene snap-top vials sealed with septa caps (Wheaton Science, Millville, N.J.) were loaded into the nanoAcquity autosampler prior to analysis. Following sample injection (2 μL, partial loop), trapping was performed for 5 min with 100% A at a flow rate of 3 μL/min. The injection needle was washed with 750 μL each of solvents A and B after injection to avoid cross-contamination between samples. The elution program consisted of a linear gradient from 25% to 30% B over 55 min, a linear gradient to 40% B over 20 min, a linear gradient to 95% B over 0.33 min, isocratic conditions at 95% B for 11.67 min, a linear gradient to 1% B over 0.33 min, and isocratic conditions at 1% B for 11.67 min, at a flow rate of 500 nL/min. The analytical column and sample compartment were maintained at 35° C. and 8° C., respectively.
Mass Spectrometry
The column was connected to a NanoEase nanoelectrospray ionization (nanoESI) emitter mounted in the nanoflow ion source of a quadrupole time-of-flight mass spectrometer (Q-Tof Premier, Waters). The nanoESI source parameters were as follows: nanoESI capillary voltage 2.3 kV, nebulizing gas (nitrogen) pressure 0.15 mbar, sample cone voltage 30 V, extraction cone voltage 5 V, ion guide voltage 3 V, and source block temperature 80° C. No cone gas was used. The collision cell contained argon gas at a pressure of 8×10-3 mbar. The Tof analyzer was operated in “V” mode. Under these conditions, a mass resolving power1 of 1.0×104 (measured at m/z=771) was routinely achieved, which is sufficient to resolve the isotopic distributions of the singly and multiply charged peptide ions measured in this study. Thus, an ion's mass and charge could be determined independently, i.e., the ion charge was determined from the reciprocal of the spacing between adjacent isotope peaks in the m/z spectrum. External mass calibration was performed immediately prior to analysis, using solutions of sodium formate. Survey scans were acquired in the positive ion mode over the range m/z=450-1800 using a 0.95 s scan integration and a 0.05 s interscan delay. In the data-dependent mode, up to five precursor ions exceeding an intensity threshold of 35 counts/second (cps) were selected from each survey scan for tandem mass spectrometry (MS/MS) analysis. Real-time deisotoping and charge state recognition were used to select 2+, 3+, 4+, 5+, and 6+ charge state precursor ions for MS/MS. Collision energies for collisionally activated dissociation (CAD) were automatically selected based on the mass and charge state of a given precursor ion. MS/MS spectra were acquired over the range m/z=50-2500 using a 0.95 s scan integration and a 0.05 s interscan delay. Ions were fragmented to achieve a minimum total ion current (TIC) of 30,000 cps in the cumulative MS/MS spectrum for a maximum of 3 s. To avoid the occurrence of redundant MS/MS measurements, real time exclusion was used to preclude re-selection of previously analyzed precursor ions over an exclusion width of ±0.25 m/z unit for a period of 180 s.
Mass Spectrometry Data Analysis
The data resulting from LC-MS/MS analysis of trypsin-digested proteins were processed using ProteinLynx Global Server software (version 2.3, Waters), which performed background subtraction (threshold 35% and fifth order polynomial), smoothing (Savitzky-Golay2 10 times, over three channels), and centroiding (top 80% of each peak and minimum peak width at half height four channels) of the mass spectra and MS/MS spectra. The processed data were searched against the N. crassa database (Broad Institute) using the following criteria: tryptic fragments with up to five missed cleavages, precursor ion mass tolerance 50 ppm, fragment ion mass tolerance 0.1 Da, and the following variable post-translational modifications: carbamylation of N-terminus and Lys side chains, Met oxidation, and Ser/Thr dehydration. The identification of at least three consecutive fragment ions from the same series, i.e., b or y-type fragment ions, was required for assignment of a peptide to an MS/MS spectrum. The MS/MS spectra were manually inspected to verify the presence of the fragment ions that uniquely identify the peptides.
Quantitative RT-PCR
The RT-PCR was performed in an ABI7300 with reagents from Qiagen (SYBR-green RT-PCR kit (Cat No. 204243)). The primers for CBHI (NCU07340) were: forward 5′-ATCTGGGAAGCGAACAAAG-3′ (SEQ ID NO: 16) and reverse 5′-TAGCGGTCGTCGGAATAG-3′ (SEQ ID NO: 17). The primers for CBHII (NCU09680) were: forward 5′-CCCATCACCACTACTACC-3′ (SEQ ID NO: 18) and reverse 5′-CCAGCCCTGAACACCAAG-3′ (SEQ ID NO: 19). Actin was used as a control for normalization. The primers for actin were: forward 5′-TGA TCT TAC CGA CTA CCT-3′ (SEQ ID NO: 20) and reverse 5′-CAG AGC TTC TCC TTG ATG-3′ (SEQ ID NO: 21). Quantitative RT-PCR was performed according to Dementhon et al., (2006).
Example 5
Discussion of Transcriptome and Secretome Studies
Degradation of plant biomass requires the production of many different enzymatic activities, which are regulated by the type and complexity of the available plant material (FIG. 9) (Bouws et al., 2008). The first systematic analyses of plant cell wall degradation by a cellulolytic fungus are described here, which include transcriptome, secretome, and mutant analyses. Profiling data showed that N. crassa coordinately expresses a host of extracellular and intracellular proteins when challenged by growth on Miscanthus or Avicel (FIG. 9). Many of the most highly expressed genes during growth on cellulosic substrates encode proteins predicted to be involved in the metabolism of plant cell wall polysaccharides, many of which were identified by MS analyses. Genome comparisons of filamentous fungi show a large number of glycosyl hydrolases ( ̃200) with varying numbers of predicted cellulases, from 10 in T. reesei (Martinez et al., 2008) to 60 in Podospora anserina (Espagne et al., 2008), a dung-degrading species closely related to N. crassa. A comparison between these results and a recent transcriptome/secretome study on the white rot basidiomycete fungus, Phanerochaete chrysosporium, (Wymelenberg et al., 2009) showed little overlap in regulated genes (18 genes) and secreted proteins (2 proteins) when both species were grown on pure cellulose. These data suggest that different fungi may utilize different gene sets for plant cell wall degradation. However, one aspect that both studies had in common was the high number of uncharacterized genes/proteins associated with cellulose degradation. Other cellulolytic fungi, including P. chrysosporium, do not have the genetic and molecular tools that are readily available with N. crassa. Using the functional genomic tools available with N. crassa, both the function and redundancy of plant cell wall degrading enzyme systems can be addressed to create optimal enzyme mixtures for industrial production of liquid fuels from lignocellulose biomass.
In this study, it was found that cellobiohydrolase(I) (CBHI) in N. crassa is the most highly produced extracellular protein during growth on Avicel or Miscanthus, and deletion of this gene caused the most severe growth deficiencies on cellulosic substrates. These results are similar to those reported in T. reesei (Suominen et al., 1993, Seiboth et al., 1997). Deletion of cellobiohydrolase(II) also caused growth deficiencies on cellulosic substrates, but to a much lesser extent than CBH(I), suggesting that exoglucanase activity in N. crassa is predominantly from CBH(I) and that cellulases and other CBHs do not compensate for the loss of CBH(I). Here, it was shown that the three most highly produced endoglucanases during growth on cellulosic substrates are the proteins encoded by NCU05057, NCU00762, and NCU07190. These proteins have homology to endoglucanases EG1, EG2, and EG6, respectively. Deletion of these genes did not affect growth on Avicel, although differences in the secreted protein levels and endoglucanase activity were observed. Unexpectedly, in the ΔNCU05057 strain, extracellular protein levels were much higher, especially CBH(I), suggesting that to maintain the wild-type growth phenotype on crystalline cellulose the mutant was forced to increase production of other cellulases or that the products of NCU05057 catalysis may repress cellulase production. It was concluded that no one endoglucanase in N. crassa is required for growth on crystalline cellulose and that the different endoglucanases have overlapping substrate specificities.
The glycoside hydrolase family 61 enzymes are greatly expanded in N. crassa compared to T. reesei (Martinez et al., 2008). These enzymes have poorly defined biological function, but their general conservation and abundance in cellulolytic fungi suggests an important role in plant cell wall metabolism. Here, genes for 10 of the 14 GH61 enzymes were identified in the N. crassa transcriptome, suggesting that these enzymes are utilized during growth on cellulosic biomass. The four GH61 deletion strains tested showed only small differences compared to wild type in the secreted protein levels, endoglucanase, and total cellulase activities. However, analyses of additional GH61 mutants and the capacity to create strains containing multiple mutations in N. crassa via sexual crosses will address redundancy and expedite functional analysis of this family.
In addition to predicted cellulase genes, genes encoding hemicellulases, carbohydrate esterases, β-glucosidases, β-xylosidases, and other proteins predicted to have activity on carbohydrates were identified in the N. crassa transcriptome from both Miscanthus and Avicel. The fact that Avicel contains no hemicellulose components suggests that cellulose is probably the primary inducer of genes encoding plant cell wall degrading enzymes in N. crassa. However, genes encoding some hemicellulases and carbohydrate esterases were only expressed during growth on Miscanthus. Similarly, in other cellulolytic fungi such as T. reesei and Aspergillus niger, genes encoding some cellulases and hemicellulases are coordinately regulated, while others are differentially regulated (Stricker et al., 2008). As expected, deletions of non-cellulase genes had little effect on growth on Avicel or cellulase activity, with the exception of NCU05137 and gh3-4. The ΔNCU05137 strain secreted more protein, had higher cellulase activity, and showed higher expression of cbh-1 (CBH(I)) and gh6-2 (CBH(II)) than wild type. NCU05137 encodes a secreted hypothetical protein that has no homology to proteins of known function, but is highly conserved in other cellulolytic fungi (FIG. 2; E value 0.0). NCU05137 also has more distant homologs, but also of unknown function, in a number of bacterial species. The protein product of NCU05137 may interfere with signaling processes associated with induction of cellulase gene expression N. crassa (FIG. 9). Similarly, mutations in gh3-4 (NCU04952) also increased CBH(I) activity. Deletion of this gene completely removed PNPGase activity and cellobiose accumulated in in vitro cellulase assays using Δgh3-4 culture filtrates. All the data together suggested that NCU04952 encodes the primary extracellular β-glucosidase in N. crassa. These data were consistent with catabolite repression of cellulase production by glucose.
Extracellular degradation of cellulose and hemicellulose results in the formation of soluble carbohydrates that are subsequently transported into the cell (FIG. 9). In this study, 10 genes encoding permeases/transporters were identified which showed significantly increased expression when N. crassa was grown on Miscanthus or Avicel, suggesting their involvement in transport of plant cell wall degradation products into the cell. The major degradation products by cellulases and hemicellulases in vitro are cellobiose, glucose, xylobiose, and xylose. Some of these transporters may be functionally redundant or capable of transporting oligosaccharides. The function of these putative transporters was further explored (see Examples 7-9). Construction of downstream processing strains capable of transporting oligosaccharides by heterologous expression of N. crassa transporters may improve industrial fermentation of biomass hydrolysis products. None of these transporters or what they may transport has been characterized at the molecular or functional level in any filamentous fungi.
Many genes that showed increased expression levels during growth on Miscanthus and Avicel encode proteins of unknown function that are conserved in other cellulolytic fungi. By assessing the phenotype of only 16 strains, a mutant in a gene encoding a protein of unknown function that significantly affects cellulase activity was identified. The well-understood genetics and availability of functional genomic resources in N. crassa make it an ideal model organism to determine the biological function of these proteins, as well as regulatory aspects of cellulase and hemicellulase production, and to dissect redundancies and synergies between extracellular enzymes involved in the degradation of plant cell walls.
Example 6
Screening of Mutants of Genes Upregulated During Growth on Miscanthus 
In order to analyze additional genes identified in the transcriptional profiling experiment, the phenotypes of mutants of 188 genes that were upregulated in Neurospora grown on Miscanthus for 16 hours were analyzed (see Example 1). A knockout mutant of each gene was grown on minimal Vogel's medium for 10-14 days. Conidia were harvested with 2 mL ddH2O and inoculated into 100 mL media in 250 mL flasks at a concentration of 106 conidia per mL. One of three different carbon sources was added to each flask: 2% sucrose, 2% Avicel, or 2% Miscanthus (1 mm particles from Calvin Laboratory, University of California, Berkeley, Calif.). Cultures were grown at 25° C. with 220 rpm of shaking for 4 days.
Table 10 lists the phenotypes of the mutants that showed a significant difference in cellulase activity and growth on Avicel or Miscanthus compared to wild-type. Growth on Avicel or Miscanthus was evaluated by eye with a “+” scoring system. Wild-type growth was set at “++”. Total protein in the culture supernatant was measured by Bradford assay (100 μl supernatant to 900 μl Bradford dye). Endoglucanase activity was measured with the Azo-CMC kit from Megazyme and indicated in Table 10 as the percentage of endoglucanase activity in the mutant compared to wild type. Total cellulase activity was measured by detecting cellobiose levels in the supernatant as described in Example 4. Results are indicated in Table 10 as a percentage of wild-type.







TABLE 10







shows mutant screening data
























% WT
% WT
% WT 
% WT
% WT
% WT







Growth 
Bradford
Bradford
endo 
endo
cellobiose 
cellobiose


NCU #
FGSC #
Broad Annotation (Domains)
Pfam*
Up-Regulation
(Avi, Mis)
(Avi)
(Mis)
(Avi)
(Mis)
(Avi)
(Mis)





















NCU00130.2
FGSC
beta-glucosidase (GH1)
Glycosyl
394.6
++, ++
203.2477947
118.3987972
152.2858578
129.3547494
n/a
n/a



11823

Hydrolase 1













(2.5e−196)










NC000248.2
FGSC
Predicted Protein
no significant hit
9.74
+, ++
86.96013289
86.04471858
30.39187506
156.5050144
93.05143946
89.27698219



12214












NC000326.2
FGSC
Conserved Hypothetical
SMP-30/
7.7
+, ++
33.02879291
144.1210486
39.91568458
227.0366809
89.76872415
79.05154639



15868
(SMP-30/gluconolactonase)
Gluconolaconase/













LRE-like 













region













(3.5e−82)










NC000762.2
FGSC
Endoglucanase-3
Cellulase
29.6
++, ++
104.3504411
84.57056944
26.92790756
39.26890058
n/a
n/a



16747
precursor (GH5, CBD1)
(1.4e−69),













Fungal













cellulose













binding













domain













(9.2e−14)










NC000810.2
FGSC
Similar to Glycosyl Hydrolase
Glycosyl
5.3
++, ++
163.805047
123.5564757
161.2908993
159.4983744
102.2745211
91.73345664



11285
(GH2, beta-galactosidase)
hydrolases













family 2













(1.7e−145)










NCU00890.2
FGSC
Similar to beta-manosidase
Glycosyl
20.45
+, +
47.57417803
101.5974441
43.25546345
164.0819718
n/a
n/a



16749
(GH2)
hydrolases













family 2 













(4.1e−06)










NCU03328.2
FGSC
Conserved Hypothetical
Glycosyl
26 .4
++, ++
100.1752848
109.9667248
142.6962073
167.0075481
n/a
n/a



16589
(GH61)
hydrolase













family 61













(2.3e−10)










NCU03415.2
FGSC
Aldehyde Dehydrogenase
Aldehyde
9.8
++, ++
104.2278204
96.61435373
96.4633125
63.45523329
76.966433943
103.1273983



12922

dehydro-













genase













family













(2.5e−267)










NCU03731.2
FGSC
Similar to HAD Superfamily
haloacid
2.7
++, ++
131.3691128
110.5801446
145.0235135
134.627995
230.14505412
100.4172375



18653
Hydrolase
dehalo-













genase-like













hydrolase













(9.2e−21)










NCU03753.2
FGSC
ccg-1 (clock controlled
no
10.5
++, ++
107.6792892
111.3481086
74.42402278
129.2196777
n/a
n/a



16379
gene)
significant













hit










NCU04197.2
FGSC
Conserved Hypothetical
no
5.04
++, ++
103.0668127
99.08305414
108.9737808
89.86128625
75.17285531
96.05075054



17499

significant













hit










NCU04249.2
FGSC
Hypothetical Protein
no
5.3
++, ++
93.29682366
106.1012167
79.0053469
84.16141236
64.07989522
100.124185



18628

significant













hit










NCU04287.2
FGSC
Predicted Protein
no
4.7
++, ++
115.5157859
102.2361065
125.5086234
127.9282577
202.516129
183.8679245



14573

significant













hit










NCU04349.2
FGSC
Similar to mitochondrial
BCDHK_A
2.9
++, ++
87.87776465
89.36205196
71.41381803
145.2415813
208.4329349
101.2993763



18634
pyruvate dehydrogenase
dom3












kinase
(4.7e−78),













HistidineA













TPase_c













(6.9e−14)










NCU04475.2
FGSC
Predicted
no
76.7
+++, ++
98.10205352
122.2034851
156.3643221
127.0676692
n/a
n/a



15386
Protein
significant













hit










NCU04997.2
FGSC
Similar to xylanase
Glycosyl
25.6
++, ++
105.3520176
114.5840184
123.3295466
231.6983895
136.4189483
102.5403983



15623
(GH10, CBD1)
hydrolase













family 10













(3.3e−148),













Fungal













cellulose













binding













domain













(2.1e−16)










NCU05057.2
FGSC
Endoglucanase EG-1
Glycosyl
8.7
++, ++
137.5316563
95.69220651
133.5226686
174.2679356
182.023775
97.81330657



13342
precursor (GH7)
hydrolase













family 7













(3.3e−189)










NCU05159.2
FGSC
acetylxylan esterase
Cutinase
34.8
+++, ++
86.18543871
39.51658235
92.2873845
67.11779449
n/a
n/a



13439
precursor (Cutinase, CBD1)
(3.4e−110),













Fungal













cellulose













binding













domain













(7.4e−14)










NCU05493.2
FGSC
Predicted Protein
no
4.5
+, ++
73.25266013
104.4102564
102.3841739
116.8954593
70.37185126
99.42837929



14625

significant













hit










NCU05519.2
FGSC
Similar to Tna1
Major











19924
(MFS transporter)
Facilitator













Superfamily
2.8
++, ++
85.31191321
101.0666667
118.8447721
87.77719113
51.6886931
97.87501655





(3.7e−40)










NCU05751.2
FGSC
Conserved Hypothetical
GDSL-like
3.9
+, ++
97.01648237
111.4051282
114.7202911
136.3780359
87.71492649
105.5920583



15757
(GDSL-like lipase)
Lipase/













Acylhydrolase 













(1.3e−11)










NCU05770.2
FGSC
Peroxidase/Catalase 2
Peroxidase 
11.9
++, ++
109.8630989
86.73412029
69.1872525
146.2155388
n/a
n/a



11532

(9.4e−195)










NCU05853
FGSC
Sugar Transporter
Sugar
130.7
+
40.27924687
24.41259790
n/a
n/a
n/a
n/a



13771

Transporter
20.9
+, ++
33.78464142
34.72754541
26.3266891
86.25954198
n/a
n/a


NCU05897.2
FGSC
Similar to l-fucose permease
Major











13717
(MFS transporter)
Facilitator













Superfamily













(3.8e−16)










NCU05932.2
FGSC
Predicted Protein
no
38.2
++, ++
70.89826428
76.87132044
80.78910753
117.9596823
58.07431478
96.7108463



19952

significant













hit










NCU06009.2
FGSC
Similar to aldo/keto
Aldo/keto
6.9
+, ++
148.6633726
74.06784413
120.602266
99.48075748
70.89513625
97.00573241



14922
reductase
reductase













family













(4.8e−63)










NCU06490.2
FGSC
Conserved Hypothetical
no
13.8
+, ++
77.46104143
80.26352677
76.95289207
79.38301772
59.91109168
99.99371385



15539

significant













hit










NCU07340.2
FGSC
Exoglucanase-1 precursor,
Glycosyl
426.4
+, ++
21.09634551
95.21973786
35.54661301
96.99134496
93.62619808
78.44902553



15630
CBH1 (GH7)
hydrolase













family 7













(1e−999),













Fungal













cellulose













binding













domain













(4.9e−18)










NCU07853.2
FGSC
Uricase
Uricase
4.3
+++, ++
n/a
n/a
120.9286562
168.2340648
65.7599456
99.14659177



19036

(1.7e−119)










NCU07997.2
FGSC
Predicted Protein
no
4.5
++, ++
n/a
n/a
148.127436
98.11912226
60.65548063
93.78704271



18273

significant













hit










NCU08114.2
FGSC
Similar to MFS hexose
Sugar
6.7
+, ++
81.69263905
79.22624054
85.18187239
92.97495418
58.83068556
93.1432252



17869
transporter (MFS transporter)
(and other)













transporter 













(5.1e−88), 













Major













Facilitator













Superfamily 













(3.8e−24)










NCU08744.2
FGSC
Predicted Protein, possible
no
2.3
++, ++
n/a
n/a
168.8527368
110.7628004
136.2451567
97.44134197



11387
TF (basic region leucine
significant












zipper)
hit










NCU08746.2
FGSC
Conserved Hypothetical
Starch
6
++, ++
98.69504624
79.11410149
111.0713576
120.2504582
447.2796518
100.5753667



18358
(starch binding domain)
binding













domain













(5.3e−54)










NCU08760.2
FGSC
Predicted Protein (CBD1)
Fungal
107.5
++, ++
158.1395349
86.17964534
208.2590783
81.00013738
97.32646961
84.34251774



15664

cellulose













binding













domain













(1.9e−11),













Glycosyl













hydrolase













family 61













(1.3e−9)










NCU09108.2
FGSC
Conseryed Hypothetical
no
4.1
++, ++
n/a
n/a
93.22148788
111.8077325
60.89420655
97.24517906



19207

significant













hit










NCU09495.2
FGSC
set-6, histone methyltransferase
SET
26.2
++, ++
109.3300111
122.5327679
129.9223915
130.8971013
152.7495439
92.25216554



12411

domain













(6.9e−5)










NCU09680.2
FGSC
Exoglucanase-2
Glycosyl
230.9
+, ++
102.7131783
95.20046261
89.54680464
102.6789394
94.61873756
83.87661343



15633
precursor, CBH2
hydrolases












(GH6, CBD1)
family 6













(1.1e−152),













Fungal













cellulose













domain













(1.2e−13)










NCU10045.2
FGSC
pectin-esterase precursor
Pectin-
10.9
+, ++ 
105.3085012
101.5138772
109.8886901
132.5290165
83.25906421
103.6151641



18480

esterase













(4.4e−22) 













*Note: All sequences were searched against Pfam Is models and hits were accepted with an e-value <.0001






Example 7
Further Analyses of Transporter Genes
As described in Example 1, ten genes encoding predicted sugar transporter proteins showed increased expression levels when Neurospora was grown on Miscanthus and Avicel: NCU00801, NCU00988, NCU01231, NCU04963, NCU05519, NCU05853, NCU05897, NCU06138, NCU08114 and NCU10021. Deletion strains for nine of these genes were available from the Fungal Genetics Stock Center. A deletion strain of NCU10021 was not available.
Deletion mutations of NCU05853, NCU05897, or NCU08114 resulted in strains that showed a growth defect on Miscanthus or Avicel and/or had a cellulase enzyme defect (see Example 6; Table 10). ΔNCU05853 showed reduced growth on Avicel and reduced endoglucanase activity compared to wild-type. ΔNCU05897 showed reduced growth on Avicel and reduced endoglucanase activity compared to wild-type, and ΔNCU08114 showed reduced growth on Avicel and reduced cellobiose levels compared to wild-type. Notably, in a comparison with expression analysis of Sporotrichum thermophile, another filamentous fungus, the homologs of NCU05853 (ST8454) and NCU08114 (ST5194) were also upregulated when S. thermophile was grown on Avicel compared to glucose (see Example 8, Table 11), further indicating their importance in cellulose utilization.







TABLE 11







shows S. thermophile expression data


















Like
Gene










Gene Name
NCU #
Length
Glu
Avi
Cot
Glu norm
Avi norm
Cot_ norm
Avi/Glu
Cot/Glu





jgi|Spoth1|108890|
NCU00988
1937
322
370
293
42.97830583
60.2250594
48.07756207
1.149068
0.9099379


estExt_fgenesh1_












pg.C_60848












jgi|Spoth1|48439|
NCU01132
1539
113
59
56
15.08244894
9.60345542
9.188885583
0.522124
0.4955752


e_gw1.3.3367.1












jgi|Spoth1|79030|
NCU01231
1776
1171
1206
469
156.2968824
196.30114
76.95691676
1.02988
0.4005124


estExt_Genewise1












Plus.C_31624












jgi|Spoth1|116270|
NCU05519
1680
103
78
54
13.74771895
12.6960936
8.860711098
0.757282
0.5242718


estExt_fgenesh1_












pm.C_50266












jgi|Spoth1|84164|
NCU05853
1706
2703
20760
14284
360.7775176
3379.11414
2343.822173
7.680355
5.2844987


estExt_Genewise1












Plus.C_62100












jgi|Spoth1|
NCU05897
1446
1510
546
322
201.5442292
88.8726553
52.8360921
0.361589
0.213245


102977|fgenesh1_












pm.5_#_763












jgi|Spoth1|84305|
NCU06138
1605
1131
1330
2376
150.9579624
216.484673
389.8712883
1.17595
2.1007958


estExt_Genewise1












Plus.C_70023












jgi|Spoth1|114107|
NCU08114
1945
2246
22423
10779
299.7803568
3649.80137
1768.696388
9.983526
4.7991986


estExt_fgenesh1_












pm.C_20669












jgi|Spoth1|112305|
NCU10021
2026
6204
5287
5619
828.0664888
860.567268
922.006216
0.852192
0.905706


estExt_fgenesh1_












kg.C_60263












jgi|Spoth1|43941|
NCU00801
1614
41
71
159
5.472392979
11.5567006
26.08987157
1.731707
3.8780488


e_gw1.2.4209.1












jgi|Spoth1|62521|
NCU04963
2204
799
1548
641
106.6449266 
251.968627
105.1799225
1.937422
0.8022528


estExt_Genewise












1.C_21757









In order to narrow down the identity of each predicted transporter's substrate, strains containing deletion mutations of NCU05853 or NCU08114 were cultured on glucose, xylose, cellobiose, xylan and Avicel (Table 12). The culturing medium contained Vogel's medium plus 2% of the carbon source. Both mutants showed greatly reduced growth on Avicel but not on xylan, glucose, xylose, or cellobiose.







TABLE 12







shows growth of deletion mutants on different sugars














Gene
Growth on
Growth
Growth
Growth on
Growth on
Growth
Growth on


Name
Sucrose
on Avicel
on Mis
Xylan
Glucose
on Xylose
Cellobiose





NCU00801
***
***
***






NCU00988
***
***
***






NCU01231
***
***
***






NCU04963
***
***
***






NCU05519
***
***
***






NCU05853
***
*
**
***
***
***
***


NCU05897
***
*
**






NCU06138
***
***
***






NCU08114
***
*
**
***
***
***
***


NCU10021
No









deletion









strain








wt
***
***
***
***
***
***
***









To investigate the role of these transporters in utilization of hemicellulose, the expression of the ten transporter genes was examined when Neurospora was grown on xylan. Methods were used as described in Example 4, except that strains were grown on Vogel's salts with 2% (w/v) xylan. Expression of all ten transporters was upregulated during growth on xylan (Table 13), suggesting that they can transport sugars derived from hemicellulose degradation (e.g., xylobiose, xylose, arabinose, xylo-oligosaccharides) as well as from cellulose degradation (e.g., cellobiose, glucose, cello-oligosaccharides). The mutant growth results and expression analyses suggested that at least two of the predicted transporters, NCU05853 and NCU08114, can transport disaccharides (cellobiose, xylobiose) and/or oligosaccharides (cellodextrins).







TABLE 13







shows expression analysis of transporter genes









Gene Name 
wt-Xylan 4 h
Fold change in St-Avice1-4 h/Glucose-4 h












NCU00801
~6
10


NCU00988.2
31.1
NO CHANGE


NCU01231.2
732.1
NO CHANGE


NCU04963.2
96.5
NO DETECT


NCU05519.2
3.9
NO CHANGE


NCU05853.2
71.2
8.5


NCU05897.2
122.3
NO CHANGE


NCU06138.2
141.0
NO CHANGE


NCU08114.2
10.0
11


NCU10021.2
44.7
NO CHANGE









Example 8
Expression Analysis of Sporotrichum thermophile Homologs of N. crassa Transporters During Growth on Various Carbon Sources
In order to compare the expression of homologous genes from a different filamentous fungus, the expression profile of Sporotrichum thermophile was analyzed from cultures grown on glucose, Avicel, or cotton. cDNA was isolated from cultures grown on minimal media with a carbon source of glucose, Avicel, or cotton for 16-30 hours.
First, in order to identify homologs of Neurospora transporter proteins in the S. thermophile genome, each Neurospora sequence was compared against a database of S. thermophile proteins with BLAST. The sequences of S. thermophile proteins found by this method were then compared to a database of Neurospora proteins with BLAST. These results are listed in FIG. 10. The amino acid sequences for all of the S. thermophile homologs of putative Neurospora transporters that were identified can be found in SEQ ID NOs: 22-32.
Next, the expression profile of the S. thermophile homologs was examined. The data is presented in Table 11. The first column contains the S. thermophile gene name from the Joint Genome Institute S. thermophile assembly. The second column contains the NCU number for the most closely related putative transporter in Neurospora. The third column contains the gene length of the S. thermophile gene in nucleotides. The fourth to sixth columns contain the expression level (number of reads, comparable to absolute expression level) during growth on Vogel's minimal media supplemented with 2% of glucose, Avicel, or cotton balls as the carbon source. The seventh to ninth columns contain the normalized expression data (the # of reads divided by the total reads in the dataset). The final two columns contain the relative expression level data for each gene as a ratio of Avicel/glucose or cotton/glucose. Homologs of NCU5853, NCU8114, and NCU0801 were upregulated when grown on both Avicel and cotton. The homolog of NCU6138 was upregulated when grown on cotton, and the homolog of NCU4963 was upregulated when grown on Avicel. These data provided further support that putative transporters NCU5853, NCU8114, NCU0801, NCU6138, and NCU4963 are important for the utilization of cellulose.
Example 9
Identification and Analysis of Cellodextrin Transporters
When grown on pure cellulose, N. crassa was shown to increase transcription of seven Major Facilitator Superfamily sugar transporters as well as an intracellular β-glucosidase (Ex. 1; also see Supplemental Data, Dataset S1, page 6 in Tian et al., PNAS, 2009). Notably, knockout strains lacking individual transporters from this set grew more slowly on crystalline cellulose, suggesting that they may play a direct role in cello-oligosaccharide uptake under cellulolytic conditions (Ex. 7; Tables 10, 12). For example, deletion of NCU08114 resulted in severely retarded N. crassa growth (FIGS. 11A-B), and reduced N. crassa consumption of cellobiose (FIGS. 12A-D and 13). In this example, transporter genes NCU00801/cbt1 and NCU08114/cbt2 were further analyzed and identified to encode transporters of cellodextrin.
To assay the function of each transporter individually, the fact that cellobiose is not catabolized by S. cerevisiae and is not accumulated in its cytoplasm was exploited (FIG. 14). It was reasoned that expression of a functional cellobiose transporter in conjunction with an intracellular β-glucosidase would allow S. cerevisiae to grow when cellobiose is presented as the sole carbon source. Yeast strains were engineered to express the transporters NCU00801 or NCU08114 fused to Green Fluorescent Protein (GFP), and the putative intracellular β-glucosidase, NCU00130. Both transporters were expressed and localized correctly to the plasma membrane (FIGS. 15A-B). The strains expressing NCU00801 or NCU08114 allowed yeast to grow with specific growth rates of 0.0341 hr−1 and 0.0131 hr−1, respectively (FIG. 16A). These growth rates correspond to 30% and 12% of the growth rate on glucose, respectively (FIG. 17). Growth could not be explained by the extracellular hydrolysis of cellobiose to glucose followed by transport, as a strain expressing only the putative intracellular β-glucosidase grew at a rate of 0.0026 hr−1 (FIG. 16A), and did not grow in large-scale cultures (FIG. 18). Based on these observations, NCU00801 and NCU08114, which were named CBT1 and CBT2, were determined to function as cellobiose transporters.
To directly assay transporter function, the uptake of [3H]-cellobiose into yeast cells was measured. Both CBT1 and CBT2 were found to be high-affinity cellobiose transporters, with Km values of 4.0±0.3 μM and 3.2±0.2 μM, respectively (FIG. 19). The expression-normalized Vmax of CBT1 was 2.2 times that of CBT2, a fact that explained differences seen in the yeast growth assays. Notably, cellodextrin molecules longer than cellobiose supported the growth of yeast expressing cbt1 and cbt2 (FIG. 20; FIG. 16B), suggesting that cellodextrin molecules are transported by CBT1 and CBT2. In agreement, cellobiose transport by CBT1 and CBT2 was inhibited by excess cellotriose, and CBT1 activity was also inhibited by cellotetraose (FIG. 21). Furthermore, upon purification, the β-glucosidase, NCU00130 (FIG. 22), was found to hydrolyze cellobiose, cellotriose, and cellotetraose (FIG. 16C).
Orthologs of cbt1 and cbt2 were identified and found to be widely distributed in the fungal kingdom (FIG. 23). Recent expression data shows their importance to various interactions between fungi and plants. For example, when the ascomycete, Tuber melanosporum, or the basidiomycete, Laccaria bicolor, interacts symbiotically with root tips to form ectomycorrihzas, the ortholog of cbt1 is upregulated in both (Martin et al., 2010). Likewise, the saprophytes, Aspergillus oryzae (Noguchi et al., 2009), Postia placenta (Vanden Wymelenberg et al., 2010), and Phanerochaete chrysosporium (Vanden Wymelenberg et al., 2010), upregulate orthologs of cbt2 when in contact with plant wall material. Certain yeasts, such as Kluveromyces lactis and Pichia stipitis grow on cellobiose (Freer, 1991; Preez et al., 1986), and cellobiose transport has been reported in Clavispora lusitaniae (Freer and Greene 1990). It was determined in this study that all of these yeasts contain orthologs of cbt1, cbt2, or both (see below for methods). Cellobiose transport has been observed in Hypocrea jecorina (Trichoderma reesei), but since the transporter was not identified, it is not clear if this activity can be ascribed to orthologs of cbt1 or cbt2 (Kubicek et al., 1993).
The use of cellobiose transporters by cellulolytic fungi suggests that they are essential for their optimal growth on cellulose. To test whether cellobiose catabolism could improve yeast ethanol production, the yeast strains constructed above were grown under fermentation conditions. With little optimization, yeast with a complete cellobiose catabolism pathway ported from N. crassa were shown to ferment cellobiose to ethanol efficiently (FIG. 24A), with an ethanol yield of 0.47, 86% of the theoretical value (Bai et al., 2008). This was comparable to industrial yields from glucose of 90-93% (Basso et al., 2008). The high affinity of CBT1 and CBT2 for cellobiose compared to the hexose transporters of S. cerevisiae (Reifenberger et al., 1997), and reported extracellular β-glucosidases (Chauve et al., 2010), suggested that a cellobiose/cellodextrin transport system would be particularly useful during SSF. For example, cellobiose/celldextrin transport would lower the requirement for full hydrolysis of cellulose to glucose, decrease cellobiose-mediated inhibition of cellulolytic enzymes, and reduce the risk of contamination by glucose-dependent organisms. Indeed, yeasts expressing a cellobiose/cellodextrin transport system markedly improved the efficiency of SSF reactions by reducing the steady state concentration of both cellobiose and glucose, and increasing the ethanol production rate (FIG. 24B, C).
Biofuel production from cellulose requires efficient and economical depolymerization of plant biomass to sugars coordinated with fuel production by improved host strains (Kumar et al., 2008). Here it was shown that cellulolytic fungi use cello-oligosaccharide transport pathways for optimal growth on plant biomass. Furthermore, reconstitution of these pathways in yeast revealed that they can be ported in a modular fashion to improve cellobiose catabolism, with a minimal pathway composed of a transporter and an intracellular cello-oligosaccharide hydrolase (FIG. 25). The use of cellodextrin transport in biofuel-producing strains of yeast and other organisms is critical for making cellulosic biofuel processes more economically viable.
Transporter and β-Glucosidase Orthologs
GenBank accession numbers or Joint Genome Institute (JGI) protein ID (PID) numbers for cellodextrin transporters are as follows: Tuber melanosporum, CAZ81962.1; Pichia stipitis, ABN65648.2; Laccaria bicolor, EDR07962; Aspergillus oryzae, BAE58341.1; Phanerochaete chrysosporium, PID 136620 (JGI) (Martinez et al., 2004); Postia placenta, PID 115604 (JGI) (Martinez et al., 2009). The GenBank accession number for Saccharomyces cerevisiae HXT1 and Kluyveromyces lactis LACP are DAA06789.1 and CAA30053.1, respectively. The P. chrysosporium and P. placenta genomes can be accessed at genome.jgi-psf.org/Phchr1/Phchr1.home.html and genome.jgi-psf.org/Pospl1/Pospl1.home.html, respectively.
GenBank accession numbers for cellodextrin hydrolases that are orthologs of NCU00130 are as follows: T. melanosporum, CAZ82985.1; A. oryzae, BAE57671.1; P. placenta, EED81359.1; and P. chrysosporium, BAE87009.1. The other organisms that contain cellodextrin transporter orthologs contain genes in the GH3 family predicted to be intracellular β-glucosidases (Bendtsen et al., 2004; Cantarel et al., 2009), as follows: Kluyveromyces lactis, CAG99696.1; Laccaria bicolor, EDR09330; Clavispora lusitaniae, EEQ37997.1; and Pichia stipitis, ABN67130.1.
Strains and Media
The yeast strain used in this study was YPH499 (Sikorski et al., 1989), which has the genotype: MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1. It was grown in YPD media supplemented to 100 mg/L adenine hemisulfate. Transformed strains (Becker et al., 2001) were grown in the appropriate complete minimal dropout media, supplemented to 100 mg/L adenine hemisulfate. Neurospora crassa stains used in this study were obtained from the Fungal Genetics Stock Center (McCluskey 2004) and include WT (FGSC 2489) and two cellobiose transporter deletion strains (FGSC 16575, ΔNCU00801.2 and FGSC 17868, ΔNCU08114.2 (Colot et al., 2006)).
Plasmids and Cloning
Transporters were cloned into the 2μ plasmid, pRS426, which was modified to include the S. cerevisiae PGK1 promoter inserted between SacI and SpeI using the primers, ATATATGAGCTCGTGAGTAAGGAAAGAGTGAGGAACTATC (SEQ ID NO: 53) and ATATATACTAGTTGTTTTATATTTGTTGTAAAAAGTAGATAATTACTTCC (SEQ ID NO: 54). (In all primers above and below, restriction sites are underlined). NCU00801 with a C-terminal Myc-tag and optimized Kozak sequence (Miyasaka 1999) was then inserted between BamHI and EcoRI using the primers, ATGGATCCAAAAATGTCGTCTCACGGCTCC (SEQ ID NO: 55) and ATGAATTCCTACAAATCTTCTTCAGAAATCAATTTTTGTTCAGCAACGATAGCTTCGGAC (SEQ ID NO: 56), and NCU08114 with a C-terminal Myc-tag and optimized Kozak sequence was inserted between SpeI and ClaI using the primers, ATACTAGTAAAAATGGGCATCTTCAACAAGAAGC (SEQ ID NO: 57) and GCATATCGATCTACAAATCTTCTTCAGAAATCAATTTTTGTTCAGCAACAGACTTGCCCTCAT G (SEQ ID NO: 58). To make GFP fusions, superfolder GFP (Pedelacq et al., 2006) with an N-terminal linker of Gly-Ser-Gly-Ser was first inserted between the ClaI and SalI sited of the PGK1 promoter-containing pRS426 plasmid with the primers, TATTAAATCGATGGTAGTGGTAGTGTGAGCAAGGGCGAGGAG (SEQ ID NO: 59) and TATTAAGTCGACCTACTTGTACAGCTCGTCCATGCC (SEQ ID NO: 60). Transporters were then fused to GFP as follows: NCU00801 was inserted between BamHI and EcoRI using the primers, GCATGGATCCATGTCGTCTCACGGCTCC (SEQ ID NO: 61) and TATAATGAATTCAGCAACGATAGCTTCGGAC (SEQ ID NO: 62), and NCU08114 was inserted between SpeI and EcoRI using the primers, TATTAAACTAGTATGGGCATCTTCAACAAGAAGC (SEQ ID NO: 63) and TTATAAGAATTCAGCAACAGACTTGCCCTCATG (SEQ ID NO: 64).
The β-glucosidase, NCU00130, was cloned into the 2μ plasmid, pRS425, modified to include the PGK1 promoter described above. NCU00130 with an optimized Kozak sequence and a C-terminal 6× His tag was inserted between SpeI and PstI using the primers, GCATACTAGTAAAAATGTCTCTTCCTAAGGATTTCCTCT (SEQ ID NO: 65) and ATACTGCAGTTAATGATGATGATGATGATGGTCCTTCTTGATCAAAGAGTCAAAG (SEQ ID NO: 66). All constructs included the Cyc transcriptional terminator between XhoI and KpnI. All N. crassa genes were amplified by PCR from cDNA synthesized from mRNA isolated from N. crassa (FGSC 2489) cultured on minimal media with pure cellulose (Avicel) as the sole carbon source.
Yeast Growth Assays
To monitor growth on cello-oligosaccharides, engineered strains were grown in 5 mL of complete minimal media with appropriate dropouts overnight. These starter cultures were washed three times with 25 mL of ddH2O, and resuspended to an OD (at 600 nm) of 0.1 in Yeast Nitrogen Base (YNB) plus the appropriate Complete Supplemental Media (CSM) and 1% (w/v) of cellobiose, or 0.5% (w/v) of either cellotriose or cellotetraose. Assays were performed in a Bioscreen CTM with constant shaking at maximum amplitude at 30° C. and a final assay volume of 0.4 mL. The change in OD was measured either at 600 nm or using a wideband filter from 450-580 nm. Growth rates were taken from the linear portion of each growth curve, and are reported as the mean of three independent experiments±the standard deviation between these experiments. Cellotriose and cellotetraose were obtained from Seikagaku Biobusiness Corporation (Tokyo, Japan).
Purification of NCU00130 and Assay of its Activity
A 1 L culture of S. cerevisiae expressing cbt1 and NCU00130 was grown to an OD of 2.0 in complete minimal media. Cells were harvested by centrifugation and resuspended in 30 mL of lysis buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 10 mM imidazole, 2 mM β-ME, CompleteTM Mini, EDTA free protease inhibitor cocktail). Cells were lysed by sonication, and the lysate was cleared by centrifugation at 15,000 g for 30 minutes. The lysate was bound to 1 mL of nickel-NTA resin by gravity flow, and washed three times with 25 mL wash buffer (identical to lysis buffer but with 20 mM imidazole). NCU00130 was eluted with 5 mL of elution buffer (identical to lysis buffer but with 250 mM imidazole), and the appropriate fractions were pooled, exchanged into storage buffer (Phosphate Buffered Saline (PBS), 2 mM DTT, 10% glycerol), aliquoted, frozen in liquid nitrogen, and stored at −80° C. Purity was determined by SDS-PAGE (FIG. 22), and protein concentration was determined from the absorbance at 280 nm, using an extinction coefficient of 108,750 M−1cm−1.
Purified NCU00130 was assayed from hydrolysis activity with different cellodextrin substrates. Activity was measured by incubating 5 pmol of enzyme with 500 μM of each sugar in 150 μL PBS plus 3 mM DTT. Reactions proceeded for 40 minutes at 30° C. before 100 μL was removed and quenched in 400 μL of 0.1 M NaOH. The results were analyzed by ion chromatography with a Dionex ICS-3000, with CarboPac PA200 column. Peaks were detected with an electrochemical detector.
Phylogenetic Analysis of Transporter Orthologs
Amino acid sequences of orthologs of CBT1 and CBT2 were obtained from online databases. Multiple sequence alignments were performed using T-Coffee (Notredame et al., 2000). A maximum likelihood phylogeny was determined using PhyML version 3.0 (Guindon and Gascuel 2003) with 100 Bootstraps. Both programs were accessed through Phylogeny.fr (webpage phylogeny.fr/). The resulting tree was visualized with FigTree v.1.2.1 (webpage tree.bio.ed.ac.uk/).
Fermentation and SSF
In fermentation and SSF experiments, comparisons were made between yeast expressing NCU00130 and either Myc-tagged cbt1, or no transporter. These strains were grown aerobically overnight in complete minimal media, washed three times with 25 mL water, and resuspended to a final OD of 2.0 in 50 mL YNB plus the appropriate CSM, and either 2% (w/v) cellobiose or 3% (w/v) pure cellulose (Avicel), in sealed serum flasks. The SSF reactions also included 50 Filter Paper Units/g cellulose of filter-sterilized Celluclast (Sigma C2730), without β-glucosidase supplementation. Reactions were carried out anaerobically at 30° C. with shaking. At indicated time points, 1 mL samples were removed and filtered through a 0.2 μm syringe filter. The ethanol, glucose, and cellobiose concentration in the filtrate was determined by HPLC with an Aminex HPX-87H column and refractive index detection.
N. crassa Growth and Alamar Blue® Assays
WT N. crassa (FGSC 2489), and the homokaryotic NCU08114 (FGSC 17868) (Colot et al., 2006) were acquired from the Fungal Genetics Research Center (McCluskey 2003), and grown at 25° C. in 50 mL of Vogel's salts plus 2% of either sucrose or pure cellulose (Avicel) in a 250 mL unbaffled flask. After 16 or 28 hours, respectively, 100 μL of Alamar Blue® was added, and cultures were incubated at room temperature for 20 minutes. At this time, 1 mL samples were removed, debris pelleted, and the fluorescence of 100 μL of the supernatant determined with excitation/emission wavelengths of 535/595 nm in a Beckman Coulter Paradigm plate reader.
N. crassa Cellobiose Transport Assays
WT N. crassa (FGSC 2489), and homokaryotic deletion lines (Colot et al., 2006) of NCU00801 (FGSC 16575) and NCU08114 (FGSC 17868) were acquired from the Fungal Genetics Stock Center (McCluskey 2003), and grown for 16 hours in 50 mL of Vogel's salts plus 2% (w/v) sucrose at 25° C., starting with an inoculum of 106 conidia/mL. Mycelia were harvested by centrifugation, washed three times with Vogel's salts, and transferred to Vogel's salts plus 0.5% (w/v) pure cellulose (Avicel) for 4 hours to induce the transporter expression. Ten mL of the culture was harvested by centrifugation, washed three times with Vogel's salts, and resuspended in 1 mL ddH2O plus cycloheximide (100 μg/mL) and 90 μM of the respective cellodextrin (cellobiose, cellotriose, or cellotetraose). To measure cellodextrin consumption, 100 μL was removed after 15 minutes, clarified by centrifugation, and transferred into 900 μL of 0.1 M NaOH. The amount of sugar remaining in the supernatant was determined by HPLC with a Dionex ICS-3000, using a CarboPac PA200 column. Peaks were detected with an electrochemical detector.
GFP Fluorescence and Confocal Fluorescence Microscopy
Bulk-cell GFP fluorescence measurements were made in a Beckman Coulter Paradigm plate reader with excitation/emission wavelengths of 485/535 nm. Confocal fluorescence microscopy was performed with cells at an OD (at 600 nm) of 0.8-1.2, using a 100× 1.4 NA oil immersion objective on a Leica SD6000 microscope attached to a Yokogawa CSU-X1 spinning disc head with a 488 nm laser and controlled by Metamorph software. Z series were recorded with a 200 nm step size and analyzed using ImageJ.
[3H] Cellobiose Transport Assays and Kinetic Parameters
Transport assays were performed using a modification of the oil-stop method (Arendt et al., 2007). Yeast strains expressing either cbt1 or cbt2 fused to GFP were grown to an OD (at 600 nm of 1.5-3.0 in selective media, washed three times with ice cold assay buffer (30 mM MES-NaOH [pH 5.6] and 50 mM ethanol), and resuspended to an OD of 20. To start transport reactions, 50 μL of cells were added to 50 μL of [3H] cellobiose layered over 100 μL of silicone oil (Sigma 85419). Reactions were stopped by spinning cells through oil for 1 minute at 17,000 g, tubes were frozen in ethanol/dry ice, and tube-bottoms containing the cell-pellets were clipped off into 1 mL of 0.5 M NaOH. The pellets were solubilized overnight, 5 mL of Ultima Gold scintillation fluid added, and CPM determined in a Tri-Carb 2900TR scintillation counter. [3H] cellobiose was purchased from Moravek Biochemicals, Inc. and had a specific activity of 4 Ci/mmol and a purity of >99%. Kinetic parameters were determined by measuring the linear rate of [3H] cellobiose uptake over 3 minutes for a range of cellobiose concentrations. Vmax and Km values were determined by fitting a single rectangular, 2-parameter hyperbolic function to a plot of rates vs. cellobiose concentration by non-linear regression in SigmaPlot®. Vmax values were normalized for differences in transporter abundance by measuring the GFP fluorescence from 100 μL of cells at OD 20 immediately before beginning transport assays. Kinetic parameters reported in the text are mean±the standard deviation from three separate experiments. Competition assays were performed by measuring transport of 50 μM [3H]-cellobiose over 20 seconds in the percent of 250 μM of the respective competitors.
Large Scale Yeast Growth
To monitor growth on different carbon sources, engineered strains were grown in 5 mL of complete minimal media with appropriate dropouts overnight. These starter cultures were washed three times with 25 mL of ddH2O and resuspended to an OD (at 600 nm) of 0.1 in 50 mL Yeast Nitrogen Base (YNB) plus the appropriate Complete Supplemental Media (CSM) and 2% (w/v) cellobiose. Cultures were grown in 250 mL unbaffled flasks at 30° C., with shaking at 200 rpm. The change in OD (at 600 nm) was monitored by periodically removing samples.
Example 10
Identification of Critical Residues for Cellodextrin Transporter Function
In this example, sequence analysis and mutagenesis studies were used to identify conserved and functionally important residues in the cellodextrin transporters. In addition, additional cellodextrin transporters were identified.
The growth rates of yeast strains expressing various mutants of the cellodextrin transporter NCU00801 (cbt1) or NCU08114 (cbt2) and the wild-type β-glucosidase NCU00130 were grown with cellobiose as the sole carbon source. Amino acid residues at 96 positions of NCU00801 and at 96 positions of NCU08114 were individually mutated to alanine using QuickChange® II Site-directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) as per the manufacturer's instructions. Strains were grown in synthetic defined media-ura-leu 100 mg/L adenine with 2% cellobiose. Cultures were started from two independent colonies.
As the results shown in FIG. 26 (a, b) indicate, mutant strains that expressed NCU00801 with substitutions at W66, L73, Y74, N87, Y89, D90, Q 104, F107, G113, F120, Y123, D139, G142, K144, M147, G150, Q169, F170, G173, R174, G178, G180, P189, Y191, E194, P198, R201, Y208, W235, R236, Q242, 257PESPRF262 (SEQ ID NO: 67), Y279, G283, E296, D307, K308, W310, D312, R325, G336, Y345, N369, D385, F462, P468, E476, T480, or G486 showed at least a 25% growth defect compared to wild-type strain.
The alanine scanning experiment on NCU08114 indicated the following residues as being functionally important: L38, Y39, G54, D56, F73, G91, P100, D104, G107, R108, M118, R139, F144, Q150, P154, E159, P163, H165, R166, Y173, N174, W199, Q214, 222PESP225 (SEQ ID NO: 68), Y244, H245, D249, E258, E268, Q302, W303, S304, N306, Y312, F359, L360, F402, Y403, S404, Y414, E417, P420, Y421, K426, N442, N446, P447, W459, K460, E482, T483, L488, E489, E490, D496, and G497 (FIG. 26b).
In particular, the motifs 73LYF75, 257PESP260 (SEQ ID NO: 69), and 278KYH280 (residue numbering of NCU00801) appeared to be functionally important in both transporters (residues 257PESP26° (SEQ ID NO: 69) of NCU00801 and residues 222PESP225 (SEQ ID NO: 68) of NCU08114), which have an amino acid sequence identity of 29% (FIG. 26b, c). Several residues that are conserved in transporters in general (italicized in FIG. 26b, c), or in β-linked transporters in particular (double-underlined), were experimentally shown to be important for transporter function (underlined), e.g., D90 (NCU00801) and D56 (NCU08114), and L73 (NCU00801) and L38 (NCU08114). Results of the mutagenesis experiment also implicated residues conserved in the NCU00801/NCU08114 clade (capped) as being functionally important, e.g., Q168 (NCU00801) and Q214 (NCU08114). Moreover, multiple residues determined to be functionally important in this experiment were previously shown to be conserved in the S. cerevisiae sugar transporters (Hxt1/Hxt3), e.g., L73 (NCU00801) and L38 (NCU08114).
Orthologs of N. crassa cellodextrin transporters from different organisms were also studied (FIGS. 27A-C). Representative orthologs were synthesized by Genescript and cloned into the expression vector, pRS426 containing the Cup1 promoter using the sites BamHI and HindIII. These constructs were transformed into the yeast strain, YPH499 along with the intracellular β-glucosidase, NCU00130. Transporter activity was determined by measuring the growth rates of these strains when cellobiose was present as the sole carbon source.
Alternatively, different fungal strains containing putative orthologs were cultivated in rich media supplemented with cellobiose. Total RNA was isolated and reverse transcribed into cDNA. Polymerase chain reaction (PCR) was used to amplify the putative transporter genes directly from cDNA. However, because the regulation mechanism and expression pattern were unknown for cellodextrin transporters in fungal species, cDNAs encoding the putative transporters were not always obtainable despite alteration of cultivation condition. In this case, primers were designed according to the corresponding cDNA sequences from GenBank and used to amplify the exons using genomic DNA as a template. Overlap-extension PCR was then used to assemble the exons into the full-length genes. The resulting PCR products were cloned into the pRS424 shuttle vector containing a HXT7 promoter and a HXT7 terminator using the DNA assembler method. Yeast plasmids isolated from transformants were retransformed into E. coli DH5α, and isolated E. coli plasmids were first checked by diagnostic PCR using the primers used to amplify the original transporter genes. The entire open reading frames were submitted for sequencing to confirm the correct construction of the plasmids. In the orthologs LAC2, LAC3, HXT2.1, and HXT2.6 from P. stipitis, one or more alternative codons (CUG) substitute Ser for Leu. Most of the cloning work was carried out using the yeast homologous recombination mediated DNA assembler method. pRS424-HXT7-GFP plasmid was used for cloning of putative cellodextrin transporters. In this plasmid, the HXT7 promoter, the GFP gene flanked with the EcoRI sites at both ends, and the HXT7 terminator were assembled into the pRS424 shuttle vector (New England Biolabs) linearized by ClaI and BamHI. PCR products of the putative transporters flanked with DNA fragments sharing sequence identity to the HXT7 promoter and terminator were co-transferred into CEN.PK2-1C with EcoRI digested pRS424-HXT7-GFP using the standard lithium acetate method. The resulting transformation mixture was plated on SC-Trp plates supplemented with 2% D-glucose to recover transformants. Yeast expressing putative cellodextrin transporter orthologs and NCU00130 were tested for growth on cellobiose as the sole carbon source.
A listing of the putative cellodextrin transporter orthologs and results obtained from the study are shown in Table 14.







TABLE 14







Listing of putative cellodextrin transporter orthologs and summary of results.














NCBI







Reference







Sequence/







NCBI GI
Aver.




N. crassa

Number/JGI
Growth
Growth



ortholog
Species
number ¥
Rate
Rate error
Seq results*





NCU00809
Chaetomium globusom
XP_001220480
—
—
OK



CBS148.51






NCU00809
Podospora anserina
XP_001912722
—
—
—


NCU00809
Nectria haematococca
EEU41662
—
—
—



mpVI77-13-4






NCU00809
Aspergillus nidulans
XP_660803
—
—
1 intron and



FGSC A4



50 bp







insertion


NCU00809
Aspergillus terreus
XP_001218592
—
—
—



NIH2624






NCU00809
Talaromyces stipitatus
XP_002341594
—
—
—



ATCC 10500






NCU00809
Aspergillus niger
XP_001395979
—
—
Ala > Val


NCU00809
Aspergillus fumigatus
XP_747891
—
—
—



Af293






NCU00809
Aspergillus terreus
XP_00120996
—
—
—



NIH2624






NCU00809
Aspergillus oryzae RIB40
XP_001817400
—
—
OK


NCU08114
Podospora anserina
XP_001908539
—
—
N/A


NCU08114
Penicillium chrysogenum
XP_002568019
—
—
N/A



Wisconsin 54-1255






NCU08114
Aspergillus terreus
XP_001209810
—
—
Wrong



NIH2624






NCU08114
Aspergillus oryzae RIB40
XP_001820343
—
—
OK


NCU08114
Aspergillus terreus
XP_001210859
—
—
N/A



NIH2624






NCU08114
Neurospora crassa
XP_001728155
—
—
N/A



OR74A






NCU08114
Aspergillus oryzae RIB40
XP_001826848
—
—
N/A


NCU08114
Aspergillus nidulans
XP_657617
—
—
OK



FGSC A4






NCU08114
Talaromyces stipitatus
XP_002487579
—
—
N/A



ATCC 10500






NCU08114
Chaetomium globosum
XP_001227497
—
—
Wrong



CBS 148.51






NCU08114
Trichoderma atroviridae
215408
0.000836364
0.00064871
I, D


NCU08114
Chaetomium globosum
XP_001220290.1
0.004036364
0.00047168
OK


NCU08114
Aspergillus nidulans
ANID_08347
0.011109091
0.000072727
Other


NCU08114
Pleurotus ostreatus
51322
0.00390303
0.00018212
—


NCU08114
Sporotrichum
114107
0.009569697
0.00216366
—



thermophile






NCU00801
Aspergillus nidulans
XP_660418.1
0.000860606
0.000438
P


NCU00801
Magnaporthe grisea
XP_364883.1
005090909
0.00138313
OK


NCU00801
Aspergillus fumigatus
XP_753099.1
0.003975758
0.00211951
OK


NCU00801
Trichoderma atroviridae
211304
0.002678788
0.00031193
D


NCU00801
Chaetomium globosum
XP_001220469.1
0.005890909
0.00010285
OK


NCU00801
Tremella mesenterica
63529
0.004381818
0.00115751
D


NCU00801
Heterobasidion. annosum
105952
0.002751515
0.00068763
D


NCU00801
Cryphonectria parasitica
252427
0.02250303
0.00021692
D


NCU00801
Trichoderma ressei
67752
0.003672727
0.00066233
D


NCU00801
Aspergillus clavatus
XP_001268541.1
0.014381818
0.00059613
OK


NCU00801
Neurospora discreta
77429
0.007060606
0.00110566
D


NCU00801
Trichoderma reesei
3405
0.003264646
0.001033998
D


NCU00801
Sporotrichum
43941
0.013654545
0.00431534
—



thermophile






NCU00801
Neurospora crassa
XP_963801.1
0.048754872
0.00354017
—


NCU05853
Chaetomium globosum
XP_001226269.1
0.003593939
0.00062306
OK


NCU05853
Trichoderma reesei
46819
0.002042424
0.000085924
D


NCU05853
Mycosphaerella
68287
0.00290101
0.00060123
D



graminicola






NCU05853
Aspergillus flavus
AFLA_000820A
0.003078788
0.00209132
—


—
None
—
0.0026
0.0001
—


NCU00809
Pichia stipitis CBS6054
XP_001383110.1/
FIGS.
—
—



(LAC1)
GI: 126133170
27A-C




NCU00809
Pichia stipitis CBS6054
XP_001387231.1/
FIGS.
—
—



(LAC2)
GI: 126276337
27A-C




NCU00809
Pichia stipitis CBS6054
XP_001383677.2/
FIGS.
—
—



(LAC3)
GI: 150864727
27A-C




NCU08114
Pichia stipitis CBS6054
XP_001386873.1/
FIGS.
—
—



(HXT2.1)
GI: 126275571
27A-C




NCU05853
Pichia stipitis CBS6054
XP_001382754.1/
FIGS.
—
—



(HXT2.3)
GI: 126132458
27A-C




NCU08114
Pichia stipitis CBS6054
XP_001387757.1/
FIGS.
—
—



(HXT2.4)
GI: 126273939
27A-C




NCU08114
Pichia stipitis CBS6054
XP_001385684.1/
FIGS.
—
—



(HXT2.5)
GI: 126138322
27A-C




NCU08114
Pichia stipitis CBS6054
XP_001384653.2/
FIGS.
—
—



(HXT2.6)
GI: 15086543
27A-C





*Wrong = difference between tested sequence and sequence in NCBI or JGI databases;


I = insertion in tested sequence;


D = deletion in tested sequence;


P = point mutation in tested sequence;


OK = no difference between tested sequence and sequence deposited in NCBI or JGI databases;


Other = other problems in sequencing, excluding insertion, deletion, and point mutations in tested sequence; “-” = results not yet available (study in progress).


¥ When accession numbers were not available, the JGI number was used. The JGI number allows access to the gene sequence via the JGI genome portal for this organism (accessible from the following page: genome.jgi-psf.org/programs/fungi/index.jsf). The A. flavus and A. nidulans identifiers allow access to the genes through their genome portals at webpage cadre-genomes.org.uk/and webpage broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html, respectively.






In certain cases, the sequences of the cloned orthologs were determined to be correct, and the yeast expressing those clones were able to utilize cellobiose. Thus, these clones, LAC2 from Pichia stipitis and XP—001268541.1 from Aspergillus clavatus were confirmed to be functional cellobiose transporters. Testing of the cellobiose transporting function of other clones is still in progress. Cloned orthologs with sequences different from the published sequences in databases (e.g., ones with insertions, deletions, etc.) (Table 14) will be re-cloned, re-sequences, and similarly tested for cellobiose transport activity by expressing them in S. cerevisiae and monitoring growth rates.
An alignment of NCU00801, NCU08114, and functional orthologs of these transporters is shown in FIGS. 28A-C. The alignment in FIG. 28a includes both putative and confirmed cellodextrin transporters, whereas the alignment in FIG. 28b includes only confirmed cellodextrin transporters. In addition, FIG. 28c shows an alignment of NCU00801 and NCU08114. The two transporters share 29% amino acid sequence identity.
Motifs critical for cellodextrin transporter function were identified by visual inspection of multiple sequence alignments between sugar transporters. Specifically, motifs common to cellodextrin transporters were identified from multiple sequence alignments produced in T-COFFEE between putative cellodextrin transporter orthologs and confirmed cellodextrin transporters. To ensure that these motifs were largely unique to cellodextrin transporters, their absence was confirmed from a multiple sequence alignment between the hexose transporters of S. cerevisiae, the human glucose transporter, Glut1, and two N. crassa monosaccharide transporters produced in T-COFFEE.
The identified motifs are described below. In the motifs, residues that were found to be critical to the function of NCU00801 are underlined. The residues that were critical for the function of NCU08114 are marked with the superscript “†”. The residues that were critical to the function of both transporters are marked with the superscript “*”. All motifs were defined using PROSITE notation. As an example of how to read a PROSITE motif, the following motif, [AC]-x-V-x(4)-{ED}, is translated as: [Ala or Cys]-any-Val-any-any-any-any-{any but Glu or Asp} (SEQ ID NO: 13)
Cellodextrin transporters, like all sugar transporters, have 12 transmembrane α-helices. The N- and C-terminus of cellodextrin transporters are both intracellular.
The sequence before transmembrane helix 1 had no distinguishing features.
Transmembrane helix 1 contained the motif, [L*IVM]-Y*-[FL]-x(13)-[YF]-D* (SEQ ID NO: 1).
Transmembrane helix 2 contained the motif, [YF]-x(2)-G†-x(5)-[PVF]-x(6)-[DQ]* (SEQ ID NO: 2).
The loop connecting transmembrane helix 2 and transmembrane helix 3 contained the motif, G*-R†-[RK]* (SEQ ID NO: 3).
Transmembrane helix 3 had no distinguishing features.
Transmembrane helix 4 had no distinguishing features.
Transmembrane helix 5 contained the motif, R*-x(6)-[YF]*-N† (SEQ ID NO: 4).
Transmembrane helix 6 contained the motif, W*R-[IVLA]-P-x(3)-Q(SEQ ID NO: 5).
The sequence between transmembrane helix 6 and transmembrane helix 7 contained the motif, P*-E*-S*-P*-R-x-L-x(8)-A-x(3)-L-x(2)-Y*-H† (SEQ ID NO: 6).
Transmembrane helix 7 contained the motif, F†-[GST]-Q*-x-S†-G-N†-x-[LIV] (SEQ ID NO: 7).
Transmembrane helix 8 had no distinguishing features.
Transmembrane helix 9 had no distinguishing features.
Transmembrane helix 10 and transmembrane helix 11 and the sequence between them contained the motif, L-x(3)-[YIV]†-x(2)-E*-x-L-x(4)-R-[GA]-K†-G (SEQ ID NO: 8).
Transmembrane helix 12 had no distinguishing features.
The sequence after transmembrane helix 12 had no distinguishing features.
Homology models of NCU00801 and NCU08114 were produced from the primary amino acid sequences of NCU00801 and NCU08114 using the I-TASSER server at: zhanglab.ccmb.med.umich.edu/I-TASSER/ (Roy et al., 2010). The top structural models produced by I-TASSER were visualized in PYMOL (webpage pymol.org/). Mapping of the motifs was also performed in PYMOL. The homology models of NCU00801 and NCU08114 with the cellodextrin transporter motifs marked are shown in FIG. 29 (a, b).
Example 11
Characterization of Novel Pentose-Specific Transporters from Neurospora crassa and Pichia stipitis in Saccharomyces cerevisiae 
In this example, a bioinformatics approach was taken to identify novel pentose-specific transporters in N. crassa and P. stipitis. 
Genome Mining of Pentose-Specific Transporters
Bioinformatics Study
To discover novel D-xylose-specific transporters, the genes encoding the D-glucose/D-xylose symporter Gxsl from C. intermedia (Leandro et al., 2006) and the uncharacterized putative L-arabinose-proton symporter Aut1 from P. stipitis (locus tag PICST—87108) were used as probes in BLAST searches (webpage ncbi.nlm.nih.gov/) against the sequenced genomes of two efficient xylose-utilizing species, N. crassa and P. stipitis (Galagan et al., 2003; Jeffries et al., 2007). Any proteins with known D-glucose transport activity or activity other than sugar transport were eliminated from the analyses. Using a cut-off of 25% minimal sequence identity, 17 putative pentose transporter genes were identified (Table 15), in addition to AUT1 from P. stipitis. These putative pentose transporter genes shared 25-50% identity with either GXS1 from C. intermedia or AUT1 from P. stipitis. All 17 putative pentose transporters were annotated as either sugar-transport proteins or hypothetical proteins with unknown activity. The D-glucose transporter genes SUT1 and SUT2 from P. stipitis were also cloned for comparison.
Table 15 shows the putative pentose transporters obtained from BLAST using (a) AUT1 from P. stipitis as a probe and (b) GSX1 from C. intermedia as a probe.







TABLE 15





shows the putative pentose transporters obtained 


from BLAST using (a) AUT1 and P. Stipitis as a probe


(b) GSX1 from C. intermedia as a probe.







a. BLAST search results using AUT1 as a probe.














% identity

Length



Name
Origin
with AUTI
Annotation from NCBT
(cDNA)
Locus Tag





Ap31/SUT2
P. stipitis
31
sugar uptake (tentative)
1653
ABN66266


Ap26/XP_001387242
P. stipitis
26
sugar transporter
1404
XP001387242


AN49/NCU01494
N. crassa
49
hypothetical protein
2025
EAA2669I





NCU01494, similar to MFS







sugar transporter




AN41/NCU09287
N. crassa
41
hypothetical protein
1968
EAA28903





NCU09287, similar to







galactose-proton symporter




AN29-2/NCU04963
N. crassa
29
hypothetical protein
1584
EAA30175





NCU04963, similar to MFS 







monosaccharide transporter




AN28-3/NCU02188
N. crassa
28
hypothetical protein
1458
EAA30346





NCU02188, conserved







hypothetical protein




AN25/NCU00821
N. crassa
25
sugar transporter
1689
EAA35128










b. BLAST search results using GSX1 as a probe.














% identity 

Length



Name
Origin
with GSX1
Annotation from NCBI
(cDNA)
Locus Tag





Xy50/NCU04537
N. crassa
50
hypothetical protein NCU04537 
1626
EAA26741





similar to monosaccharide







transporter




Xy31/NCU06138
N. crassa
31
hypothetical protein NCU06138, 
1757
EAA30764





similar to MFS monosaccharide 







transporter




Xy33/NCU00988
N. crassa
33
hypothetical protein NCU00988, 
1614
EAA34662





similar to MFS quinate transporter




Xyp37/SUT3
P. stipilis
37
sugar uptake (tentative)
1653
ABN67990


Xyp33/XUT3
P. stipilis
33
sugar transporter, putative xylose 
1656
EAZ63115





transporter uptake (tentative); 







predicted (major facilitator 







superfamily)




Xyp32/XUT1
P. stipilis
32
sugar transporter, high affinity, 
1701
ABN67554





putative; xylose uptake (tentative)




Xyp30/STL1
P. stipilis
30
sugar transporter, strongly conserved
1590
ABN65745


Xyp31/XUT
P. stipilis
31
sugar transporter, xylose transporter 
1407
AAVQOIOOOO02





(tentative) similarly to GXSI(STL1)




Xyp29/STL12/XUT6
P. stipilis
29
sugar transporter, putative (STL12); 
1641
ABN68560





.xylose uptake (tentative)




Xyp30-I/HGT3
P. stipilis
30
high affinity xylose transporter (putative),
1587
ABN68686





xylose uptake (tentative)




Xyp28/XUT7
P. stipilis
28
xylose transporter, high affinity, putative 
1257
EAZ63044





similarity to STL13, high affinity sugar 







transporters











Cloning of Putative Pentose Transporters
N. crassa and P. stipitis were cultivated in rich media supplemented with either D-xylose or L-arabinose as carbon sources. Total RNA was isolated and reverse transcribed into cDNA. Polymerase chain reaction (PCR) was used to amplify the putative transporter genes directly from cDNA. However, because the regulatory mechanism and expression patterns of pentose transporters in fungal species were unknown, cDNAs encoding the putative pentose transporters were not always obtainable despite alteration of cultivation conditions. In those cases, primers were designed according to the corresponding cDNA sequences from GenBank and used to amplify the exons with genomic DNA as templates. Overlap-extension PCR was then used to assemble the exons into full length genes. The resulting PCR products were cloned into the pRS424-HXT7-GFP shuttle vector using the yeast homologous recombination-mediated DNA assembler method (Shao et al., 2009). In this plasmid, an HXT7 promoter, a GFP gene flanked with the EcoRI sites at both ends, and an HXT7 terminator were assembled into the pRS424 shuttle vector (New England Biolabs) linearized by ClaI and BamHI. PCR products of the putative pentose transporters flanked with DNA fragments, sharing sequence identity with the HXT7 promoter and terminator (FIG. 30a) were co-transferred into S. cerevisiae CEN.PK2-1C strain (MATα leu2-3,112 ura3-52, trp1-289, his3-Δ1 MAL2-8c) purchased from Euroscarf (Frankfurt, Germany) with EcoRI digested pRS424-HXT7-GFP using the standard lithium acetate method. The resulting transformation mixture was plated on SC-Trp plates supplemented with 2% D-glucose.
Yeast plasmids isolated from transformants using Zymoprep Yeast Plasmid Miniprep II (Zymo Research, Orange, Calif.) were re-transferred into Escherichia coli DH5α cells (Cell Media Facility, University of Illinois at Urbana-Champaign, Urbana, Ill.). The plasmids were isolated using the QIAprep Spin Miniprep Kit (QIAGEN, Valencia, Calif.) and then checked by diagnostic PCR with the primers used to amplify the original transporter genes. The entire open reading frames were also submitted for DNA sequencing to confirm correct construction (Core Sequencing Facility, University of Illinois at Urbana-Champaign, Urbana, Ill.). The DNA sequencing results were compared to gene sequences in databases using Sequencher 4.7 (Gene Codes Corporation, Ann Arbor, Mich.). All sequences of cloned putative transporters are listed in SEQ ID NOs: 33-52.
Yeast strains were cultivated in synthetic dropout media to maintain plasmids (0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, 0.05% amino acid drop out mix). YPA media supplemented with 2% of sugar was used to grow yeast strains harboring no plasmids (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate). S. cerevisiae strains were cultured at 30° C. and 250 rpm for aerobic growth and at 30° C. and 100 rpm for oxygen-limited conditions. Yeast strains were grown under aerobic conditions for cell manipulation unless specified otherwise. E. coli strains were cultured at 37° C. and 250 rpm in Luria broth (LB) (Fisher Scientific, Pittsburgh, Pa.). All restriction enzymes were purchased from New England Biolabs (Ipswich, Mass.). All chemicals were purchased from Sigma Aldrich (St. Louis, Mo.) or Fisher Scientific.
Transporter Activity Assay for Cloned Putative Transporters
Intracellular Accumulation of Pentose Sugars
The cloned putative pentose transporters were over-expressed in an S. cerevisiae sugar transporter deletion strain, and uptake of pentose sugars was measured. The D-xylose-uptake ability of putative pentose transporters was determined by summation of intracellular D-xylose and xylitol concentrations. D-xylose accumulated within S. cerevisiae cells can be partially converted to xylitol due to the presence of endogenous aldose reductase. Both D-xylose and xylitol were extracted using osmosis and analyzed using high performance liquid chromatography (HPLC).
The sugar transporter knock-out S. cerevisiae strain EBY.VW4000 (CEN.PK2-1c Δhxt1-17, Δstl1, Δagt1, Δydl247w, Δyjr160c, Δgal2), which was a gift from Professor E. Boles' laboratory (Institut für Mikrobiologie, Heinrich-Heine-Universität, Universitätsstr. 1, Geb. 26.12.01, D-40225 Düsseldorf, Germany), had concurrent knock-outs of more than 20 sugar transporters and sensors including HXT1-17 and GAL2. Growth on D-glucose as the sole carbon source was completely abolished in this strain, whereas uptake of maltose through a different sugar transport system was retained. The EBY.VW4000 strain also exhibited minimal pentose-uptake under HPLC assay conditions, which made it a suitable host for testing recombinant D-xylose uptake. Plasmids over-expressing the cloned putative pentose transporter genes were transferred into the EBY.VW4000 strain using the standard lithium acetate method, and single colonies were used for measuring sugar uptake activity.
Cells were first cultured in 2 mL SC-Trp medium supplemented with 2% maltose. Seed culture was then used to inoculate a 50 mL culture in a 250 mL flask. The cells were harvested by centrifugation after 24 hours of growth and re-suspended in YPA medium supplemented with 2% D-xylose or L-arabinose to a final OD600 of 10. At 30 min, 60 min, 120 min, and 24 hours, 5 mL cultures were taken for measuring intracellular sugar concentrations. Culture samples were washed twice with ice-cold water and re-suspended in 3 mL of deionized water. Cell suspensions were incubated at 37° C. with 250 rpm agitation for 2 days to extract intracellular sugars. The resulting cell suspension was filtered through a 0.22 μm PES filter (Corning, Lowell, Mass.) before HPLC analysis. The concentrations of sugar and corresponding sugar alcohol (discussed below) were determined using Shimadzu HPLC equipped with a BioRad HPX-87C column (BioRad Laboratories, Hercules, Calif.) and Shimadzu ELSD-LTII low temperature-evaporative light scattering detector (Shimadzu) following the manufacturer's protocol. The sugar-uptake activity was calculated as mg of sugar extracted through osmosis per mL of cell culture at OD ̃10.
Several putative pentose transporters were identified to be active in uptake of D-glucose or D-xylose or both. Since D-glucose can be metabolized once inside yeast, the D-glucose transport activity could not be determined by measuring intracellular D-glucose concentration. However, because the EBY.VW4000 strain normally cannot grow on media containing D-glucose as the sole carbon source, growth of the strain transformed with a putative pentose transporter on D-glucose indicated that the putative transporter has D-glucose transport activity.
Introduction of SUT3 (Xyp37), XUT3 (Xyp33), SUT2 (Ap31), NCU04963 (An29-2), and NCU06138 (Xy31) restored growth of the EBY.VW4000 strain on D-glucose and, thus, enabled glucose transport activity. SUT3, XUT3, SUT2, and NCU04963 also had xylose transport activity, whereas NCU04963 and NCU06138 showed arabinose transport activity (FIG. 31). The rest of the putative transporters failed to enable growth on D-glucose, and most of them also did not show any pentose transport activity. However, NCU00821 and STL12/XUT6 showed xylose transport activity, and XUT1 exhibited arabinose transport activity, indicating they may be sugar transporters specific for pentoses (FIG. 32).
To further confirm that STL12/XUT6 and XUT1 from P. stipitis and NCU00821 from N. crassa were actually pentose-specific transporters with no D-glucose-uptake activity, the sugar-uptake assay was performed using 14C-labeled D-glucose, D-xylose, and L-arabinose as substrates. It was found that D-glucose- and L-arabinose-uptake activities of the EBY.VW4000 strain over-expressing only STL12/XUT6 and NCU00821 were too low to be measured under assay conditions used to determine D-xylose-uptake kinetics of both transporters.
14C-labeled D-glucose, L-arabinose, and D-xylose were purchased from American Radiolabeled Chemicals (St. Louis, Mo.) as solutions in 90% ethanol. Radiolabeled sugars were first dried in a chemical hood and then re-suspended in water. Sugar solutions at concentrations of 1.33 M and 1 M with specific radioactivity of approximately 40,000 dpm/μL, and at concentrations of 500 mM, 350 mM, 250 mM, 100 mM, and 50 mM with specific radioactivity of about 20,000 dpm/μL were used for the sugar-uptake assay. Cell culture at the exponential phase was harvested and washed twice with ice-cold water and re-suspended to about 60 mg dry cell weight (DCW) per mL in 100 mM Tris-citrate buffer at pH 5. Three aliquots of 160 μL cell suspension were dried at 65° C. for 24 hours to determine the DCW. The rest of the cell suspension was kept on ice before use. For the sugar-uptake assay, cell suspension was equilibrated at 30° C. for 5 min before the assay. In a 50 mL conical tube, 160 μL of cell suspension was mixed with 40 μL of radio-labeled sugar solution for 40 or 60 seconds (accurately timed). The reaction was stopped by adding 10 mL of ice-cold water delivered by a syringe. The zero-time-point sample was obtained by adding ice-cold water and cell suspension simultaneously in a culture tube containing the radio-labeled solution. The mixture was then filtered immediately through a Whatman GF/C filter (Whatman, Florham Park, N.J.) pre-soaked in 40% sugar solution and washed with 15 mL of ice-cold water. The filter was placed in 3 mL of Econo I scintillation cocktail (Fisher Scientific) and counted using a Beckman LS6500 scintillation counter (Beckman Coulter, Brea, Calif.) for 1 min. All data points were measured in three independent experiments. The sugar-uptake rate was calculated as mmol sugar transported per hour per gram of dry cell weight.
Intracellular accumulation of both D-xylose and L-arabinose in EBY.VW4000 strains over-expressing STL12/XUT6, NCU00821, or XUT1 was also measured using HPLC. Cell cultures incubated with pentose sugars for 30 min, 60 min, 120 min, and 24 hours were analyzed by HPLC. The EBY.VW4000 strains over-expressing STL12/XUT6 or NCU00821 exhibited D-xylose uptake activity, whereas the strain over-expressing XUT1 exhibited L-arabinose-uptake activity after a 24-hour incubation (FIGS. 33A-B).
The 14C-labeled sugar uptake assay together with HPLC analysis of intracellular sugar accumulations confirmed that among the three most abundant monosaccharides in lignocellulosic hydrolysates, D-glucose, D-xylose, and L-arabinose, STL12/XUT6 and NCU00821 were responsible for D-xylose uptake and XUT1 was responsible for L-arabinose uptake. Of note, most sugar transporters studied in yeast for D-xylose uptake have higher uptake activity towards D-glucose than towards D-xylose. Only Trxlt1 from Trichoderma reesei after adaptive evolution exhibited D-xylose-specific uptake activity (Saloheimo et al., 2007). This data indicated that STL12/XUT6 from P. stipitis, NCU00821 from N. crassa are the first two experimentally confirmed naturally-occurring D-xylose-specific transporters introduced into S. cerevisiae. Similarly, XUT1 from P. stipitis is the first experimentally confirmed naturally-occurring L-arabinose-specific transporter introduced into S. cerevisiae. 
Kinetic Parameters
Using the 14C-labeled sugar-uptake assay, kinetic parameters of D-xylose transport through NCU00921, STL12/XUT6, and XUT1 were determined. It was observed that under the assay conditions, sugar uptake was within a linear range for the first 60 seconds (FIG. 34). The EBY.VW4000 strains over-expressing NCU00821, STL12/XUT6, or XUT1 were incubated with labeled D-xylose or L-arabinose for 40 or 60 seconds followed by addition of ice-cold water to stop further sugar uptake. The reaction mixture was then filtered and washed before measurement using a liquid scintillation counter. The sugar-uptake rates and substrate concentrations were fitted into a Michaelis-Menten equation by non-linear regression using the Origin software (OriginLab Corporation, Northampton, Mass.). The Km values for D-xylose uptake by the EBY.VW4000 strain harboring only NCU00821 or STL12/XUT6 were 175.7±21.4 mM and 56.0±9.4 mM, respectively. The corresponding Vmax values were 36.7±2.9 and 41.5±2.3 μmol/h/gram DCW, respectively. Similarly, the Km and Vmax values for L-arabinose uptake by the EBY.VW4000 strain harboring XUT1 were 48.0±13.2 mM and 5.6±1.6 μmol/h/gram DCW respectively.
In naturally-occurring D-xylose-assimilating fungal species, both the high affinity D-xylose-proton symport system and the low affinity D-xylose facilitated diffusion system are present. The Km values of these two systems were determined to be 0.4-4 mM for the symport system and around 140 mM for the facilitated diffusion system (Leandro et al., 2006; Stambuk et al., 2003). These values are close to the affinity of the D-glucose-uptake system in S. cerevisiae, which has a Km of 1.5 mM for the high affinity system and 20 mM for the low affinity system (Lang and Cirillo 1987; Ramos et al., 1988). Unfortunately, the D-xylose uptake affinity of wild-type S. cerevisiae is two orders of magnitude lower than its affinity for D-glucose. The Km values for D-xylose uptake in S. cerevisiae are only 190 mM for the high affinity system and 1.5 M for the low affinity system (Kötter and Ciriacy, 1993). The affinities of the newly discovered D-xylose-specific transporters were lower when compared to the high affinity D-xylose-uptake system in naturally occurring D-xylose-assimilating yeasts. However, compared to the D-xylose-uptake system in wild-type S. cerevisiae, NCU00821 and STL12/XUT6 showed higher affinity towards D-xylose. In particular, the Km of D-xylose uptake by STL12/XUT6 and XUT1 were only one-fourth of the Km of xylose uptake by the transporter in wild-type S. cerevisiae. The Km values of the D-xylose-specific transporters were also close to those of Gxfl (Km 88 mM) and Sut1 (Km 145 mM), which have been shown to improve D-xylose fermentation in recombinant S. cerevisiae (Runquist et al., 2009; Katahira et al., 2008). Thus, D-xylose fermentation may be improved by introducing these newly discovered D-xylose-specific transporters into S. cerevisiae. 
Cellular Localization of Sugar Transporters
Sugar transporters are transmembrane proteins, and correct folding and localization in the cell membrane is required for them to be functional. Since no signal peptide was specifically added when the putative pentose transporters were cloned, it was important to ensure that the D-xylose-specific transporters were correctly localized to the cell membrane. This was particularly true for putative pentose transporters like NCU00821 cloned from the filamentous fungi N. crassa, which exhibits a very different physiology compared to S. cerevisiae. To study the cellular localization of D-xylose-specific transporters in S. cerevisiae, NCU00821, STL12/XUT6, and XUT1 were fused with Green Fluorescent Protein (GFP) at the C-termini via linkers, and their localization was monitored by fluorescent imaging.
The fusion proteins of the pentose-specific transporters with the GFP at the C-terminus were constructed for the transporter localization study. A GS-linker (Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 70)) was introduced between the transporter and the GFP. The GS-linker was added to the N-terminus of the GFP open reading frame by a PCR primer, resulting in a PCR product of GS-linker-GFP flanked with nucleotide sequence homologous to the transporters at the 5′-end and the HXT7 terminator at the 3′-end. Transporter genes were amplified from the original pRS424-HXT7-transporter constructs to generate DNA fragments of the transporters flanked with nucleotide sequence identical to the HXT7 promoter at the 5′-end and GS-linker-GFP at the 3′-end. These two fragments were then co-transferred into the S. cerevisiae strain CEN.PK2-1C with pRS424-HXT7-GFP digested with EcoRI (FIG. 30b). The resulting transformation mixture was plated on SC-Trp plates supplemented with 2% D-glucose.
Single colonies were inoculated into 2 mL of SC-Trp liquid medium supplemented with 2% maltose. Cell culture was harvested at the exponential phase. In a centrifuge tube, 250 μL of cell culture was stained with 10 μL Hoechst 33342 nuclei dye (Invitrogen, Carlsbad, Calif.) for 10 minutes at room temperature. A small droplet of cell culture was then transferred onto a piece of cover glass and fluorescent images were taken using an Andor Technology Revolution System Spinning Disk Confocal Microscope (Core facilities, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Ill.). Images were processed using Imaris image analysis and visualization software (Bitplane, Saint Paul, Minn.).
Yeast strains over-expressing pentose-specific transporters showed a distinctive fluorescent halo at the cell periphery (FIG. 35). For NCU00821 and XUT1, almost all the GFP fluorescence appeared in the cell membrane, while a large portion of fluorescence in STL12/XUT6-over-expressing cells remained in the cytoplasm. This could indicate inefficient export of the STL12/XUT6 transporter due to elevated expression of the membrane protein. It was also noticed that not all the cells showed fluorescence, indicating that expression of the transporter was not optimal. Further improvements of transporter expression can be achieved through altering the expression level and/or integrating the transporter genes into the genome of recombinant S. cerevisiae. 
Determination of the Type of Pentose Transporters
There are two types of sugar transporters in S. cerevisiae, symporters and facilitators. For symporters, sugar uptake is coupled to proton uptake. Sugar symporters usually exhibit high affinity towards sugar. Meanwhile, sugar uptake through facilitators is not coupled to proton transport, and facilitators usually exhibit low sugar-uptake affinities (Leandro et al., 2006). Symporter assays were performed for NCU00821, STL12/XUT6, and XUT1 expressed in the EBY.VW4000 strain.
To determine the type of transporters, pH change of the EBY.VW4000 over-expressing pentose-specific transporters was measured in un-buffered cell suspension containing D-xylose, L-arabinose, or maltose using a Seven Multi pH meter equipped with an USB communication module and Direct pH software (Mettler Toledo, Columbus, Ohio). Plasmids encoding pentose-specific transporters were transferred into EBY.VW4000 strain followed by plating on the SC-Trp plates supplemented with 2% maltose. Single colonies were inoculated in 2 mL SC-Trp medium supplemented with 2% maltose. Seed culture was then used to inoculate a 400 mL culture in 2 L flasks. The culture was harvested at OD ̃1 and washed twice with ice-cold water. Cell pellets were re-suspended in 4 mL of water and kept on ice before use. For the symporter assay, the pH electrode was immersed in a water-jacketed beaker of 50 mL capacity kept at 25° C. and provided with magnetic stirring. To the beaker, 23 mL of deionized water and 1 mL of cell suspension equilibrated at 25° C. was added. The pH was adjusted to 5, and a base line was obtained. The pH change was recorded with addition of 1 mL of 50% sugar solution at pH 5.
FIGS. 36A-C show pH changes in un-buffered cell suspension after the addition of maltose. As was reported, pH in un-buffered S. cerevisiae cell suspension went up with the addition of maltose. One mL of 50% maltose solution was added to the un-buffered cell suspension to ensure that the pH recording system was functional. The pH elevations observed in all samples indicated that the pH recording system could monitor transient pH changes in the experimental setting.
No elevation of pH in un-buffered cell suspensions was observed for any of the pentose-specific transporters, indicating that pentose uptake through these transporters is not coupled with proton transport (FIGS. 37A-F). Thus, NCU00821, STL12/XUT6, and XUT1 were determined to be pentose facilitators.
This result was consistent with the fact that the kinetic parameters of NCU00821 and STL12/XUT6 were similar to those of the low affinity D-xylose facilitated diffusion system in naturally-occurring D-xylose-assimilating yeasts. Despite the fact that symporters have higher affinities towards D-xylose, over-expression of symporters may not always facilitate sugar utilization by D-xylose-assimilating strains due to the ATP requirement to create the proton gradient. In fact, most of the transporters shown to be beneficial for D-xylose fermentation are facilitators (Runquist et al., 2009; Katahira et al., 2008).
Heterologous Over-Expression of D-Xylose-Specific Transporters
The over-expression of active heterologous D-xylose-specific transporters in S. cerevisiae strains containing the D-xylose utilization pathway was also investigated to determine whether their over-expression could improve xylose utilization. Xylose utilization was studied using a shake-flask under aerobic conditions. Plasmids expressing the xylose transporters NCU00821, NCU04963, XUT1, STL12/XUT6, and Hxt7 were introduced into strain HZE63 (CEN.PK2 ura3::xylose utilization pathway). This strain had a xylose utilization pathway integrated into the URA3 site onto the chromosome. It was constructed using a plasmid from previous work that contained xylulose reductase (XR) and xylitol dehydrogenase (XDH) from N. crassa and xylulokinase (XKS) from P. stipitis. This plasmid was digested with ApaI and transformed into yeast strain CEN.PK2 to yield the strain HZE63.
The HZE63 strain transformed with the xylose transporter-encoding plasmids was selected by plating on SC-Ura plates supplemented with 2% glucose. The transformed strain was pre-cultured in SC-Trp-Ura with 2% glucose and then inoculated into SC-Trp-Ura supplemented with 0.5% or 5% of xylose to an initial OD600=1.0. Cell cultures were grown in a 125 mL shake-flask containing 50 mL of culture at 30° C. and 250 rpm (FIGS. 38A-G).
Yeast plasmids of transformants were transformed into E. coli DH5α cells. The plasmids were then isolated and checked by diagnostic PCR and submitted for sequencing to confirm correct construction. Plasmid maps can be found in FIG. 39.
Unfortunately, the advantage of pentose-specific transporter over-expression could not be observed despite alteration of expression strategies, cultivation conditions, and choice of the D-xylose utilization pathway. There are several possible reasons. Firstly, the over-expression of membrane proteins, such as sugar transporters, could affect the integrity of the cell membrane and consequently hamper cell growth (Wagner et al., 2006). It was observed that transporter over-expression strains displayed a slower growth rate even when D-glucose was used as a carbon source. The final OD of 2-day cultures of strains carrying transporters grown in glucose-containing SC-ura media was only 4, whereas the OD of the negative control was around 6. Secondly, the D-xylose-uptake activity of the wild-type S. cerevisiae through hexose transporters is much higher than the D-xylose-uptake activity of a certain D-xylose transporter over-expressed in a hexose transporter knockout strain. The low sugar transport activity of newly discovered D-xylose-specific transporters may make it hard to observe the improvement of sugar uptake ability. Thirdly, even if the introduction of new D-xylose-specific transporters could improve the uptake of D-xylose into S. cerevisiae cells, the benefit of D-xylose utilization can only be observed when the D-xylose utilization pathway is efficient enough to make sugar-uptake the limiting step. It was shown that the effect of over-expression of sugar transporters depends on the strain background and cultivation conditions (Runquist et al., 2010). Examples 12-15 below describe the optimization of the xylose utilization pathway in yeast.
Cloning of Additional Pentose-Specific Transporters
Orthologs of NCU00821, STL12/XUT6, and XUT1 were cloned and tested for pentose uptake. Different fungal strains were cultivated in rich media supplemented with glucose or pentoses. Total RNA was isolated and reverse transcribed into cDNA. Polymerase chain reaction (PCR) was used to amplify the putative transporter genes directly from cDNA. However, because the regulation mechanism and expression pattern were unknown for pentose transporters in fungal species, cDNAs encoding the putative pentose transporters were not always obtainable despite alteration of cultivation condition. In this case, primers were designed according to the corresponding cDNA sequences from GenBank and used to amplify the exons using genomic DNA as a template. Overlap-extension PCR was then used to assemble the exons into the full-length genes. The resulting PCR products were cloned into the pRS424 shuttle vector containing a HXT7 promoter and a HXT7 terminator using the DNA assembler method. Yeast plasmids isolated from transformants were retransformed into E. coli DH5α, and isolated E. coli plasmids were first checked by diagnostic PCR using the primers used to amplify the original transporter genes. The entire open reading frames were submitted for sequencing to confirm the correct construction of the plasmids.
Most of the cloning work was carried out using the yeast homologous recombination mediated DNA assembler method. pRS424-HXT7-GFP plasmid was used for cloning of putative pentose transporters. In this plasmid, the HXT7 promoter, the GFP gene flanked with the EcoRI sites at both ends, and the HXT7 terminator were assembled into the pRS424 shuttle vector (New England Biolabs) linearized by ClaI and BamHI. PCR products of the putative pentose transporters flanked with DNA fragments sharing sequence identity to the HXT7 promoter and terminator were co-transferred into CEN.PK2-1C with EcoRI digested pRS424-HXT7-GFP using the standard lithium acetate method. The resulting transformation mixture was plated on SC-Trp plates supplemented with 2% D-glucose. Transformants were then tested for pentose transport activity.
The results are shown below in FIG. 40 and Table 16. Among the eight putative pentose specific transporters [XP—960000 (NC52), CAG88709 (DH48), XP—457508 (DH61), XP—681669 (32-10), XP—001487429 (29-6), XP—001727326 (29-9), XP—657854 (32-8), XP—720384 (29-4)], only NC52 enabled cell growth on a glucose plate, which suggested that the other seven transporters may be pentose-specific or inactive. Using the HPLC-based pentose uptake assay, four xylose-specific transporters were found, including XP—457508 (DH61), XP—001727326 (29-9), XP—720384 (29-4), and XP—681669 (32-10). In addition, one arabinose-specific transporter, XP—657854 (32-8) was identified (FIG. 40; Top). Five additional putative pentose specific transporters (XP—002488227, AB070824.1, XP—001389300, XP—002488227, EEQ43601.1) were also tested, none of which enabled cell growth in a glucose plate. Further pentose uptake assays indicated that XP—002488227 and AB070824.1 were xylose specific transporters (FIG. 40; Bottom). The summary of these results are shown in Table 16D.







TABLE 16A







Cloning of xylose-specific transporter NCU00821 orthologs











NCBI Reference

Sequence
Uptake



Sequence
Origin
Results*
Assay
Status





XP_002488227
Talaromyces stipitatus
Correct
Yes
Cloned


XP_001400900
Aspergillus niger
Correct
Yes
Cloned


XP_001220481
Chaetomium globosum
No
No
Sequenced, 



CBS 148.51


one intron


XP_001912725
Podospora anserina
No
No
OE-PCR, 






no PCR 






product


XP_660079
Aspergillus nidulans
Correct
Yes
Cloned



FGSCA4





AAL89823
Aspergillus niger
Correct
Yes
Cloned


XP_002382573
Aspergillus flavus
Wrong
Yes
Cloned



NRRL3357





XP_459386
Debaryomyces
No
No
Genomic 



hansenii CBS767


DNA,






no PCR 






product


XP_001825132
Aspergillus oryzae
Correct
Yes
Cloned



RIB40





XP_001389300
Aspergillus niger
Correct
Yes
Cloned





*“Correct” = Sequence of clone matched sequence in database(s); “Wrong” = Sequence of clone did not match sequence in database(s); “No” = Results not available (work in progress)













TABLE 16B







Cloning of xylose-specific transporter STL12/XUT6 orthologs











NCBI Reference

Sequence
Uptake



Sequence
Origin
Results*
Assay
Status





XP_457508
Debaryomyces hansenii
Correct
No
Cloned


(DH61)
CBS767





XP_002551364
Candida tropicalis MYA-
Wrong
No
No



3404





XP_001523322
Lodderomyces
Wrong
No
No



elongisporus NRRL





XP_720384 
Candida albicans
Correct
No
Cloned


(29-4)
SC5314





XP_456868
Debaryomyces hansenii
Wrong
No
No



CBS767





XP_001487429
Pichia guilliermondii
Wrong
No
Cloned


(29-6)
ATCC 6260





XP_961039
Neurospora crassa
Wrong
No
No


CAG88709 
Debaryomyces hansenii
Correct
No
Cloned


(DH48)
CBS767





XP_001727326
Aspergillus oryzae
Correct
No
Cloned


(29-9)






XP_001816757
Aspergillus oryzae
Correct
No
Cloned





*“Correct” = Sequence of clone matched sequence in database(s); “Wrong” = Sequence of clone did not match sequence in database(s); “No” = Results not available (work in progress)













TABLE 16C







Cloning of arabinose-specific transporter XUT1 orthologs











NCBI Reference

Sequence
Uptake



Sequence
Origin
Results *
Assay
Status





XP_002545773
Candida tropicalis
Correct
Yes
Cloned



MYA-3404





EEQ43601
Candida albicans
Correct
Yes
Cloned



WO-1





XP_001818631
Aspergillus oryzae
No
No
No PCR 



RIB40


product


XP_002558275
Penicillium
Wrong
Yes
Cloned



chrysogenum






Wisconsin 54-1255





XP_001390883
Aspergillus niger
No
No
No PCR 






product


XP_750103
Aspergillus fumigatus
Wrong
No
No



Af293





XP_960000
Neurospora crassa
Wrong
No
Cloned


(NC52)
OR74A





XP_657854 
Aspergillus nidulans
Correct
No
Cloned


(32-8)
FGSC A4





XP_001825068
Aspergillus oryzae
Correct
No
Cloned



RIB40





XP_681669 
Aspergillus nidulans
Correct
No
Cloned


(32-10)
FGSC





* “Correct” = Sequence of clone matched sequence in database(s); “Wrong” = Sequence of clone did not match sequence in database(s); “No” = Results not available (work in progress)













TABLE 16D







Listing of new xylose-specific transporters and one 


arabinose-specific transporter.










NCBI Reference

Xylose-
Arabinose-


Sequence
Origin
specific
specific





XP_457508
Debaryomyces hansenii 
Yes



(DH61)
CBS767




XP_001727326
Aspergillus oryzae
Yes



(29-9)





XP_720384 
Candida albicans 
Yes



(29-4)
SC5314




XP_681669 
Aspergillus nidulans 
Yes



(32-10)
FGSCA4




XP_657854 
Aspergillus nidulans 

Yes


(32-8)
FGSCA4




XP_002488227
Talaromyces stipitatus
Yes



AB070824.1
Aspergillus oryzae
Yes









The orthologs with sequences inconsistent with the sequences in databases (e.g., ones with mutations) will be re-cloned, sequenced, expressed in yeast strains, and tested for sugar uptake function. Similarly, the orthologs for which there is no sequencing results will also be tested for transporter function.
Sequence alignments of the pentose transporter orthologs were analyzed to identify conserved residues, which could have potential roles in transporter function. Alignments of a sample of xylose transporters (NCU0821, STL12/XUT6, XP—002488227.1, and XP—002382573.1) and arabinose transporters (XUT1 and EEQ43601.1) are shown in FIG. 41 (a, b) respectively. Several residues are specifically conserved in xylose transporters whereas others are specifically conserved in the arabinose transporters. These residues may have critical roles in transporting the specific pentose. An overall comparison of the sequences of the xylose and arabinose transporters (FIG. 41c) shows that there are also residues that are conserved in both types of pentose transporters, indicating functional roles in uptake of pentoses in general.
Examples 12-15 relate to optimization of the xylose utilization pathway in yeast.
Example 12
Engineering Pentose-Utilizing S. cerevisiae Strain
An efficient xylose metabolic pathway was reconstituted by exploiting the concept of isoenzymes. Isoenzymes catalyze the same chemical reaction with different kinetic or regulatory properties, and are known to confer fine-tuned control of metabolic fluxes in response to dynamic changes in the cytosolic environment. However, no prior metabolic engineering approaches had employed isoenzymes to increase fluxes of interest. This study demonstrated that simultaneous expression of both wild-type and mutant xylulose reductase (XR) isozymes could decrease xylitol accumulation and increase the overall xylose fermentation rate.
Inspired by the prevalence of isoenzymes in living systems, wild type XR and mutant XR (R276H) were co-expressed in S. cerevisiae along with xylitol dehydrogenase (XDH) and xylulokinase (XK) in order to construct a functional xylose metabolic pathway in S. cerevisiae. The XR mutant had been reported to exhibit much lower preference for NADPH over NADH whereas wild type XR showed 116 two-fold higher preference for NADPH over NADH (Watanabe et al., 2007).
The xylose-metabolizing genes (wild-type XYL1, 2, and 3 and mutant XYL1) from P. stipitis were PCR-amplified and placed under the control of constitutive promoters (PGK1 and TDH3) to construct expression cassettes. These integration cassettes were integrated into the genome of the D452-2 strain.
Transformation of expression cassettes for constructing xylose metabolic pathways was performed using the yeast EZ-Transformation kit (BIO 101, Vista, Calif.). To select transformants using an amino acid auxotrophic marker, yeast synthetic complete (YSC) medium was used, which contained 6.7 g/liter yeast nitrogen base plus 20 g/liter glucose, 20 g/liter agar, and CSM-Leu-Trp-Ura (BIO 101), which supplied appropriate nucleotides and amino acids. Yeast strains were routinely cultivated at 30° C. in YP medium 234 (10 g/liter yeast extract, 20 g/liter Bacto peptone) with 20 g/liter glucose.
The effect of S. cerevisiae strain background on xylose-metabolizing efficiency was also tested by expressing identical constructs containing optimized xylose utilization pathway enzymes in several different yeast strains. The three laboratory strains used were D452-2 (MATα, leu2, his3, ura3, can1), L2612 (MATα, leu2-3, leu2-112, ura3-52, trp1-298, can1, cyn1, gal+), and CEN.PK. Production of xylitol, acetate, and ethanol was monitored together with use of xylose and OD600. The results indicated that the D452-2 strain was the best amongst the three tested strains (FIG. 42-44). S. cerevisiae D452-2 was used for engineering of the xylose-metabolizing enzymes in yeast. Strains and plasmids used in this study are described in Table 17.







TABLE 17







Strain and plasmids used in study









Strain or plasmid
Description
Reference










Strain









D452-2
MATa, leu2, his3,ura3,can1
Hosaka et al., (1992)


D801-130
D452-2 expressing β-glucosidase 
In this study



(NCU00130) and cbt1 (NCU00801)



D809-130
D452-2 expressing β -glucosidase
In this study



(NCU00130) and NCU00809



D8114-130
D452-2 expressing β -glucosidase
In this study



(NCU00130) and cbt2 (NCU08114)



DA24
D452-2 expressing XYL1, mXYL1,
In this study



XYL2, and XKS1 (Isogenic of D452-2




except for leu2::TDH3P-XYL1-TDH3T,




ura3::URA3-PGKP-mXYL1-PGKT-




PGKP-XYL2-PGKT, Ty3::neo-TDHP-




XKS1-TDHT )



DA24-16
Evolved strain of DA24 in xylose
In this study



containing media




DA24-16 expressing β-glucosidase



DA24-16BT3
(NCU00130) in a multi-copy plasmid
In this study



and cbt1 (NCU00801) though single-




copy integration



DA24-16BT-M
DA24-16 expressing β-glucosidase
In this study



(NCU00130) and cbt1 (NCU00801) in




multi-copy plasmids








Plasmid









pRS425
LEU2, a multi copy plasmid
Christianson et al., (1992)


pRS426
URA3, a multi copy plasmid
Christianson et al., (1992)


pRS403
HIS3, an integrative plasmid
Sikorski et al., (1989)


pRS405
URA3, an integrative plasmid
Sikorski et al., (1989)


pRS425-β-
β-glucosidase (NCU00130) under the
Submitted


glucosidase
control of PGK promoter in pRS425



pRS426-cbt1
cbt1 under the control of PGK promoter
Submitted



n pRS426



pRS426-cbt2
cbt2 under the control of PGK promoter
Submitted



n pRS426



pRS426-NCU00809
NCU00809 under the control of PGK
Submitted



promoter in pRS426



pRS403-cbt1
cbt1 under the control of PGK promoter 
In this study



in pRS403









The engineered xylose-fermenting S. cerevisiae strain (DA24) consumed xylose and produced ethanol with negligible amounts of xylitol accumulation. When 40 and 80 g/L of xylose were used as a sole carbon source, the DA24 strain produced ethanol with consistent yields (YEthanol/Xylose=0.31 ̃0.32 g/g) in both shaker-flask and bioreactor fermentation experiments (FIGS. 45A-C). However, the DA24 strain consumed xylose slower than the naturally existing xylose-fermenting yeast, P. stipitis. Xylose fermentation capability of DA24 was further improved using an evolutionary engineering approach (Sauer 2001). One of the strains (DA24-16) isolated after repeated sub-cultures of the DA24 on xylose-containing medium showed much faster xylose fermentation rates as compared to the parental strain under various culture conditions (Table 18).










Table 18 shows the comparison of fermentation parameters 


of the two S. cerevisiae strains DA24 and DA24-16 


under different sugar conditions.















Sugar






Produced
consumption 

Product-


Carbon

Ethanol
rate
Yield
ivity


source
Strains
(g/L)
(g/L/h)
(g/g)
(g/L· h)















Xylose 
DA24
24
1.16
0.34
0.40


(80 g/L)
DA24-16
28
1.32
0.35
0.47


Glucose (70 
DA24
34
1.45
0.39
0.74


g/L) and 
DA24-16
45
1.78
0.42
0.96


xylose 







(40 g/L)









Interestingly, the DA24-16 strain consumed xylose as fast as P. stipitis, the fastest xylose-fermenting yeast known. However, ethanol yield by DA24-16 was slightly lower than that by P. stipitis (FIGS. 46A-B).
A screen was set up using S. cerevisiae strain L2612 expressing the xylose-utilizing enzymes (strain YSX3) transformed with a genomic library. Transformation was followed by serial culture transfer in 40 g/L xylose under oxygen-limiting conditions to enrich for strains that are efficient in utilizing xylose. Fermentations were performed in 50 mL YPX media under oxygen-limited conditions and 0.1% (50 μL) of a fully grown cell culture was transferred to the next serial culture when OD600, 10 was reached. After 10 serial cultures, cells were spread with serial dilution on YPX (40 g/L) agar media. Through fermentation experiments using 5 mL of YPX media, colonies were screened for low xylitol and high ethanol formation. DNA sequencing revealed that the two most efficient strains contained integrated copies of XYL2, which was then cloned into a multi-copy plasmid through homologous recombination and transformed into YSX3 cells.
The XYL2 gene was placed in integration vectors under the control of promoters of different strength, e.g., TDHp or PGKp, and transformed into YSX3 cells (FIG. 47). Studies were conducted to monitor the effect of these plasmids on xylitol and ethanol formation in the transformed yeast cells. The results indicated that the YSX3 cells expressing higher levels of XYL2 (under the PGKp) were more efficient at ethanol production and in addition, produced lower amounts of xylitol (FIG. 48). When additional XYL3 was expressed in these cells (termed SR1 strain), the amount of xylitol produced was further decreased in the resulting strain SRu-23 (FIG. 49). Therefore, it appeared that XYL2 expression level in engineered S. cerevisiae strains is a key factor for implementing xylose fermentation, and when expression is under a strong promoter, the strain has less xylitol accumulation as well as high ethanol yield. Simultaneous over-expression of XYL2 and XYL3 can further decrease the amount of xylitol accumulation. However, when XYL1 was further over-expressed in a strain over-expressing XYL2 and XYL3, there was considerable xylitol accumulation and consequently decreased xylose fermentation (FIGS. 50-51). Therefore, it appeared that there was an optimal level of XYL1 for efficient xylose fermentation.
Experiments were also carried out to test if over-expression of endogenous GRE3 in S. cerevisiae expressing XYL2 and XYL3 could facilitate xylose fermentation. For the construction of pRS403-GRE3, GRE3 gene was amplified from S. cerevisiae D452-2 and inserted into pR403 vector with TDH3 promoter and CYC terminator. After linearization of pRS403-GRE3, it was integrated into the genome of D452-2. The xylose-utilizing genes were introduced into the yeast strain D452-2 (FIG. 52), and xylose fermentation parameters were monitored. The results indicated that over-expression of GRE3 was as effective as the over-expression of XYL1 in ethanol production and xylitol accumulation, particularly when cells were grown in 80 g/L of xylose at high OD inoculations (FIGS. 53-54).
Example 13
Engineering LAD and XDH
L-arabinitol and xylitol accumulation, thought to be caused by cofactor imbalance between NADPH-dependent XR and NAD+-dependent XDH and LAD, has been regarded as a major bottleneck during xylose fermentation in engineered S. cerevisiae expressing the pentose-utilizing enzymes. While the imbalance between XR and XDH has been corrected by engineering enzymes with reversed cofactor preferences (Watanabe et al., 2007; Matsushika et al., 2008; Bengtsson et al., 2009), this approach resulted in reduced flux, as the modified enzymes had reduced specific activities. The P. stipitis XR mutant had been reported to exhibit much lower preference for NADPH over NADH whereas wild type psXR showed two-fold higher preference for NADPH (Watanabe et al., 2007).
In this study, similar studies were done on L-arabinitol 4-dehydrogenase (LAD) and XDH from N. crassa to alter cofactor specificity and hence improve xylose fermentation in engineered S. cerevisiae. 
Identification of Putative LAD-Encoding Genes
Methods of identifying putative LAD-encoding genes and of cloning LAD-encoding and putative LAD-encoding genes are described.
Identification of Putative LAD-Encoding Genes
From a protein BLAST search using ncLAD (EAA36547.1) as a probe, two putative genes were identified in P. chrysogenum (XP—002569286.1) and P. guilliermondii (EDK37120.2), respectively. The amino acid sequence identities of these two proteins with ncLAD were 71% and 46%, respectively.
Cloning LAD-Encoding and Putative LAD-Encoding Genes
A. niger (NRRL 326), P. guilliermondii (NRRL Y2075), and P. chrysogenum (NRRL 807) were obtained from the United States Department of Agriculture Agricultural Research Service Culture Collection (Peoria, Ill.). T. longibrachiatum (T. reesei, YSM 768) was obtained from the German Resource Centre for Biological Material (DSMZ).
A. niger, T. longibrachiatum, P. chrysogenum, and P. guilliermondii were grown in liquid media or on agar plates containing 1% yeast extract, 2% peptone, and 2% L-arabinose. Cells were frozen in liquid nitrogen for the isolation of total RNA or genomic DNA. Reverse transcription-PCR (RT-PCR) was performed on mRNAs isolated from T. longibrachiatum, P. chrysogenum, and P. guilliermondii to obtain cDNA, and PCR was used to obtain the genes encoding (putative) LADs. For A. niger, the putative LAD gene could not be amplified from cDNA due to unknown reasons. Thus, overlap extension-PCR (OE-PCR) was used to clone this intron-containing gene from the isolated genomic DNA. Note that all primer sequences used to clone these genes are listed in Table 19.







TABLE 19







Primers used for the cloning of wild type LADs. 


Restriction enzyme sites are in bold and italicized.











Restriction





Enzyme
Primer
Sequence





anLAD
NdeI
Fwd-fragment1a
5′-GACATCGATGA GCTACCGCAAC-3′





SEQ ID NO: 71




Rev-fragment1
5′-GTGCACGTCGGACCCGCAGATTCC-3′





SEQ ID NO: 72


 



BamHI
Fwd-fragment2b
5′-GGAATCTGCGGGTCCGACGTGCAC-3′





SEQ ID NO: 73




Rev-fragment2
5′-CAGAAGATTTAA TGAACGTAGA-3′





SEQ ID NO: 74


 


tlLAD
NdeI
Fwd
5′-GACATCAGTGA TCGCCTTCC-3′





SEQ ID NO: 75



BamHI
Rev
5′-CCTGGATTGA TGAACGTATA-3′





SEQ ID NO: 76


 


pcLAD
NdeI
For
5′-GACATCGATGA GCTTCCGCAAC-3′





SEQ ID NO: 77



EcoRI
Rev
5′-CCAGAAGTATTGA TGAACGTAGA-3′





SEQ ID NO: 78


 


pgLAD
NdeI
Fwd
5′-GACATCGATGA GCGACTCTGC-3′





SEQ ID NO: 79



BamHI
Rev
5′-GGATACAGAATGA TGAACGTAGA-3′





SEQ ID NO: 80





a,bFragment 1 and 2 indicate the upstream and downstream exons flanking the intron.


cSequences in bold (italicized) indicate restriction enzyme sites.






PCR products were subcloned into pET-28a vector and the constructs were used to transform into two E. coli strains, DH5α and BL21 (DE3), by electroporation for cloning and expression, respectively. NdeI/BamHI restriction sites were used for the subcloning of the predicted genes from A. niger, T. longibrachiatum, and P. guilliermondii, and NdeI/EcoRI sites were used for P. chrysogenum. The constructs encoded (putative) LADs as N-terminal His6-tagged fusions. Plasmids were sequenced using BIGDYETM Terminator sequencing method and analyzed with 3730xL Genetic Analyzer (Applied Biosystems, Foster City, Calif.) at the Biotechnology Center at the University of Illinois at Urbana-Champaign (Urbana, Ill.).
Protein Expression and Purification
Genes encoding pcLAD (XP—002569286.1), pgLAD (EDK37120.2), anLAD (CAH69383.1), and tlLAD (AAL08944.1) were cloned into the pET-28a vector and expressed in E. coli BL21 (DE3). E. coli BL21 (DE3) containing the LAD genes were grown overnight at 30° C. on a rotary shaker at 250 rpm. Overnight culture (50 μL) was used to inoculate a fresh culture (5 mL), which was grown at 30° C. with shaking at 250 rpm until the optical density at 600 nm (OD600) reached 0.6-1.0. The cultures were then induced with 0.3 mM IPTG at 30° C. for 3-4 hrs or at 18° C. for 20 hrs.
The induced cells (1 mL) were lysed by re-suspending them in 1 mL of 50 mM potassium phosphate buffer (pH 7.0) with 1 mg/mL lysozyme and shaking at 30° C. and 250 rpm for 30 min. Cells were kept at −80° C. overnight and thawed at room temperature. The resulting cell lysates were centrifuged at 13,200 rpm for 15 min, and the supernatant and precipitate were analyzed for protein expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
For protein purification, the induced cells (400 mL) were treated with 15 mL of Buffer A (20 mM Tris, 0.5 M NaCl, 20% glycerol, pH 7.6) with 1 mg/mL lysozyme and shaken at 30° C. and 250 rpm for 30 min. After a freeze-thaw cycle, the resulting product was further lysed by sonication followed by centrifugation for 20 min at 12,000 rpm to remove cell debris. The supernatants were applied to a column packed with Co2+-immobilized metal affinity chromatography resin to purify His6-tagged proteins following the manufacturer's instructions. The purified proteins were desalted by ultrafiltration (Amicon Ultra, Millipore, Billerica, Mass.) and washed with HEPES buffer (pH 7.0) containing 150 mM NaCl and 15% glycerol and kept at −20° C. Protein concentrations were determined by the Bradford method (Bradford 1976) according to the manufacturer's protocol.
Characterization of LAD Proteins
The steady-state kinetics, molecular weight, quaternary structure, temperature dependence, pH dependence, L-arabinitol dehydrogenase activity, and metal content of LAD enzymes were analyzed.
L-Arabinitol Dehydrogenase Activity
Lysates were prepared from host cells expressing LAD from P. chrysogenum, P. guilliermondii, A. niger, and T. longibrachiatum. Ten microliters of cell lysate were used for an activity assay with 200 mM L-arabinitol and 2 mM NAD+ as the substrates in 50 mM potassium phosphate buffer (pH 7.0). NADH production was monitored by measuring absorbance at 340 nm (ε=6.22 mM−1cm−1) using a Cary 300 Bio UV-vis spectrophotometer (Varian, Cary, N.C.).
Steady-State Kinetics
Kinetic parameters of different LAD enzymes were determined. Initial rates were determined by measuring the absorbance change at 340 nm using a UV-vis spectrophotomer at room temperature in 50 mM potassium phosphate buffer (pH 7.0). Initial rates were measured at various concentrations of the substrate (L-arabinitol) and cofactors (NAD+/NADP+) (5 to 320 mM for L-arabinitol, 0.5 to 3.2 mM for cofactors). Enzyme kinetics for the substrate and cofactors were analyzed using Michaelis-Menten kinetics, and kinetic parameters were determined by fitting data to the Lineweaver-Burk plot. The parameters for substrate were determined by measuring initial rates at saturated cofactor concentrations (3.2 mM) and those for cofactors were determined at saturated substrate concentrations (320 mM). Assays were performed in triplicate.
The cloned LADs showed different binding affinities and catalytic activities for L-arabinitol: Km differed by two fold and kcat by about three fold amongst the LADs. For L-arabinitol, the Km values of anLAD, tlLAD, and pcLAD were 25±1, 18±1, and 37±2 mM, and the kcat values were 507±22, 346±41, and 1085±71 min−1, respectively (Table 20). The tlLAD enzyme had the lowest Km while pcLAD showed the highest catalytic activity (kcat) and efficiency (kcat/Km) despite having the highest Km (Table 20). For cofactor NAD+ kinetics, the cloned LADs showed Km values in the range of 0.2-0.3 mM and catalytic efficiencies in the range of 2526 to 3460 mM−1·min−1 (Table 21). All cloned LADs showed minimal activities toward NADP+ (Tables 20, 21). The initial rates were not saturated at highest substrate and cofactor concentration (320 mM for L-arabinitol and 3.2 mM for NADP+) due to the large Km. Therefore, only the catalytic efficiency of the enzyme was determined using 0.1 or 0.2 mM for NADP+ and 10 or 20 mM for L-arabinitol (Km>>[S]) (Tables 20, 21).







TABLE 20







Kinetic parameters of LADs for L-arabinitol at 


saturated cofactor concentrations.














Specific







activity
Km
kcat
kcat/Km




(U/mg protein)
(mM)
(min−1)
(mM−1 · min−1)





anLAD
NAD+
 11.7 ± 0.3a
25 ± 1
507 ± 22 
20.0 ± 0.8



NADP+
—b
—
—
 0.04 ± 0.01


tlLAD
NAD+
8.7 ± 0.1
18 ± 1
346 ± 41 
19.0 ± 0.8



NADP+
—
—
—
 0.13 ± 0.02


pcLAD
NAD+
25.3 ± 1.4 
37 ± 2
1085 ± 71 
29 ± 1



NADP+
—
—
—
 0.04 ± 0.02





aError indicates standard deviation from the mean, n = 3


bDash indicates not determined due to high Km for indicated cofactor













TABLE 21







Kinetic parameters of LADs for NAD+ and NADP+


at saturated L-arabinitol concentration.













Km
kcat
kcat/Km




(mM)
(min−1)
(mM−1 · min−1)





anLAD
NAD+
 0.20 ± 0.01a
494 ± 11 
2526 ± 83 



NADP+
—b
—
20 ± 9 


tlLAD
NAD+
0.2 ± 0.1
436 ± 96 
2689 ± 646 



NADP+
—
—
17 ± 9 


pcLAD
NAD+
0.3 ± 0.1
1039 ± 165 
3460 ± 505 



NADP+
—
—
15 ± 4 





aError indicates standard deviation from the mean, n = 3


bDash indicates not determined due to high Km for indicated cofactor






Molecular Weight and Quaternary Structure
Calculated molecular weights of the subunits of the four proteins were 43 kDa (anLAD), 41 kDa (tlLAD), 42 kDa (pcLAD), and 42 kDa (pgLAD). The molecular weights of the proteins were determined using a Bio-Sil SEC-250 column (300×7.8 mm, Bio-Rad, Hercules, Calif.) on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). The mobile phase consisted of 50 mM Na2HPO4, 50 mM NaH2PO4, 150 mM NaCl, and 10 mM NaN3 (pH 6.8) and the flow rate was 1.0 mL/min. The molecular weights were calculated by comparing the retention times with those of protein molecular weight standard.
The quaternary structures were determined based on the molecular weights observed by HPLC and the molecular weights of monomeric subunits which were determined by SDS-PAGE analysis. Molecular weights of an-, tl-, and pcLAD were determined to be 178, 194, and 173 kDa, respectively. Comparing to the molecular weights of the subunits determined by SDS-PAGE, results suggested that the LADs were non-covalently linked tetramers in their native forms.
Temperature and pH Dependence
The optimal temperatures of the proteins were determined by assaying enzyme activities at temperatures ranging from 10 to 70° C. Thermal inactivation was determined by measuring enzyme activity after various incubation times at 50° C. in phosphate buffer. Enzyme activity was measured with 2 mM NAD+ and 200 mM L-arabinitol. Half-life of enzyme activity was determined using a first-order exponential decay function. Temperature was controlled by a Cary temperature controller connected to the UV-vis spectrophotometer (Varian, Cary, N.C.). pH-dependent enzyme activity was determined by measuring activity at pH between 5.0 and 11.0 at saturated concentrations of NAD+ (2 mM) and L-arabinitol (200 mM) in a universal buffer (50 mM morpholineethanesulfonic acid/50 mM Tris/50 mM glycine) (Ellis and Morrison 1982).
The optimal temperatures of anLAD and pcLAD were between 40 and 50° C., whereas tlLAD showed higher optimal temperature between 55 and 65° C. (FIG. 55a). Catalytic activities of the LADs exponentially decreased with the length of incubation time at 50° C. and were almost completely deactivated after 100 min (FIG. 55b). tlLAD was the most thermally stable with a half-life of 20 min at 50° C., and anLAD was least stable with a half-life of less than 5 min at 50° C. All characterized LADs showed activity in the pH range of 7 to 11 with maximum activity around pH 9.4 (FIG. 55c). In the pH range outside of 9 to 10, activity was significantly reduced and approximately 20% of activity remained at pH 7.0 (FIG. 55c). No activity was detected at or below pH 5.0.
Metal Analysis
Duplicate samples for metal analysis were prepared in phosphate buffered saline (PBS) by buffer exchange and lyophilization. Each sample contained 1-2 mg of protein in 1 mL buffer solution. The identity and content of the metal were analyzed by inductively coupled plasma atomic emission spectrometry (OES Optima 2000 DV, Perkin Elmer, Boston, Mass.) in the Microanalytical Laboratory at the University of Illinois at Urbana-Champaign (Urbana, Ill.).
Measured weight percentages of Zn2+ were close to those calculated based on the 1:1 molar ratio (Table 22).







TABLE 22







Calculated and measured Zn2+ contents.












Calculated Weighta (%)
Measured weight (%)















anLAD
0.027
0.027 ± 0.003b



tlLAD
0.047
0.048 ± 0.003 



pcLAD
0.048
0.061 ± 0.013 







aCalculated molecular weights were determined based on the buffer composition, protein concentration, and 1:1 molar ratio of LAD monomer subunit and Zn2+. Buffer solution (1 L) contained NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.44 g), and KH2PO4 (0.24 g).



bAll samples were analyzed in duplicate and errors were standard deviations.






Engineering of LAD Enzymes with Altered Cofactor Specificity
Methods of altering the cofactor specificity of LADs were determined, and mutated LADs were analyzed for altered cofactor specificity and other characteristics.
Development of LADs with Altered Cofactor Specificity
Site-directed mutagenesis was performed to alter the cofactor specificity of anLAD, tlLAD, and pcLAD from NAD+ to NADP+. Amino acid numbers 224, 225, and 362 of naturally occurring tlLAD were substituted with serine, arginine, and threonine, respectively, to generate the tlLAD with altered cofactor specificity. The amino acid sequences of cloned anLAD and pcLAD were aligned with the T. longibrachiatum LAD (tlLAD) sequence, and the amino acids that correspond to tlLAD amino acid numbers 224, 225, and 362 were mutated. For all of the LADs with altered cofactor specificity, two amino acid residues within the β-α-β motif of the coenzyme binding domain were replaced with serine and arginine, respectively: D213 and 1214 for anLAD, D224 and 1225 for tlLAD, and D212 and 1213 for pcLAD (Korkhin et al., 1998; Pauly et al., 2003; Watanabe et al., 2005), and the third mutation was introduced at A359 for anLAD, A362 for tlLAD, and 5358 for pcLAD and replaced with threonine (For primer sequences, see Table 23). Megaprimer PCR method was used to introduce site-specific mutations using wild type LAD constructs as the templates (Sarkar and Sommer 1990). Correct mutations were confirmed by DNA sequence analysis.







TABLE 23





Primers used for site directed 


mutagenesis by the megaprimer PCR method.a


















Fwd-T7-pro
5′-TAATACGACTCACTATAGGG-3′ SEQ ID NO: 81



Rev-T7-term
5′-GCTAGTTATTGCTCAGCGG-3′ SEQ ID NO: 82


 


anLAD
Fwd-D213S/I214R
5′-CCTATCGTCATTACCTCACGTbGACGAGGGGCGGCTG-3′




SEQ ID NO: 83



Rev-D213S/I214R 
5′-CAGCCGCCCCTCGTCACGTGAGGTAATGACGATAGG-3′




SEQ ID NO: 84



Fwd-A359T
5′- CCT TCGAAACGGCTACAAACCCCAAGACG-3




SEQ ID NO: 85


 


tlLAD
Fwd-D214S/I215R
5′-GCTTGTCATCACATCACGTTCAGAGAGCCGTCTG-3′




SEQ ID NO: 86



Rev-D214S/I215R
5′-CAGACGGCTCTCTGAACGTGATGTGATGACAAGC-3′




SEQ ID NO: 87



Fwd-S362T
5′-GCATTTGAGACGTCAACAGATCCCAAGAGC-3′




SEQ ID NO: 88


 


pcLAD
Fwd-D212S/I213R 
5′-CCTATTGTCATCACTTCACGTGACGAGGGCCGCTTG-3′




SEQ ID NO: 89



Rev-D212S/I213R 
5′-CAAGCGGCCCTCGTCACGTGAAGTGATGACAATAGG-3′




SEQ ID NO: 90



Fwd-S358T
5′-CCTTTGAGACTGCCACAAACCCTAAGACCGGTG-3′




SEQ ID NO: 91





aTo create mutant LADs, fragments 1 and 2 were amplified using Fwd-T7-pro and Rev-D213S/I214R and Fwd-A359T and Rev-T7-term primers, respectively. Fragment 3 was amplified using Fwd-D123S/I214R and fragment 2 (Rev megaprimer). Full mutant genes were amplified by overlap extension of fragment 1 and 3. Template DNA was pET-28a plasmid.


bSequences underlined were the mutation sites.






Kinetic Analysis of LADs with Altered Cofactor Specificity
In this example, “tlLAD mutant” is defined as tlLAD with the mutations D224S/I225R/A362T; “anLAD mutant” is defined as anLAD with the mutations D213S/1214R/A359T; and “pcLAD mutant” is defined as pcLAD with the mutations D212S/1213R/S358T. The tlLAD mutant showed significantly altered cofactor specificity from NAD+ to NADP+. It also demonstrated the highest catalytic activity. The Km and kcat of the tlLAD mutant for L-arabinitol with NADP+ were 46±4 mM and 170±9 min−1, respectively (Table 24). In all assays including the tlLAD mutant with saturated NAD+, a plateau of reaction rate was not observed in the tested concentration range, so catalytic efficiencies were determined at 0.8 mM for NAD+ and 80 mM for L-arabinitol (Tables 24, 25). For cofactors, anLAD and tlLAD mutants showed significantly higher preference for NADP+ over NAD+ (Table 25). The Km values of the anLAD and tlLAD mutants were 0.46±0.09 and 0.10±0.01 mM, and the kcat values were 55.7±6.4 and 90.5±9.2 min−1, respectively (Table 25). The catalytic efficiencies of anLAD and tlLAD mutants were 130±32 and 934±72 mM−1·min−1, and the ratios of the catalytic efficiencies with NADP+ to NAD+ were 100 and 161, respectively. For the tlLAD mutant, the ratio of catalytic efficiency for NADP+ to NAD+ was increased by 2.5×104 fold (Tables 21, 25). The pcLAD mutant showed no activity with NAD+.







TABLE 24







shows kinetic parameters of LAD mutants for L-arabinitol at 


saturated cofactor concentrations.














Specific







activity
Km
kcat
kcat/Km




(U/mg protein)
(mM)
(min−1)
(mM−1 · min−1)





anLAD
NAD+
−a
−
−
0.010 ± 0.002b


mutant
NADP+
−
−
−
0.45 ± 0.20  


t1LAD
NAD+
−
−
−
0.050 ± 0.007  


mutant
NADP+
3.9 ± 0.2
46 ± 4 
170 ± 9 
3.7 ± 0.2 


pcLAD
NAD+
−
−
−
−


mutant
NADP+
−
−
−
0.02 ± 0.02 





aDash indicates not determined due to high Km for indicated cofactor


bError indicates standard deviation from the mean, n = 3













TABLE 25







shows kinetic parameters of LAD mutants for NAD+ and 


NADP+ at saturated L-arabinitol concentration.













Km
kcat
kcat/Km




(mM)
(min−1)
(mM−1 · min−1)





anLAD 
NAD+
−a
−
 1.3 ± 0.3b


mutant
NADP+
0.46 ± 0.09
55.7 ± 6.4 
130 ± 32 


tlLAD 
NAD+
−
−
5.8 ± 0.8


mutant
NADP+
0.097 ± 0.011
90.5 ± 9.2 
934 ± 72 


pcLAD 
NAD+
−
−
−


mutant
NADP+
−
−
3.6 ± 1.0





aDash indicates not determined due to high Km for indicated cofactor


bError indicates standard deviation from the mean, n = 3






Engineering of N. crassa XDH (ncXDH) with Altered Cofactor Specificity
Cloning and Characterization of Putative ncXDH
A putative N. crassa xylitol dehydrogenase (ncXDH) sequence was found using a protein BLAST search on the National Center for Biotechnology Information website (webpage ncbi.nlm.nih.gov) using the P. stipitis xylitol dehydrogenase (psXDH) enzyme as a query sequence. The two enzymes were aligned fully using a ClustalW algorithm and found to share 44% identity and 60% similarity (FIG. 56). The whole-genome sequence of Neurospora crassa has been published (Galagan et al., 2003) and it was utilized to design primers for cloning of the putative xylitol dehydrogenase (XDH) gene.
RT-PCR performed on total RNA isolated from D-xylose-induced N. crassa 10333 showed the expected size of gene product ( ̃1.1 kb). The RT-PCR product was cloned into the pET-28a vector using NdeI and SacI restriction sites and was transformed into E. coli BL21 (DE3). This construct (pET-28a ncXDH) expressed ncXDH as an N-terminal His6-tagged fusion with a thrombin cleavage site. Cell lysates of IPTG-induced cultures of these cells were prepared, analyzed by SDS-PAGE, and assayed for XDH activities. The XDH was then purified by immobilized metal ion affinity chromatography (IMAC) using Talon® Co2+ Superflow resin (Clontech, Mountain View, Calif.) according to manufacturer's protocol. The purified protein was desalted by ultrafiltration with several washes of 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer (pH 7.25)+15% glycerol and stored frozen at −80° C. Protein concentrations were determined by the Bradford method (Bradford 1976).
ncXDH is a strictly NAD+-preferring enzyme. ncXDH also displays high stability (half-life of  ̃200 min at 50° C.) and expression. Previous work by Watanabe et al. (2005b) was aimed at reversing the cofactor specificity of psXDH.
Development of ncXDH with Altered Cofactor Specificity
Through sequence alignment, residues D204, 1205, and V206 of ncXDH were targeted for site-directed mutagenesis to alanine, arginine, and serine, respectively, to create ncXDH-ARS. Table 26 shows that ncXDH-ARS has completely reversed cofactor specificity, now preferring NADP+. The affinity for substrate xylitol did not suffer very much from the affinity-change for the co-factor.







TABLE 26







shows kinetic parameters for N. crassa and P. stipitis XDH and XDH-ARS 


with nicotinamide cofactors NAD+ and NADP+ at saturated xylitol concentrations.








NAD+
NADP+

















kcat/Km


kcat/Km




kcat
Km
(mM−1 
kcat
Km
(mM−1 



Enzyme
(min−1)
(mM)
min−1)
(min−1)
(mM)
min−1)
Source

















ncXDH-wt
2160
0.127
17000
—a
~5.6
~68
This work


ncXDH-ARS
—a
~3.5
~165
2080
0.325 
6400
This work


psXDH
1050
0.381
2760
110
170
0.65
Watanabe et









al. (2005b)


psXDH-ARS
240
1.3
181
2500
0.897
2790
Watanabe et









al. (2005b)





aNot determined, cofactor saturation not reached.


All assays were performed at 25° C. in 50 mM Tris, pH 8.0.






Kinetic Analysis of ncXDH Mutant
The mutant ncXDH had a dramatic reversal of cofactor specificity. The Km of the mutant ncXDH for NADP+ was only about 2.5-fold higher than the Km of wild-type ncXDH for NAD+ whereas the kat values were similar (Table 27).







TABLE 27







shows kinetic parameters of ncXDH mutants 


for substrate xyitol.











kcat
Km
kcat/Km


Enzyme
(min−1)
(mM)
(mM−1 min−1)













ncXDH-wt
2170 ± 135 
6.6 ± 2.0
330


ncXDH-ARS
2090 ± 35 
4.3 ± 0.3
490





a Not determined, cofactor saturation not reached.


All assays were performed at 25° C. in 50 mM Tris, pH 8.0.


All enzymes were purified and characterized with N-His6-tag






As shown in FIG. 57, XDH activity exhibits a higher tolerance to more acidic conditions with activity extending down to pH 4.0, whereas LAD activity is abolished at pH 5.0 in the in vitro activity assay.
Example 14
Expression of Xylose Isomerase from Bacteroides stercoris in S. cerevisiae 
Bacterial xylose isomerase (XI) is involved in converting xylose into xylulose. Recently, three successful cases of expressing active XI from two species of anaerobic fungi (Piromyces sp. and Orpinomyces sp.) and from the anaerobic bacteria (Clostridium phytofermentans) have been reported. A fungal XYLA gene from Piromyces sp. E2 was functionally expressed in S. cerevisiae and a maximum 1.1 U/mg-protein of XI activity was obtained at 30° C. (Kuyper et al., 2003). The second fungal XYLA gene from Orpinomyces, which has 94% identity with that from Piromyces sp., was also functionally expressed in S. cerevisiae (Madhavan et al., 2009). Recently, the first prokaryotic xylA gene from Clostridium phytofermentans was functionally expressed in S. cerevisiae (Brat et al., 2009).
The isomerase gene xylA from the anaerobic bacteria Bacteroides stercoris (BtXI) shares high sequence identity with the isomerase gene from Piromyces sp. (82%). BtXI was cloned into the pRS424TEF vector and transformed into the S. cerevisiae L2612 strain. The gene was also integrated into the S. cerevisiae D452-2 strain by using the pRS403TEF vector. Ethanol production was observed in both strains expressing BtXI (5 g/L in L2612 and 7.8 g/L in D452-2) (FIG. 58-59). However, rates of production were relatively low compared to that of engineered strains expressing the XYL genes.
The low ethanol production could be attributed to the inhibitory effect of any accumulated xylitol (formed from xylose by endogenous yeast aldose reductase). To decrease xylitol accumulation, XDH and XK were expressed in BTXI-expressing yeast strain (DBtXI). The resulting strain had slightly improved ethanol yield and decreased xylitol production (FIG. 60). Co-expression of these two XYL genes in DBtXI resulted in ethanol production even under aerobic conditions.
Example 15
Over-Expression of Enzymes in Pentose Phosphate Pathway (PPP)
The PPP enzymes glucose-6-phosphate dehydrogenase (ZWF1), 6-phosphogluconate dehydrogenase (GDN1), transaldolase (TAL1), and transketolase (TKT1) from P. stipitis were cloned into an integration vector (pRS406) under the control of a strong promoter (PGPD). The plasmid was linearized by the enzyme StuI and integrated into the chromosome of S. cerevisiae. 
However, to get the beneficial effects of over-expressing the PPP enzymes, there also had to be over-expression of XYL3 (XK) (FIG. 61). Expression of XYL3 and the PPP enzymes also improved ethanol production in YP-xylulose media.
Example 16
Expression of Aldose-1-Epimerase
Hydrolysis of cellobiose by β-glucosidase releases β-D-glucose. However, yeast hexokinases prefer (or exclusively use) α-D-glucose, and the rate of mutaroation of β-D-glucose to α-D-glucose could effectively slow down metabolic rate. One way of enhancing the conversion was to over-express the predicted aldose-1-epimerase NCU09705. This hypothesis was tested by over-expressing NCU09705 homologs: galM in E. coli; GAL10, YHR210C, and YNR071C in S. cerevisiae; and GAL 10 in P. stipitis. The strains were then tested for cellobiose consumption and ethanol production (FIGS. 62A-B). The results indicated that over-expression of the homologs in S. cerevisiae caused a slight increase in cellobiose consumption and ethanol production.
Example 17
Co-Fermentation of Xylose and Cellobiose
In this example a new strategy was used to overcome glucose repression in which a dimer of glucose, cellobiose, was co-fermented with xylose (a pentose). Cellobiose is an intermediate product from enzymatic hydrolysis of cellulose, which is further converted to glucose by β-glucosidases in the cocktail of cellulases including exocellulases, endocellulases, and β-glucosidases, whereas pentose sugars are the products of dilute acid hydrolysis of hemicellulose. Wild type S. cerevisiae cannot assimilate cellobiose because it lacks both a cellobiose transporter and a β-glucosidase capable of hydrolyzing cellobiose into glucose. Hence, the newly discovered cellodextrin transporter genes described in Example 9 and a β-glucosidase gene from N. crassa were co-expressed in S. cerevisiae and a mixture of xylose and cellobiose was used as carbon source (FIGS. 63A-C). Similar approaches have employed either secretion, or cell surface display, of β-glucosidases to allow cellobiose fermentation by S. cerevisiae (van Rooyen et al., 2005; Skory et al., 1996; Kotaka et al., 2008; Katahira et al., 2006). In those cases, cellobiose was hydrolyzed into glucose extracellularly before being transported by the endogenous hexose transport system of S. cerevisiae. In contrast, in this strategy, cellobiose was hydrolyzed intracellularly following transport.
In the conventional methods for mixed sugar fermentation in S. cerevisiae, a mixture of glucose and pentose sugars derived from lignocellulose is used. However, in this new strategy, a mixture of cellobiose and pentose sugars was used. The cellobiose was transported inside yeast cells via the heterologous cellodextrin transporters while pentose sugars were transported inside yeast cells by endogenous hexose transporters, thus removing the direct competition between glucose and pentose sugars for the same transporters, a phenomenon that is partly responsible for glucose repression. Once inside yeast cells, cellobiose was converted to glucose by β-glucosidase and immediately consumed by yeast cells, which resulted in low intracellular glucose concentration, thereby further alleviating glucose repression.
The engineered xylose-utilizing yeast strain L2612 was used as a host to co-express cellodextrin transporter and β-glucosidase genes. In this strain, the D-xylose utilization pathway consisting of xylose reductase, xylitol dehydrogenase, and xylulokinase from Pichia stipitis was integrated into the chromosome. The cellodextrin transporters from Neurospora crassa including NCU008011, NCU08114, and, NCU00809, and two β-glucosidase genes, one from Neurospora crassa and the other from Aspergillus aculeatus, were evaluated.
S. cerevisiae L2612 (MATα, leu2-3, leu2-112, ura3-52, trp1-298, can1, cyn1, gal+) was cultivated in synthetic dropout media to maintain plasmids (0.17% of Difco yeast nitrogen base without amino acids and ammonium sulfate, 0.5% of ammonium sulfate, 0.05% of amino acid dropout mix). YPA medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate) with 2% of sugar was used to grow yeast strains.
To integrate the D-xylose utilization pathway consisting of D-xylose reductase, xylitol dehydrogenase, and xylulokinase from Pichia stipitis, the corresponding genes were PCR-amplified and cloned into the pRS416 plasmid using the DNA assembler method (Shao et al., 2009). BamHI and HindIII were used to remove the DNA fragment encoding the D-xylose utilization pathway and then ligated into the pRS406 plasmid digested by the same two restriction enzymes. The resulting plasmid was then linearized by ApaI and integrated into the URA3 locus on the chromosome of L2612.
The pRS425 plasmid (New England Biolabs, Ipswich, Mass.) was used to co-express a cellodextrin transporter gene and a β-glucosidase gene. As shown in FIG. 64, the pRS425 plasmid was digested by BamHI and ApaI. The PYK1 promoter and the ADH1 terminator were added to N-terminus and C-terminus of the cellodextrin transporter, respectively, while the TEF1 promoter and the PGK1 terminator were added to the N-terminus and C-terminus of the β-glucosidase, respectively. These DNA fragments were assembled into the linearized pRS425 shuttle vector using the DNA assembler method (Shao et al., 2009). Three cellodextrin transporter genes NCU00801 (XM—958708), NCU08114 (XM—958780), and NCU00809 (XM—959259) from Neurospora crassa and two β-glucosidase genes NCU00130 (XM—951090) from Neurospora crassa and BGL1 (D64088) from Aspergillus aculeatus were used. There were six combinations in total, each with one cellodextrin transporter gene and one β-glucosidase gene.
Yeast plasmids were then transferred into E. coli DH5α, which were used for recombinant DNA manipulation. The transformants were plated on Luria broth plates containing 00 mg/L ampicillin. Single colonies of E. coli transformants were then inoculated into the liquid Luria broth media (Fisher Scientific, Pittsburgh, Pa.) and grown at 37° C. and 250 rpm. Plasmids were isolated from E. coli using the QIAprep Spin Miniprep Kit (QIAGEN). These plasmids were transformed into the L2612 strain individually to yield the following strains: SL01 (contained the plasmid harboring the NCU00801 cellodextrin transporter gene and the NCU00130β-glucosidase gene from Neurospora crassa), SL02 (contained the plasmid harboring the NCU00809 cellodextrin transporter gene and the NCU00130β-glucosidase gene from Neurospora crassa), SL03 (contained the plasmid harboring the NCU08114 cellodextrin transporter gene and the NCU00130 β-glucosidase gene from Neurospora crassa), SL04 (contained the plasmid harboring the NCU00801 cellodextrin transporter gene and the BGL1 gene from Aspergillus aculeatus), SL05 (contained the plasmid harboring the NCU00809 cellodextrin transporter gene and the BGL1 gene from Aspergillus aculeatus), and SL06 (contained the plasmid harboring the NCU08114 cellodextrin transporter gene and the BGL1 gene from Aspergillus aculeatus). The empty pRS425 plasmid was transformed into the L2612 strain to yield the SL00 strain, which was used as a negative control. Yeast transformation was carried out using the standard lithium acetate method (Gietz et al., 1995). The resulting transformation mixtures were plated on SC-Ura-Leu medium supplemented with 2% D-glucose.
To confirm the proper construction of plasmids using the DNA assembler method, plasmids were isolated from yeast cells using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research, Orange, Calif.) and then transferred into E. coli DH5α cells. The resulting cells were spread on LB plates containing 100 mg/L ampicillin. Single E. coli colonies were inoculated into the LB liquid media. Plasmids were isolated from E. coli using the QIAprep Spin Miniprep Kit (QIAGEN, Valencia, Calif.) and checked by diagnostic PCR or restriction digestion using ClaI and HindIII. All restriction enzymes were obtained from New England Biolabs (Ipswich, Mass.). All chemicals were purchased from Sigma Aldrich or Fisher Scientific.
For each yeast strain, single colony was first grown up in 2 mL SC-Ura-Leu medium plus 2% glucose, and then inoculated into 50 mL of the same medium in a 250 mL shake flask to obtain enough cells for mixed sugar fermentation studies. After one day of growth, cells were spun down and inoculated into 50 mL of YPA medium supplemented with 4% cellobiose and 5% D-xylose, or 4% cellobiose, 5% xylose, and 0.5% glucose, or 4% cellobiose, 5% xylose, and 1% glucose in a 250 mL unbaffled shake-flask. Starting from an initial OD600 ̃1, cell culture was grown at 30° C. at 100 rpm for fermentation under oxygen limited condition. OD600 reading and cell culture sample were taken at various time points. Sugar concentrations were analyzed using HPLC, while ethanol formation was analyzed using the Ethanol Kit (R-biopharm, Darmstadt, Germany). For each data point, triplicate samples were taken. The mixed sugar fermentation results for the strains ranging from SL00 to SL06 are shown in FIGS. 65A-G. The best strain SL01 was selected for further characterization.
A total of six different strains, ranging from SL01 to SL06, were constructed by introducing a pRS425 plasmid harboring one of the cellodextrin transporter genes and one of the β-glucosidase genes into the L2612 strain. In each plasmid, the cellodextrin transporter gene and the β-glucosidase gene were added with a yeast promoter and terminator, respectively, and assembled into the pRS425 multi-copy plasmid by the DNA 10 assembler method (Shao et al., 2009) (FIG. 64). The empty pRS425 plasmid was introduced into the L2612 strain to yield the SL00 strain, which was used as a negative control. All strains were cultivated with a mixture of 40 g/L cellobiose and 50 g/L D-xylose in shake-flasks, and their sugar consumption rates, cell growth rates, and ethanol titers were determined (FIG. 65). Amongst all strains, the SL01 strain containing the β-glucosidase from Neurospora crassa and the cellodextrin transporter NCU00801 showed the highest sugar consumption rate and ethanol productivity. Thus, this strain was selected for further characterization.
Both SL01 and SL00 were cultivated using a mixture of 40 g/L cellobiose and 50 g/L D-xylose in both shake-flasks and bioreactors (FIGS. 66A-D). In the shake-flask cultivation (FIG. 66a-b), 83% cellobiose was consumed in 96 hours by SL01, with 41.2% higher average D-xylose consumption rate compared to SL100 (from 0.33 g/L/h to 0.46 g/L/h). Consistent with the enhanced sugar consumption rate, 1.32-fold increased average biomass growth rate was observed (from 0.031 g dry cell weight/L/h to 0.072 g dry cell weight/L/h). The ethanol productivity was increased by more than 2.1-fold, from 0.07 g/L/h to 0.23 g/L/h. The highest ethanol yield of 0.31 g per g sugar was reached in 48 hours, and the average ethanol yield was 0.28 g per g sugar, representing a 23% increase compared to the SL00 strain. In the SL01 cultivation, a faster D-xylose consumption rate was observed, without the lag phase that is the hallmark of glucose repression in co-fermentation of glucose and D-xylose. Moreover, enhanced biomass growth and ethanol production were also observed.
The Multifors system (Infors-HT, Bottmingen, Switzerland) was used for mixed sugar fermentation in bioreactors. Each vessel had a total capacity volume of 750 mL. For each vessel, there was one individual set of pO2 sensor, air sparger, exit gas cooler, temperature sensor, inoculation port, spare port, dip tube, antifoam sensor, pH sensor, drive shaft, heater block, rotameter, and peristaltic pumps system. The whole bioreactor system was equipped with a cooling system, ThermoFlex900 (Thermo Scientific, Waltham, Mass.).
Single colonies of yeast strains were first grown up in 2 mL SC-Ura-Leu medium plus 2% glucose, and then inoculated into 50 mL of the same medium in a 250 mL shake flask to obtain enough cells for mixed sugar fermentation studies. After one day of growth, 10 mL saturated culture were inoculated in 400 mL YPA medium supplemented with 4% cellobiose and 5% D-xylose, or 4% cellobiose, 5% xylose, and 0.5% glucose, or 4% cellobiose, 5% xylose, and 1% glucose. The temperature was maintained at 30° C. and the pH was maintained at 5.5, adjusted by addition of either 2 N H2SO4 or 4 N NaOH. In the first 48 hours, the air flow rate was maintained at 0.5 L/min, with the impeller speed at 250 rpm. Afterwards, the air flow rate was adjusted to 0.2 L/min to achieve high ethanol production under oxygen limited condition. Triplicate samples were taken at various time points and the OD600, sugar concentration, and ethanol concentration were determined as described above.
In the bioreactor cultivation (FIG. 66c-d), almost all cellobiose and 66% D-xylose were consumed in 48 hours, representing 44% increased D-xylose consumption rate (from 0.47 g/L/h to 0.68 g/L/h) and 1.1-fold increased biomass growth rate (from 0.08 g dry cell weight/L/h to 0.17 g dry cell weight/L/h). The ethanol productivity was increased by more than 4.3-fold (from 0.09 g/L/h to 0.50 g/L/h), and the ethanol yield was 0.39 g per g sugar. Compared to shake-flask cultivations, sugar consumption rates in the first 24 hours were lower, which was due to the low cell density used in the beginning of batch cultivation.
Unexpectedly, a small amount of glucose was detected even though there was no glucose added in fermentation (FIG. 66a-b). The maximum glucose concentration was reached in approximately 24 hours in both shake-flasks (12.1 g/L) and bioreactors (17.5 g/L) and then dropped to a very low level. However, no obvious glucose repression was observed even in the presence of such glucose. Because no glucose was detected in the SL00 strain, the extracellular glucose may result from the slow conversion of β-glucose to its epimer α-glucose, the main form of glucose used in glycolysis. Typically, β-glucose can be efficiently converted to α-glucose either enzymatically or chemically because of its relatively low concentration in glucose (Bouffard et al., 1994). However, in the engineered SL01 strain, catalyzed by β-glucosidase, an excess amount of β-glucose is produced from cellobiose intracellularly and a small fraction may be secreted outside cells, similar to what was observed with β-galactose (Bouffard et al., 1994).
Because a small amount of glucose (less than 10% of total sugars) is typically present in lignocellulosic hydrolysates in industrial settings, the fermentation performance of the engineered SL01 strain was also investigated using a mixture of cellobiose, D-xylose, and glucose. Two concentrations of glucose, 5 g/L or 10 g/L, were combined with 40 g/L cellobiose and 50 g/L D-xylose as mixed carbon source in bioreactors. With 5 g/L glucose (FIG. 67a-b), 81.7% cellobiose was consumed by SL01, with 67.8% D-xylose consumed at 48 hours in batch cultivation. The D-xylose consumption rate was increased by 1.19-fold, from 0.32 g/L/h to 0.69 g/L/h. The ethanol productivity was increased by 3.3-fold (from 0.11 g/L/h to 0.46 g/L/h) while the ethanol yield was increased from 0.26 g per g sugar to 0.33 g per g sugar. With 10 g/L glucose (FIG. 67c-d), 83.8% cellobiose was consumed by SL01, with 74.7% D-xylose consumed at 48 hour in batch cultivation. The D-xylose consumption rate was increased by 68%, from 0.45 g/L/h to 0.76 g/L/h. The ethanol productivity was increased by 2.1-fold (from 0.16 g/L/h to 0.50 g/L/h) and the ethanol yield was increased from 0.30 g per g sugar to 0.33 g per g sugar. As expected, the engineered SL01 strain showed both a higher efficiency of sugar consumption and a higher rate of ethanol production than the SL00 wild type strain. More importantly, there was no significant glucose repression in the co-fermentation of three sugars even with glucose up to 10% of total sugars (FIG. 67c-d) suggesting that this approach may be viable for industrial applications.
A similar study was carried out in the S. cerevisiae strain D452-2, where the three N. crassa cellodextrin transporters NCU00801, NCU08114, and NCU00809 were introduced together with the β-glucosidase NCU00130. The transformants were selected on YSC medium containing 20 g/liter cellobiose expressing an intracellular β-glucosidase (NCU00130). Strains and plasmids used in this work are described in Table 17 (Ex. 12). The primers used are listed in Table 28.







TABLE 28







shows the synthetic


oligonucleotides used in the study.








Name
Sequences





NCU00801-F
ATGGATCCAAAAATGTCGTCTCACGGCTCC



SEQ ID NO: 92


 


NCU00801-R
ATGAATTCCTACAAATCTTCTTCAGAAATCAATTTTTGT



TCAGCAACGATAGCTTCGGAC



SEQ ID NO: 93


 


NCU08114-F 
ATACTAGTAAAAATGGGCATCTTCAACAAGAAGC



SEQ ID NO: 94


 


NCU08114-R
GCATATCGATCTACAAATCTTCTTCAGAAATCAATTTTT



GTTCAGCAACAGACTTGCCCTCATG



SEQ ID NO: 95


 


NCU00130-F
GCATACTAGTAAAAATGTCTCTTCCTAAGGATTTCCTCT



SEQ ID NO: 96


 


NCU00130-R
ATACTGCAGTTAATGATGATGATGATGATGGTCCTTCTT



GATCAAAGAGTCA AAG



SEQ ID NO: 97









Yeast were grown in YP medium containing 20 g/L of glucose or 20 g/L of cellobiose to prepare inoculums for xylose or cellobiose fermentation experiments, respectively. Cells at mid-exponential phase from YP media containing 20 g/L of glucose or cellobiose were harvested and inoculated after washing twice with sterilized water. All of the flask fermentation experiments were performed using 50 mL of YP medium containing 40 g/L or 80 g/L of xylose in 250 mL flask at 30° C. with initial OD600 of 1.0 under oxygen limited conditions. Bioreactor fermentations were performed in 400 mL of YP medium containing appropriate amounts of sugars using Sixfors Bioreactors (Appropriate Technical Resources, Inc) at 30° C. with an agitation speed of 200 rpm under oxygen limited 250 conditions. Initial cell densities were adjusted to OD600=1.0.
Cell growth was monitored by optical density (OD) at 600 nm using UV-visible Spectrophotometer (Biomate 5, Thermo, N.Y.). Glucose, xylose, xylitol, glycerol, acetate, and ethanol concentrations were determined by high performance liquid chromatography 264 (HPLC, Agilent Technologies 1200 Series) equipped with a refractive index detector using 265 a Rezex ROA-Organic Acid H+(8%) column (Phenomenex Inc., Torrance, Calif.). The column was eluted with 0.005 N of H2SO4 at a flow rate of 0.6 mL/min at 50° C.
All three transformants were able to grow and produce ethanol when cellobiose was the sole carbon source (FIGS. 68A-C), but the three transformants exhibited different cellobiose fermentation rates (NCU00801>NCU08114>NCU00809). The fastest cellulose-fermenting transformant (D801-130), expressing both NCU00801 and NCU00130, consumed 40 g/L of cellobiose within 4 hours, producing 16.8 g/L of ethanol. The volumetric productivity of cellobiose fermentation (PEthanol/Cellobiose=0.7 g/L/h) was lower than that of glucose fermentation (PEthanol/Glucose=1.2 g/L/h), and ethanol yield from cellobiose (YEthanol/Cellobiose=0.42 g/g) was about the same as ethanol yield from glucose (YEthanol/Glucose=0.43 g/g) under the same culture conditions. However, the observed cellobiose consumption rate and ethanol yield by D801-130 were an improvement over S. cerevisiae strains engineered to ferment cellobiose through surface display of β-glucosidase (Kotaka et al., 2008; Nakamura et al., 2008). These results suggest that simultaneous expression of NCU00801 and NCU00130 in S. cerevisiae can result in efficient cellobiose fermentation.
After developing the efficient xylose fermenting strain DA24-16 (described in Example 13), genes coding cellodextrin transporter and β-glucosidase (NCU00801 and NCU00130) enzyme were introduced into the strain enabling it to consume cellobiose and xylose simultaneously. It was hypothesized that glucose repression of xylose utilization may be alleviated in this strain, due to the intracellular hydrolysis of cellobiose. The NCU00801 gene was integrated into the genome of DA24-16, and NCU00130 was expressed from a multi-copy plasmid. The resulting transformant, DA24-16-BT3, was selected on an agar plate containing cellobiose as the sole carbon source.
The DA24-16-BT3 strain grown in media containing various amounts of cellobiose and xylose co-consumed cellobiose and xylose, and produced ethanol with yields of 0.38-0.39 g/g in all conditions tested (FIGS. 69A-C). The potential synergistic effects of co-fermentation were tested by culturing DA2416-BT3 under three different conditions: 40 g/L of cellobiose, 40 g/L of xylose, and 40 g/L of both sugars (total 80 g/L of sugars). Surprisingly, DA24-16BT3 was able to co-consume 80 g/L of a cellobiose/xylose mixture within the same period that was required to consume 40 g/L of cellobiose or 40 g/L xylose separately (FIGS. 70A-C). Moreover, DA24-16BT3 produced ethanol with a higher yield (0.39 g/g) from a mixture of cellobiose and xylose as compared to ethanol yields (0.31 ̃0.33 g/g) from single sugar fermentations (cellobiose or xylose). Ethanol productivity also drastically increased from 0.27 g/L/h to 0.65 g/L/h during co-fermentation. These results demonstrated that co-fermentation of cellobiose and xylose can enhance overall ethanol yield and productivity. Fermentation experiments were also done to compare this engineered S. cerevisiae strain (DA24-16BT3) to P. stipitis, which is capable of co-fermenting cellobiose and xylose efficiently.
A simulated hydrolysate (10 g/L of glucose, 80 g/L of cellobiose, 40 g/L of xylose) based on the composition of energycane was used. The composition of different lignocellulosic plants varies in a broad range. For instance, the US Department of Energy biomass database lists the composition of more than 150 biomass samples (webpage eere.energy.gov/biomass/m/feedstock_databases.html). The cellulose-to-hemicellulose ratios of these samples are between 1.4 and 19, and the average is 2.3. Energy crops typically have higher hemicellulose content than woody biomass. The average cellulose to hemicellulose ratios of sugarcane bagasse, corn stover, sorghum are 2.0, 1.85 and 2.14, respectively. We therefore used a glucan/xylan ratio of 2 in our simulated sugar experiment design. The engineered yeast will likely be used in conjunction with traditional cellulase cocktails that are deficient in β-glucosidase activities for the biofuels production. The biomass hydrolysis process may result in small amounts of glucose in the lignocellulosic hydrolysates as 6-30% glucan-to-glucose conversions with incomplete cellulase cocktails were reported (Medve et al., 1998). Considering all the above factors, a sugar combination of 10 g/L glucose, 80 g/L cellobiose, and 40 g/L xylose was chosen in the simulated sugar experiments.
The DA24-16BT3 consumed glucose first before co-consuming cellobiose and xylose rapidly. A total of 130 g/L of sugars was consumed within 60 hours even though small inoculums were used (OD600=1). In contrast, P. stipitis could not finish fermenting the sugar mixture within the same period under identical culture conditions (FIGS. 71A-B). DA24-16BT3 produced 48 g/L of ethanol within 60 hours (YEthanol/Sugars=0.37 g/g and PEthanol/Sugars=0.79 g/L/h).
A transient accumulation of cellodextrins in the medium during cellobiose consumption was observed (FIG. 72-73). The accumulated cellotriose and cellotetraose were again consumed after depletion of cellobiose. It is likely that the accumulated cellodextrins were generated by the trans-glycosylation activity (Christakopoulos et al., 1994) of β-glucosidase (NCU00130), and secreted by the cellodextrin transporter (NCU00801), which might facilitate the transport of cellodextrins in both directions (intracellularextracellular). This transient cellodextrin accumulation would probably not reduce product yields since the accumulated cellodextrins would eventually be consumed by the engineered yeast. However, it might decrease productivity because the transport rates of cellotriose and cellotetraose might be slower than that of cellobiose.
Small amounts of glucose were constantly detected in the medium during co-fermentation. Since even low amounts of glucose accumulation can repress xylose fermentation, glucose levels have to be kept at a minimum. It can be hypothesized that the relative expression levels of the cellodextrin transporter and β-glucosidase are likely to affect glucose accumulation. In support of this, it was observed that more glucose was accumulated in the medium when NCU00801 was introduced on a multi-copy plasmid than when NCU00801 was integrated into the yeast genome. The strain (DA24-16-BT). containing both NCU00801 and NCU00130 on multi-copy plasmids, had relatively slower xylose utilization rates than those observed in DA24-16-BT3, a potential reason being glucose repression (FIGS. 74A-B). Further adjustments of the cellodextrin transporter and β-glucosidase expression levels, or the identification of β-glucosidases with reduced trans-glycosylation activities, may be able to reduce the accumulation of glucose and cellodextrin during co-fermentation.
Co-fermentation of xylose and cellobiose could also be achieved by mixed cultivation of two different yeast strains: the xylose-fermenting DA24-16 strain and the cellobiose-fermenting DA452BT (FIG. 75). As explained above, the yeast strain DA24-16 expressed the xylose-utilizing enzymes wild type xylose reductase (XYL1), mutant xylose reductase R276H (mXYL1), xylitol dehydrogenase (XYL2), and xylulokinase (XKS1) (Ex. 12; Table 17). D452BT was formed by engineering D452 to express the cellodextrin transporter NCU00801 and the β-glucosidase NCU00130. In the mixed culture, the DA24-16 strain took up xylose (xylose molecule shown as a green pentagon in FIG. 75a) and metabolized it using the enzymes XYL1 (wild type and mutant), XYL2, and XYL3, whereas the other strain D452BT was able to take up cellobiose (cellobiose molecule shown as two red hexagons in FIG. 75a) using the transporter NCU00801 and convert the cellobiose into glucose using the enzyme NCU00130. Hence, the mixed culture was able to co-ferment both xylose and cellobiose to produce ethanol (FIG. 75b).
This study demonstrated a novel strategy to allow co-fermentation of hexose and pentose sugars by S. cerevisiae. By combining an efficient xylose utilization pathway with a cellodextrin transport system, the problem caused by glucose repression was over-come. As a result, the engineered yeast co-fermented two non-metabolizable sugars in cellulosic hydrolysates synergistically into ethanol. The new co-fermentation method described herein advances lignocellulosic technologies on both the saccharification and fermentation fronts. Most traditional fungal cellulase cocktails are deficient in β-glucosidase and end the cellulose hydrolysis with cellobiose that is not fermented efficiently by yeast. As a result, extra β-glucosidase enzyme must be added to convert cellobiose into glucose. The cellobiose/xylose co-fermentation yeast makes it possible to use these cellulase cocktails with limited β-glucosidase activities, lowering enzyme usage and cost associated with the cellulose saccharification process. Further, the synergy between cellobiose and xylose co-fermentation significantly increases ethanol productivity, thus improving fermentation economics. The presence of a small amount of glucose from the pre-treatment and hydrolysis of lignocellulosic materials does not affect the capacity of the engineered yeast to convert hexose and pentose sugar mixtures into ethanol.
This study involved measuring the capacity of an engineered S. cerevisiae strain to ferment various mixtures of sugars meant to mimic hydrolysates from plant biomass. The ability of this strain to co-ferment cellodextrins and xylose is particularly useful during the simultaneous saccharification and co-fermentation (SSCF) of pre-treated plant biomass. During SSCF, hemicellulose would first be hydrolyzed by acid pre-treatment, resulting in formation of xylose and still-crystalline cellulose. Then, fungal cellulases and the yeast strain described herein would be added, allowing the cellulases to co-convert xylose and cellobiose into ethanol. Because of the limited extracellular glucose production in this scheme, there will be reduced repression of xylose utilization and co-fermentation will proceed rapidly and synergistically.
Although the S. cerevisiae strain used in this study was a laboratory strain, the fermentation performance of the engineered strain was very impressive when compared to published results. The key fermentation parameters (yield and productivity) may be further improved by the use of industrial yeast strains as a platform. Applications of this co-fermentation strategy would not be limited to ethanol production. Since it is a foundational technology, the strategy presented here can be combined with any other product diversification technologies to produce commodity chemicals and advanced biofuels.
Example 18
Transcriptome Analysis of N. crassa Grown on Xylan
Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin. Examples 1-3 describe the discovery of genes critical for growth on cellulose through transcriptome and secretome analysis of N. crassa. In this example the expression profile of the N. crassa genome was examined during growth on xylan to determine which genes are important for utilization of hemicellulose.
Ten day old conidia of WT or ΔxlnR strains were inoculated at 106 conidia/mL on 100 mL 1× Vogel's salts minimal medium (2% sucrose), grown for 16 hours at 25° C. with constant light, and washed with 1× Vogel's only medium. Conidia were then transferred into 100 mL 1× Vogel's salts with 2% sucrose or 2% Beechwood xylan as the sole carbon source in the medium and allowed to grow for 4 hours. Mycelia were harvested by filtration and immediately flash frozen in liquid nitrogen. Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions and treated with DNase (Turbo DNA-free kit; Ambion) (Kasuga, Townsend et al., 2005).
For cDNA synthesis and labeling, the Pronto kit (Catalog No. 40076; Corning) was used according to the manufacturer's specifications except that the total RNA used was 10 μg per sample.
Microarray hybridization and data analysis were performed as previously described (Tian, Kasuga et al., 2007). A GenePix 4000B scanner (Axon Instruments) was used to acquire images, and GenePix Pro6 software was used to quantify hybridization signals and collect the raw data. Normalized expression values were analyzed by using the BAGEL (Bayesian analysis of gene expression levels) software program (Townsend and Hartl 2002; Townsend 2004). 354 genes were found to be induced greater than 2-fold in N. crassa grown on xylan. The list is shown in FIG. 76.
Example 19
Secretome Analysis of N. crassa Grown on Xylan
The secretome of N. crassa during growth on xylan was analyzed using a shotgun proteomics approach. Supernatants from xylan cultures were digested with trypsin and analyzed by liquid chromatography nano-electrospray ionization tandem mass spectrometry.
Mass spectrometry samples were prepared as follows. N. crassa wild type strain was grown on 2% xylan media for 4 or 7 days. Culture supernatants were isolated by centrifugation, filtered through 0.22 μm filters, and concentrated 10 times with 10 kDa MWCO PES spin concentrators. 3.36 mg of urea, 5 μL of 1M Tris pH 8.5, and 5 μL of 100 mM DTT were then added to 100 μL of concentrated culture supernatant, and the mixture was heated at 60° C. for 1 hour. After heating, 700 μL of 25 mM ammonium bicarbonate and 140 μL of methanol were added to the solution followed by treatment with 50 μL of 100 μg/mL trypsin in 50 mM sodium acetate pH 5.0. The trypsin was left to react overnight at 37° C. with inverting for about 8-9 hours at basal pH. After digestion the volume was reduced to dryness by speedvac and washed with 300 μl MilliQ water three times. The final volume was 100 μl. TFA was added at 0.1-0.3% v/v. Residual salts in the sample were removed by using OMIX microextraction pipette tips according to the manufacturer's instructions. The acetonitrile was removed by evaporation. The sample solution was an aqueous solution with 0.1%-1% TFA, and the final volume was 10 microliters or greater.
Example 20
Analysis of Xylan-Induced Genes Predicted to Encode Secreted Proteins
The transcriptome and secretome analysis results indicated a total of 71 genes, of which 55 were predicted to be secreted. The list of these genes is in Table 29. Deletion strains were available for 46 out of 69 genes. Out of these 46, six of the strains were heterokaryons, thus the remaining 40 deletion strains were analyzed for total secreted protein, amount of xylose present, and azo-endo-xylanase activity. Results are shown in FIG. 77.







TABLE 29







shows xylan-induced N. crassa genes










Gene Name
Signal P
Data
Annotation





NCU00642
Y
Transcription
probable beta-galactosidase


NCU00695
Y
Transcription
putative protein


NCU00798

MS
hypothetical protein


NCU00937
Y
Transcription
conserved hypothetical protein


NCU01517
Y
Transcription
glucan 1,4-alpha-glucosidase


NCU02136

MS
probable transaldolase


NCU02252

MS
probable phosphoglyceromutase


NCU02343
Y
Transcription
related to alpha-L- 





arabinofuranosidase A precursor


NCU02455
Y
Transcription
FK506-binding protein 2 precursor





(Peptidyl-prolyl cis-trans isomerase)


NCU02583
Y
Transcription
probable Alpha-glucosidase 





precursor (Maltase)


NCU03013
Y
Transcription
related to cytosolic Cu/Zn 





superoxide dismutase


NCU03222
Y
Transcription
putative protein


NCU03636
Y
Transcription



NCU03639
Y
Transcription
probable triacylglycerol lipase 





precursor


NCU04202

MS
nucleoside-diphosphate kinase


NCU04265
Y
Transcription
related to beta-fructofuranosidase


NCU04388
Y
Transcription
probable phosphatidylglycerol/





phosphatidylinositol transfer





protein


NCU04395

MS
beta-1,6-glucanase Neg1 NEG-1


NCU04415
Y
Transcription
related to brefeldin A resistance 





protein


NCU04431
Y
MS
related to endo-1,3-beta-glucanase


NCU04475
Y
Transcription
probable lipase B precursor


NCU04482

MS
hypothetical protein


NCU04623
Y
Transcription
related to beta-galactosidase


NCU04674
Y
Transcription
related to alpha-glucosidase b


NCU04675
Y
Transcription
putative protein


NCU04930
Y
Transcription
related to triacylglycerol lipase


NCU05137
Y
Transcription
conserved hypothetical protein


NCU05143
Y
Transcription
related to Rds1 protein


NCU05159
Y
Transcription
probable acetylxylan esterase 





precursor


NCU05275

MS
probable ubiquitin fusion protein





(ubiquitin/ribosomal protein)


NCU05315
Y
Transcription
hypothetical protein


NCU05395
Y
Transcription
conserved hypothetical protein


NCU05686
Y
MS
probable cell wall protein UTR2


NCU05751
Y
Transcription
related to acetylxylan esterase


NCU05924
Y
Transcription 
probable endo-beta-1,4-D-xylanase


NCU05965
Y
Transcription
related to putative arabinase


NCU05974

MS
related to cell wall protein 





(putative glycosidase)


NCU06364
Y
Transcription
hypothetical protein


NCU06380
Y
Transcription 
related to catecholamines up protein


NCU06650
Y
Transcription
conserved hypothetical protein


NCU06781

MS
probable beta (1-3) 





glucanosyltransferase


NCU06961
Y
Transcription
probable exopolygalacturonase


NCU07067

MS
related to class I alpha-mannosidase 





1B


NCU07143
Y
Transcription



NCU07190
Y
Transcription
related to cellulose 





1,4 betacellobiosidase II precursor


NCU07200
Y
MS
related to metalloprotease MEP1


NCU07225
Y
Transcription
probable endo-1,4-beta-xylanase B





precursor


NCU07281

MS
probable glucose-6-phosphate 





isomerase


NCU07787
Y
MS
probable SnodProt1 precursor


NCU08131
Y
Transcription
probable alpha-amylase precursor


NCU08171
Y
MS
conserved hypothetical protein


NCU08189
Y
Transcription
related to endo-1,4-beta-xylanase


NCU08384

MS
probable D-xylose reductase


NCU08418

MS
related to tripeptidyl-peptidase I


NCU08457
Y
Transcription
hydrophobin Ccg-2 CCG-2


NCU08516
Y
Transcription
related to aldose 1-epimerase


NCU08750
Y
Transcription
related to isoamyl alcohol oxidase


NCU08752
Y
Transcription
related to esterase


NCU08755
Y
Transcription
hypothetical protein


NCU08909
Y
MS
probable beta (1-3) 





glucanosyltransferase gel3p


NCU08936

MS
related to sporulation-specific 





gene SPS2


NCU09024
Y
MS
related to choline dehydrogenase


NCU09133
Y
Transcription
putative protein


NCU09170
Y
MS
probable alpha-N-





arabinofuranosidase


NCU09175
Y
Transcription
related to glucan 1,3-beta-





glucosidase precursor


NCU09267

MS
related to glyoxal oxidase precursor


NCU09491

MS
feruloyl esterase B precursor 





(subclass of the carboxylic acid





esterases)


NCU09923
Y
Transcription
related to xylan 1,4-beta-xylosidase


NCU09924
Y
Transcription 
conserved hypothetical protein


NCU10040
Y
Transcription



NCU10045
Y
Transcription









Samples were prepared as follows. 10 day old conidia were grown in 100 mL 2% xylan Vogel's media at 106 conidia/mL. Two replicates were prepared for each strain. Cultures were grown at 25° C. with constant light and 220 rpm. Samples were harvested on day 4. Supernatants were isolated by centrifugation and used in assays.
Bradford protein concentrations were measured to determine the total amount of secreted protein. Stocks were prepared with BSA standards: 0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL. Bradford solution was diluted 1:4. A multichannel pipette was used to pipette 200 μL of Bradford solution into a 96-well plate. 10 μL of sample and 10 μL of each standard were added. Samples were incubated at room temperature for 10 minutes. The absorbance was read at 595 nm, and the protein concentration was determined.
The assay used to measure xylose was modified from Bailey et al., 1992 (J Biotech 23: 257-270). Xylose standards were prepared in H2O. For concentrated 0.8 M xylose (1.2 g in 10 mL), the standards included 0 mM, 8 mM (1:100 dilution; 990 μl+10 μl), 20 mM (1:100 dilution; 975 μl+25 μl), 40 mM (1:100 dilution; 950 μl+50 μl), 80 mM (1:100 dilution; 900 μl+100 μl), and 160 mM (1:100 dilution; 800 μl+200 μl). A multichannel pipette was used to add 900 μL of substrate solution to a deep well 96-well plate. The substrate was allowed to incubate at 50° C. for 10 minutes. One hundred μL of culture supernatant and the standards were added and allowed to incubate at 50° C. for 5 minutes. Samples were centrifuged for 10 minutes at 3,400 rpm. A multichannel pipette was used to pipette 75 μL DNS solution into a 96-well PCR plate. Five μL of solution was removed from the reaction and added to the PCR plate containing DNS solution. The plate was heated at 99° C. in the PCR machine for 5 min. After the samples cooled, they were transferred to clear flat-bottomed plates, and the absorbance was read at 540 nm. Substrate solution (500 mL) contained beechwood xylan (5 g; 10 mg/mL), 3M NaOAc, pH 5.0 (8.33 mL; 50 mM), water (491 mL), and was autoclaved for 20 minutes. DNS solution (100 mL) contained 3,5-dinitrosalicylic acid (707 mg), NaOH (1.32 g), Rochelle salts (Na K tartrate) (20.4 g), Sodium meta-bisulfate (553 mg), phenol (507 μL), and water (94.4 mL).
Azo-endo-xylanase activity was measured with a kit from Megazyme. This assay indirectly measures the amount of endo-xylanase activity in a sample by spectrophotometrically measuring the amount of dye liberated from a xylan chain complexed with the dye. The more enzymes that are present, the more dye will be released. All supernatant samples were diluted 1:10 by adding 50 μL of supernatant to 450 μL of Na Acetate buffer (50 mM, pH 4.5) in separate 15 mL Falcon tubes. Next, Falcon tubes were pre-warmed about 10 minutes. Substrate solution was added for all samples (500 μL/sample) to the tubes. Samples and substrate solutions were added into a 40° C. water bath for 10 minutes to pre-equilibrate them. Five hundred μL substrate solution was added to each 1:10 diluted sample, vortexed for 10 seconds, and incubated at 40° C. for 10 minutes. The reaction was terminated by adding 2.5 mL of precipitant solution (95% ethanol) to each sample and vortexing for 10 seconds. Tubes were allowed to stand at room temperature for 10 minutes. Tubes were vortexed for 10 seconds and then centrifuged at room temperature for 10 minutes at 1,000 g. One mL of supernatant solution from each tube was placed directly into a cuvette, and the absorbance was measured at 590 nm. The blank used for this procedure was the supernatant from 500 μL substrate solution added to 2.5 mL of precipitant solution.
In conclusion, it is anticipated that the modulation of genes identified here that affect the degradation of hemicellulose in N. crassa will facilitate engineering strains that have enhanced capacity for plant cell wall breakdown and growth on plant cell wall components such as hemicellulose. Genes of interest include NCU01517, which encodes a predicted glucamylase; NCU02343, which encodes a predicted arabinofuranosidase; NCU05137, which encodes a conserved hypothetical protein; NCU05159, which encodes a predicted acetylxylan esterase precursor; NCU09133, which encodes a conserved hypothetical protein; and NCU10040, which encodes a hypothetical protein.
The growth of a cell on hemicellulose will be increased by providing a host cell that contains a recombinant polynucleotide that encodes a polypeptide encoded by NCU01517, NCU09133, or NCU10040. The host cell will be cultured in a medium that contains hemicellulose such that the recombinant polynucleotide is expressed. The host cell will grow at a faster rate in this medium than a cell that does not contain the recombinant polynucleotide.
Example 21
Further Analysis of the ΔNCU05137 Strain
As described in Examples 1-3 and 18-20, NCU05137 is a predicted secreted protein that was overexpressed during growth of N. crassa on any of Miscanthus, Avicel, or xylan. A deletion strain of N. crassa lacking NCU05137 grown on Avicel showed increased endoglucanase, β-glucosidase, and Avicelase activity. An NCU05137 deletion strain grown on xylan showed increased azo-endo-xylanase activity. As described in this example, the complementation of ΔNCU05137 was performed in order to verify that the phenotypes observed in the ΔNCU05137 strain were due to the loss of the NCU05137 gene.
A plasmid containing NCU05137 with a C-terminal GFP tag under the control of the ccg1 promoter was generated. N. crassa conidia were transformed with the NCU05137-GFP construct. Experiments were performed according to standard Neurospora procedures (webpage fgsc.net/Neurospora/NeurosporaProtocolGuide.htm).
The total secreted protein and carboxymethyl cellulase (CMC) activity of wild-type, ΔNCU05137, and ΔNCU05137-NCU05137-GFP strains was measured. Total secreted protein was measured by taking 100 μL of supernatant from a culture of each strain, adding it to 900 μL Bradford Dye, and measuring absorbance at 595 nm. CMC activity was measured with 20× diluted supernatant from each strain culture and an azo-CMC kit (Megazyme SCMCL). ΔNCU05137 knockout strains displayed increased levels of secreted protein and CMC activity. Introduction of the GFP-tagged NCU05137 into ΔNCU05137 strains reduced these levels back to wild-type levels (FIG. 78).
In addition, the localization of NCU05137-GFP in complemented strains was observed. NCU05137-GFP localized to the cell wall of conidia and to the hypha tip (FIG. 79-80). These data indicate that the GFP-tagged NCU05137 protein is fully functional and can be used for purification and experiments addressing the biochemical activity of this protein.
Thus, the normal function of NCU05137 may be to inhibit signaling processes associated with induction of cellulase and hemicellulase gene expression. Reduction of expression of NCU05137 or a homolog of NCU05137 in a cell is likely to increase cellulase and hemicellulase activity in that cell and, consequently, growth of the cell on cellulose or hemicellulose. The growth of a cell on cellulose or hemicellulose will be increased by providing a host cell that contains an endogenous polynucleotide that encodes a polypeptide encoded by NCU05137. The expression of the endogenous polynucleotide will be inhibited, and the cell will be cultured in a medium containing cellulose and/or hemicellulose. The host cell will grow at a faster rate in the medium than a cell in which expression of the endogenous polynucleotide is not inhibited.
Example 22
Further Analysis of NCU07705
Expression of NCU07705 was found to be upregulated during growth of N. crassa on cellulose. BLAST analysis of the polypeptide encoded by NCU07705 revealed that the polypeptide has high similarity to many C6 zinc finger domain containing transcription factors (FIG. 1). To further investigate the role of NCU07705 in the utilization of cellulose, the phenotype of a deletion strain lacking NCU07705 was evaluated.
The ΔNCU07705 strain was unable to grow on 2% cellulose (Avicel), PASC, or CMC as a sole carbon source (Table 30) but grew with similar kinetics to wild-type strain on sucrose, xylan, and xylose. In order to determine whether NCU07705 plays a role in regulating expression of cellulases, the expression of cellulase and hemicellulase genes was examined during growth of ΔNCU07705 on cellulose. Ten-day-old conidia from wild-type (FGSC 2489) and ΔNCU07705 strains were inoculated into Vogel's liquid MM (2% sucrose) (Vogel 1956) and grown for 16 hours. Mycelia were centrifuged, washed with 1× Vogel's salts, and then transferred into either Vogel's media with 2% sucrose or 2% Avicel and grown in constant light for 4 hours. They were harvested by filtration and immediately frozen in liquid nitrogen. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions and treated with DNase (Turbo DNA-free kit, Ambion/Applied Biosystems, Foster City, Calif.) (Kasuga et al., 2005). ChipShotTM Indirect Labeling/Clean-Up System (Catalog No. Z4000, Promega, Madison, Wis.) and CyDye Post-Labeling Reactive Dye Pack (Catalog No. RPN5661, GE Healthcare, Piscataway, N.J.) were used to synthesize and label cDNA according to the manufacturer's instructions except the amount of RNA used was 10 μg. The Pronto! Hybridization Kit (Catalog No. 40076, Corning, Lowell, Mass.) was used for microarray hybridization according to the manufacturer's specifications.
Data analyses were performed as previously described (Tian et al., 2007). A GenePix 4000B scanner (Axon Instruments, Union City, Calif.) was used to acquire images, and GenePix Pro6 software was used to quantify hybridization signals and collect the raw data. Normalized expression values were analyzed by using BAGEL (Bayesian Analysis of Gene Expression Levels) (Townsend and Hartl, 2002). None of the predicted cellulase genes were induced in the ΔNCU07705 strain, whereas induction of predicted hemicellulase genes was unaffected (see Table 30 below). Thus, NCU07705 has been named cdr-1, cellulose degradation regulator 1.
Therefore, the growth of a cell on cellulose will be increased by providing a host cell that contains a recombinant polynucleotide that encodes a polypeptide encoded by NCU07705. The host cell will be cultured in a medium that contains cellulose such that the recombinant polynucleotide is expressed. The host cell will grow at a faster rate in this medium than a cell that does not contain the recombinant polynucleotide.







TABLE 30







shows expression profile of genes in N. crassa ANCU07705 strain












7705-switch2
WT-switch1 
Gene/locus name 
GH Family
Class
up in Avi3















No
15
NCU00762
5
endo-
31.5


No
No
NCU03996
6
CBHII like



No
168
NCU07190
6
CBHII like
119


No
26
NCU09680
6
CBHII
251.3


No
18
NCU04854
7
CBHI like
10.8


No
3.8
NCU05057
7
CBHI like
7.4


No
No
NCU05104
7
CBHI like



No
93
NCU07340
7
CBHI
382.2


No
2
NCU05121
45
endo-
17.2


No
5.8
NCU00836
61
endo-
31


No
3.7
NCU01050
61
endo-
382.1


No
No
NCU01867
61
endo-



No
49
NCU02240
61
endo-
84


No
No
NCU02344
61
endo-
4.1


No
6.1
NCU02916
61
endo-
17.7


No
No
NCU03000
61
endo-



No
17
NCU03328
61
endo-
23.8


No
No
NCU05969
61
endo-
12.7


No
No
NCU07520
61
endo-



No
No
NCU07760
61
endo-



No
103
NCU07898
61
endo-
230


No
No
NCU07974
61
endo-



No
25
NCU08760
61
endo-
44.7





1Expression levels of predicted cellulase genes from an N. crassa (NCU07705) culture grown in Vogel’s/sucrose for 16 hours, filtered, and resuspended in Vogel’s/Avicel for 4 hours prior to RAN extraction.


2Expression levels of predicted cellulase gene from an N. crassa (wild type FGSC 2489) culture grown in Vogel’s/sucrose for 16 hours, filtered, and resuspended in Vogel’s/sucrose for 4 hours prior to RNA extraction.


3Expression levels derived from microarray analyses of wild type (FGSC 2489) cells grown for 30 hours in Avicel (Tian et al., 2009).






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## Claims

1. A method of increasing transport of cellodextrin into a cell, comprising:
culturing a host cell which comprises a recombinant polynucleotide encoding a cellodextrin transporter polypeptide in a medium such that the recombinant polynucleotide is expressed, said cellodextrin transporter having the structure of a Major Facilitator Superfamily protein and comprising transmembrane α-helix 1, α-helix 2, α-helix 3, α-helix 4, α-helix 5, α-helix 6, α-helix 7, α-helix 8, α-helix 9, α-helix 10, α-helix 11, α-helix 12, said transmembrane α-helix 5 characterized by:
an arginine at the position corresponding to amino acid 1 of SEQ ID NO:4;
a tyrosine or phenylalanine at a position corresponding to amino acid 7 of SEQ ID NO:4; and
an asparagine at a position corresponding to amino acid 8 of SEQ ID NO:4;

wherein expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared with a cell that does not comprise the recombinant polynucleotide.

2. The method of claim 1 wherein the polypeptide comprises an amino acid sequence having at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to NCU00801 or NCU08114.

3. The method of claim 1 wherein the host cell further comprises a second recombinant polynucleotide encoding at least a catalytic domain of a β-glucosidase.

4. The method of claim 3 wherein the β-glucosidase is from Neurospora crassa.

5. The method of claim 4 wherein the β-glucosidase is encoded by NCU00130.

6. The method of claim 1 wherein the host cell further comprises one or more recombinant polynucleotides wherein the one or more polynucleotides encode one or more enzymes selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase.

7. The method of claim 1, wherein the host cell further comprises a second recombinant polynucleotide wherein the second recombinant polynucleotide encodes a pentose transporter.

8. The method of claim 7, wherein the pentose transporter is selected from the group consisting of NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3.

9. The method of claim 1 wherein the medium comprises a cellulase-containing enzyme mixture from an altered organism, wherein the cellulase-containing mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism.

10. The method of claim 1, wherein the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, and Bacillus subtilis.

11. The method of claim 1 wherein the cellodextrin is selected from one or more of the group consisting of cellobiose, cellotriose, and cellotetraose.

12. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 85% amino acid identity to NCU00801.

13. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 85% amino acid identity to NCU08114.

14. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 90% amino acid identity to NCU00801.

15. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 90% amino acid identity to NCU08114.

16. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 95% amino acid identity to NCU00801.

17. The method of claim 2, wherein the polypeptide comprises an amino acid sequence having at least 95% amino acid identity to NCU08114.