# Patent

## Title
Increased ethanol production by genetic engineering of microorganisms to express hemoglobin

## Abstract

The present disclosure describes novel bacterial strains which express a pyruvate decarboxylase gene and at least one alcohol dehydrogenase gene from a bacteria of the genus Zymomonas and also express a hemoglobin gene from a bacteria of the genus Vitreoscilla. The present disclosure further describes methods for producing fermentation products with a microorganism which expresses a pyruvate decarboxylase gene and at least one alcohol dehydrogenase gene from a bacteria of the genus Zymomonas and also express a hemoglobin gene from a bacteria of the genus Vitreoscilla. Further the present disclosure describes methods for increasing production of a fermentation product comprising genetically engineering a microorganism which expresses a xylose isomerase gene to also express a hemoglobin gene from a bacteria of the genus Vitreoscilla.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part application of U.S. application Ser. No. 14/340,129, filed on Jul. 24, 2014, which issued as U.S. Pat. No. 9,045,779 on Jun. 2, 2015, entitled “Increased Ethanol Production by Genetic Engineering of Microorganisms to Express Hemoglobin,” which is a divisional application of U.S. application Ser. No. 13/098,189, filed on Apr. 29, 2011, which issued as U.S. Pat. No. 8,790,911, on Jul. 29, 2014, entitled “Increased Ethanol Production by Genetic Engineering of Microorganisms to Express Hemoglobin,” which claims the benefit of U.S. Provisional Application Ser. No. 61/329,796, filed on Apr. 30, 2010, entitled “Increased Ethanol Production by Genetic Engineering of Bacteria to Express Hemoglobin,” each of which are incorporated herein by reference in their entirety.


SEQUENCE LISTING
Incorporated by reference herein in its entirety is the Sequence Listing entitled “Sequence-Listing.txt”, created Aug. 8, 2013, size of 28 kilobytes.
FIELD OF THE INVENTION
The invention relates to the fields of microbiology and genetic engineering. More specifically, the present disclosure describes that expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla was found to increase production of fermentation products in microorganisms grown under substantially anaerobic conditions.
BACKGROUND
The following description provides a summary of information relevant to the present disclosure and is not a concession that any of the information provided or publications referenced herein is prior art to the claimed invention.
Production of fermentation products, including ethanol, by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research.
The pyruvate decarboxylase and alcohol dehydrogenase enzymes of Zymomonas mobilis provide a more efficient biochemical pathway for the production of ethanol in comparison to for example the pyruvate formate lyase pathway of E. coli (FIG. 14). Dien, B. S., et al. “Bacteria engineered for fuel ethanol production: current status.” Applied Microbiology and Biotechnology 63 (2003): 258-266. The efficiency of the pyruvate decarboxylase and alcohol dehydrogenase II biochemical pathway to ethanol results at least in part from the necessary investment of only one NADH in the conversion of pyruvate to ethanol whereas other ethanol pathways such as the pyruvate formate lyase pathway require the investment of two NADH in the conversion of pyruvate to ethanol.
In the context of fermentation of the sugar xylose, ethanol production efficiency may be gained by expression of the enzyme xylose isomerase. Xylose is commonly fermented to ethanol through the intermediate xylulose-5-phosphate which is then fed into the pentose phosphate pathway. The conversion of xylose to xylulose may be a less efficient two-step conversion from xylose to xylitol then to xylulose or a more efficient one-step conversion, if xylose isomerase is present, directly from xylose to xylulose.
Vitreoscilla is a genus of filamentous gram-negative bacteria found in freshwater sediments, stagnant ponds, cow dung and decaying vegetable matter where oxygen availability is low. Vitreoscilla C1 is an obligate aerobe which synthesizes a soluble hemoglobin (VHb). VHb is a dimer of two identical subunits each having a relative mass of 15.8 kDa and a b heme. VHb is the best characterized member of the family of bacterial hemoglobin proteins.
VHb has been expressed in Saccharomyces cerevisiae (S. cerevisiae), baker's yeast, and increased ethanol production was demonstrated in comparison to non-VHb expressing controls on synthetic dextrose medium supplemented with 0.1% glucose. Chen, W., et al. “Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae.” Biotechnology Progress 10 (1994): 308-313. Also, the VHb expressing strain was shown to grow to a lower cell culture density indicating a redirection of carbon from biomass production to ethanol. The metabolic changes to S. cerevisiae were attributed to changes in respiration and addition of respiration inhibitor antimycin A was shown to eliminate the effect of VHb on ethanol production.
VHb expression has been studied in E. coli from an isopropyl-β-D-thiogalactopyranoside (IPTG) inducible plasmid for the resulting effects on metabolism under low oxygen conditions in 0.4% glucose supplemented complex medium. Tsai, P. et al. “Effect of Vitreoscilla Hemoglobin Dosage on Microaerobic Escherichia coli Carbon and Energy Metabolism.” Biotechnology and Bioengineering 49 (1996): 139-150. It was found that increased concentrations of VHb (induced by increased concentrations of IPTG) increased the final cell culture density as measured by increases in the grams dry cell weight per liter. However, it was found that concentrations of ethanol were decreased monotonically with increasing VHb dosage. According to this study, VHb in E. coli redirected carbon away from ethanol production toward biomass production.
Most recently, VHb expression in S. cerevisiae was found to increase ethanol production efficiency on yeast synthetic complete media with 5% xylose. Ruohonen L., et al. “Expression of Vitreoscilla hemoglobin improves the metabolism of xylose in recombinant yeast Saccharomyces cerevisiae under low oxygen conditions.” Enzyme and Microbial Technology 39 (2006): 6-14. In both yeast and E. coli, xylose is metabolized to ethanol via the pentose phosphate pathway (PPP). The wild-type yeast pathway for preparation of xylose for entry into PPP comprises a two-step reductive and oxidative conversion of xylose to xylulose requiring the enzymes xylose reductase and xylitol dehydrogenase. First xylose is reduced to xylitol by xylose reductase with NADPH→NADP+ as cofactor, and second xylitol is oxidized to xylulose by xylitol dehydrogenase with NAD+→NADH as cofactor. Inefficient xylose metabolism in yeast has been attributed in part to the redox cofactor imbalance in pre-PPP xylose preparation between the reduction of xylose, which causes NADP+ accumulation, and the oxidation of xylitol, which causes NADH accumulation. One of the consequences of this imbalance is believed to be the build-up of xylitol, as low oxygen conditions limit regeneration of NAD+. In the study by Ruohonen, VHb expression was found to reduce xylitol production by as much as 40% and increase ethanol production by as much as 30%. The primary explanation for these improvements proposed by the authors was that VHb facilitated conversion of NADH to its oxidized form, NAD+, thus driving xylose from the xylitol intermediate to xylulose and thus facilitating entry of xylose into PPP.
In contrast to wild-type yeast, wild-type E. coli convert xylose to xylulose in one step with the enzyme xylose isomerase. Consequently, xylose preparation for PPP in E. coli does not have a redox cofactor imbalance and xylitol is not an intermediate. If the primary explanation proposed by the authors of the yeast VHb expression-xylose fermentation study accounts for most of the increase in ethanol production, then it would be expected that VHb would not similarly increase ethanol production from E. coli xylose fermentation.
There remains a need to develop novel microorganisms and methods which can increase the efficiency of the production of fermentation products, such as ethanol.
SUMMARY
The present disclosure describes novel microorganisms and methods for producing fermentation products with such novel microorganisms. In particular, the disclosure describes novel microorganisms which utilize a carbon source to produce a fermentation product wherein said microorganism expresses a pyruvate decarboxylase gene (e.g. pdc) and at least one alcohol dehydrogenase gene (e.g. adhb) from a bacteria of the genus Zymomonas, and further wherein said microorganism comprises at least one genetic modification which provides for expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla. Said microorganism is a prokaryote or eukaryote. In one embodiment, said microorganism is a prokaryote. In an embodiment, said microorganism is a bacteria of a genus selected from the group consisting of Escherichia and Zymomonas. In one embodiment, said microorganism is a bacteria of the genus Escherichia. In an embodiment, said microorganism is Escherichia coli. In another embodiment, the microorganism is a bacteria of the genus Zymomonas. In an embodiment, said microorganism is Zymomonas mobilis. Said microorganisms have improved fermentation performance compared to an essentially genetically identical microorganisms which lack at least one genetic modification which provides for expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla. In an embodiment, the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla in said microorganism produces a concentration of intracellular hemoglobin greater than 0 and less than about 125 nmoles per gram wet weight of cells. In another embodiment, the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla in said microorganism produces a concentration of intracellular hemoglobin greater than 0 and less than about 100 nmoles per gram wet weight of cells. In an embodiment, the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla in said microorganism produces a concentration of intracellular hemoglobin greater than 0 and less than about 75 nmoles per gram wet weight of cells.
In addition, the present disclosure describes a method for producing a fermentation product comprising: a) providing a microorganism which utilizes a carbon source to produce a fermentation product wherein said microorganism expresses a pyruvate decarboxylase gene and at least one alcohol dehydrogenase gene from a bacteria of the genus Zymomonas; b) modifying the genetics of said microorganism wherein said modifying comprises at least one genetic modification which provides for expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla; and c) contacting the genetically modified microorganism of step b) with at least one carbon source under substantially anaerobic conditions. Said microorganism of the method for producing a fermentation product is a prokaryote or eukaryote. In an embodiment, said microorganism is a prokaryote. In one embodiment, said microorganism is a bacteria of a genus selected from the group consisting of Escherichia and Zymomonas. In one embodiment, said method for producing a fermentation product wherein the fermentation product is ethanol. In an embodiment, said method for producing a fermentation product wherein the at least one carbon source is selected from the group consisting of glucose and xylose. In one embodiment, said method for producing a fermentation product wherein the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla produces a concentration of intracellular hemoglobin in said microorganism greater than 0 and less than about 125 nmoles per gram wet weight of cells. In an embodiment, said method for producing a fermentation product wherein the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla produces a concentration of intracellular hemoglobin in said microorganism greater than 0 and less than about 100 nmoles per gram wet weight of cells. In one embodiment, said method for producing a fermentation product wherein the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla produces a concentration of intracellular hemoglobin in said microorganism greater than 0 and less than about 75 nmoles per gram wet weight of cells.
Further, the disclosure describes a method for increasing production of a fermentation product comprising: a) providing a microorganism which utilizes a carbon source comprising xylose to produce a fermentation product wherein said microorganism expresses at least one xylose isomerase gene; b) modifying the genetics of said microorganism wherein said modifying comprises at least one genetic modification which provides for expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla; and c) contacting the genetically modified microorganism of step b) with at least one carbon source comprising xylose under substantially anaerobic conditions wherein production of the fermentation product is increased compared to fermentation under equivalent conditions with an essentially genetically identical microorganism which lacks at least one genetic modification which provides for expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla. In one embodiment, said method for increasing production of a fermentation product wherein the fermentation product is ethanol. Said microorganism of the method for increasing production of a fermentation product is a prokaryote or eukaryote. In an embodiment, said microorganism is a prokaryote. In an embodiment, said microorganism is a bacteria of the genus Escherichia. In one embodiment, said method for increasing production of a fermentation product wherein the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla produces a concentration of intracellular hemoglobin in said microorganism greater than 0 and less than about 125 nmoles per gram wet weight of cells. In an embodiment, said method for increasing production of a fermentation product wherein the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla produces a concentration of intracellular hemoglobin in said microorganism greater than 0 and less than about 100 nmoles per gram wet weight of cells. In one embodiment, said method for increasing production of a fermentation product wherein the expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla produces a concentration of intracellular hemoglobin in said microorganism greater than 0 and less than about 75 nmoles per gram wet weight of cells. In an embodiment, said method for increasing production of a fermentation product wherein the carbon source is derived from cellulosic biomass.
In one embodiment, the present disclosure describes microorganisms which are bacteria of the genus Escherichia wherein the genes pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhb) from a bacteria of the genus Zymomonas are heterologously expressed in the microorganisms. The expression of pdc and adhb is provided by insertion of one or both of such genes into the chromosome of the bacteria of the genus Escherichia or is provided for by presence of one or both such genes on an plasmid. The expression of a hemoglobin gene from a bacteria of the genus Vitreoscilla is provided by insertion of such hemoglobin gene into the chromosome of the bacteria of the genus Escherichia, is provided for on an plasmid which is the same plasmid carrying pdc and/or adhb or is provided on a plasmid which does not carry either pdc or adhb.
In another embodiment, the present disclosure describes the microorganism is a bacteria of the genus Zymomonas which has endogenous expression of at least one pyruvate decarboxylase and at least one alcohol dehydrogenase gene wherein a hemoglobin gene from a bacteria of the genus Vitreoscilla is provided by insertion of such hemoglobin gene into the chromosome of such microorganism or is provided on a plasmid.
In another embodiment, the present disclosure describes a microorganism which utilizes a carbon source comprising xylose to produce a fermentation product wherein said microorganism expresses a xylose isomerase enzyme and said microorganism is genetically modified such that a hemoglobin gene from a bacteria of the genus Vitreoscilla is provided by insertion of such hemoglobin gene into the chromosome of such microorganism or is provided on a plasmid. The xylose isomerase gene may be endogenously expressed or heterologously expressed. The xylose isomerase gene may be a wild-type gene, a mutated gene or a purposefully modified gene. In one embodiment, said microorganism produces a concentration of intracellular hemoglobin greater than 0 and less than about 125 nmoles per gram wet weight of cells. In an embodiment, said microorganism produces a concentration of intracellular hemoglobin greater than 0 and less than about 100 nmoles per gram wet weight of cells. In one embodiment, said microorganism produces a concentration of intracellular hemoglobin or greater than 0 and less than about 75 nmoles per gram wet weight of cells.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates a diagram of novel plasmid pTS3 which carries the Vitreoscilla hemoglobin gene (vgb) on a broad host range, relatively low copy number plasmid (in comparison to for example pUC plasmids), pKT230.
FIG. 2 illustrates a diagram of novel plasmid pTS4 which carries the Vitreoscilla hemoglobin gene (vgb) on a broad host range plasmid, pBBR1-MCS #5.
FIG. 3 illustrates a diagram of novel plasmid pTS5 which carries the pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhb) genes from Zymomonas mobilis and the Vitreoscilla hemoglobin gene (vgb).
FIG. 4 illustrates a gel confirming development of novel E. coli strain FBR5/pTS3 with two stable plasmids: (1) pLOI297 (which carries pdc and adhb) and (2) pTS3. DNA from each clone was separately PCR amplified for pdc+adhb in one reaction and vgb+TcR in another reaction (tetracycline resistance was used to select for clones with vgb). PCR products for each clone were combined.
FIG. 5 illustrates a gel confirming development of novel E. coli strain FBR5/pTS4 with two stable plasmids: (1) pLOI297 (which carries pdc and adhb) and (2) pTS4. DNA from each clone was separately PCR amplified for pdc+adhb in one reaction and vgb+TcR in another reaction. PCR products for each clone were combined.
FIG. 6 illustrates a gel confirming development of novel E. coli strain FBR5/pTS5 (pdc, adhb and vgb all on one plasmid) by restriction endonuclease digestion with XhoI, BamHI, and PstI as well as PCR of the Vitreoscilla hemoglobin gene (vgb) and pyruvate decarboxylase (pdc)+alcohol dehydrogenase B (adhb) cassette.
FIG. 7 illustrates a graph which consolidates data from carbon monoxide difference spectra (used for VHb protein expression measurement) which indicated that each strain of ethanol producing E. coli expressing VHb ((1) FBR5/pTS3, (2) FBR5/pTS4 and (3) NZN111/pTS5) produced different concentrations of VHb on phosphate buffered LB enriched with 8% (w/v) sugar (aerobic conditions with antibiotics at early stationary phase; measured in nmoles VHb/g wet weight of cells). n equals between 4 and 7; standard deviations indicated.
FIG. 8 illustrates a graph which provides data from carbon monoxide difference spectra which indicated that under microaerobic conditions with antibiotics that ethanol producing E. coli expressing VHb, FBR5/pTS3 and FBR5/pTS4, produced different concentrations of VHb on phosphate buffered LB enriched with 8% (w/v) sugar; measured in nmoles VHb/g wet weight of cells.
FIG. 9 illustrates a graph of consolidated ethanol assay data ([EtOH] on LB with 8% (w/v) glucose, under microaerobic conditions with antibiotics) which shows that at the 44-47 hour time point the strain FBR5/pTS3 (referred to in the graph as just “pTS3”) exceeded the ethanol concentration produced by the FBR5 control, which lacked vgb, by 15% (t-test P-value 0.68%—i.e. approximately 99% confidence). n equals 3; standard deviations indicated.
FIG. 10 illustrates a graph of consolidated ethanol assay data ([EtOH] on LB with 8% (w/v) xylose, microaerobic conditions, no antibiotics) which shows that at the 18-22 hour time point the strain FBR5/pTS3 (referred to in the graph as just “pTS3”) exceeded the ethanol concentration produced by the FBR5 control, which lacked vgb, by 138% (t-test P-value 1.52%—i.e. approximately 98% confidence). n equals 3; standard deviations indicated.
FIG. 11 illustrates a graph of consolidated ethanol assay data ([EtOH] on LB with 8% (w/v) xylose, microaerobic conditions, no antibiotics) which shows that at the 44-47 hour time point the strain FBR5/pTS3 (referred to in the graph as just “pTS3”) exceeded the ethanol concentration produced by the FBR5 control, which lacked vgb, by 119% (t-test P-value 1.16%—i.e. approximately 99% confidence). n equals 3; standard deviations indicated.
FIG. 12 illustrates a graph of ethanol assay data divided by optical density of cultures (600 nm) indicates that at the 44-47 hour time point FBR5/pTS3 (referred to in the graph as just “pTS3”) produced a 31% higher (t-test P-value 8.07%—i.e. approximately 92% confidence) concentration of ethanol per unit measure of cell biomass than the FBR5 control on LB with 8% (w/v) glucose. n equals 3; standard deviations indicated. This graph indicates more efficient production of ethanol on a cell mass basis (i.e. a gram of cells with vgb expression produce more ethanol than a gram of control cells).
FIG. 13 illustrates a graph of ethanol assay data divided by optical density of cultures (600 nm) indicates that at the 44-47 hour time point FBR5/pTS3 (referred to in the graph as just “pTS3”) produced a 44% higher (t-test P-value 2.04%—i.e. approximately 98% confidence) concentration of ethanol per unit measure of cell biomass than the FBR5 control on LB with 8% xylose. n equals 3; standard deviations indicated. This graph also indicates more efficient production of ethanol on a cell mass basis (i.e. a gram of cells with vgb expression produce more ethanol than a gram of control cells).
FIG. 14 illustrates the microaerobic pathways knocked out in E. coli strain NZN111 causing NZN111 to be unable to grow microaerobically because the lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) enzymes have been knocked out resulting in inability to reduce pyruvate via fermentation and regenerate NAD+. Heavy bars indicate knock out of pathway.



DETAILED DESCRIPTION
The practice of the invention disclosed herein employs, unless otherwise indicated, conventional methods of microbiology, molecular biology, and recombinant DNA techniques within the level of skill in the art. Such techniques are explained fully in the literature. See, e.g., (Sambrook, J., and D. W. Russell. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001.
All publications, published patent documents, and patent applications cited in this specification are indicative of the level of skill in the art(s) to which the invention pertains. All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
As used in this specification, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”
As used herein, the term “about” represents an insignificant modification or variation of the numerical values such that the basic function of the item to which the numerical value relates is unchanged.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.
As used herein, the term “gene” refers to a nucleic acid fragment that expresses a specific protein, which may include regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence.
As used herein, the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a gene. Expression may also refer to translation of mRNA into a polypeptide.
As used herein, the term “fermentation product” includes for instance ethanol, glycerol, acetone, n-butanol, butanediol, isopropanol, butyric acid, methane, citric acid, fumaric acid, lactic acid, propionic acid, succinic acid, itaconic acid, acetic acid, acetaldehyde, 3-hydroxypropionic acid, glyconic acid, tartaric acid and amino acids such as L-glutaric acid, L-lysine, L-aspartic acid, L-tryptophan, L-arylglycines or salts of any of these acids.
As used herein, the term “microorganism” has its conventional meaning in the art and includes bacteria, protozoa, yeasts, molds, and viruses.
As used herein, the term “substantially anaerobic” or “microaerobic” has its conventional meaning in the art and includes low oxygen conditions under which fermentative metabolism is favored over aerobic metabolism.
As used herein, the term “substantially genetically identical” refers to a level of genetic identity greater than about 95%.
As used herein, the term “carbon source” has its conventional meaning in the art and includes a nutrient comprising at least one carbon molecule. Examples include but are not limited to glucose, xylose, galactose, fructose, mannose, arabinose, and the like.
As used herein, the term “messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell.
As used herein, the term “genetic modification” refers to the introduction of one or more heterologous nucleic acid sequences into one or more cells, to provide for expression of a gene or protein of interest.
As used here, the term “transformation”, refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. The transferred nucleic acid may be in the form of a plasmid maintained in the host cell, or some transferred nucleic acid may be integrated into the genome of the host cell. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms as well as “transformants”.
As used herein, the terms “plasmid” and “vector”, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
As used herein, the term “lignocellulosic” refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose.
As used herein, the term “cellulosic” refers to a composition comprising cellulose and additional components, including hemicellulose.
As used herein, the term “biomass” refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, polysaccharides, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover or fiber, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, corn fiber, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
This disclosure describes increased fermentation product production in general, and increased ethanol production in particular, from fermentation of a carbon source, such as glucose or xylose, by genetically engineering ethanol producing microorganisms to express a hemoglobin from a bacteria of the genus Vitreoscilla. 
In one embodiment, E. coli strain FBR5 was genetically engineered to express Vitreoscilla hemoglobin (VHb). FBR5 is an industrially significant ethanol producing E. coli strain because it harbors plasmid pLOI297 (FBR5 is E. coli strain NZN111 plus pLOI297) with the pdc and adhb of Z. mobilis, providing a more efficient ethanol pathway, as well as having knockouts of ldh (gene for lactate dehydrogenase) and pfl (gene for pyruvate formate lyase), impeding formation of the undesirable fermentation products lactate and acetate (FIG. 14). The VHb gene, vgb, was maintained under the control of the native oxygen sensitive Vitreoscilla promoter and pdc and adhb were both under control of the lac promoter.
Three novel strains of E. coli were developed to test the effects of VHb on ethanol production.
In two of the strains, vgb was expressed on a second plasmid, in addition to pLOI297. The plasmid of FBR5, pLOI297, carrying pdc and adhb is a pUC18 based plasmid. Ingram, L. O., et al. “Genetic Engineering of Ethanol Production in Escherichia coli.” Applied and Environmental Microbiology 53.10 (1987): 2420-2425. pUC based plasmids are derived from ColE1 plasmids and have negative feedback control of replication initiation and thus incompatibility with other ColE1 plasmids. Consequently, it is generally not possible to stably propagate two plasmids of the same incompatibility group (e.g. two pUC plasmids) in the same cell. However, plasmids from different incompatibility groups, i.e. having different origins of replication, can stably propagate in the same cell. Lengeler, J. W., et al. Biology of the Prokaryotes. New York, N.Y.: Blackwell Science, 1999.
Thus, the ability of plasmids from different incompatibility groups to co-exist within the same cell was used to develop two of the three novel E. coli strains. Strain FBR5/pTS3 was developed by introduction of novel plasmid pTS3 (FIG. 1) into strain FBR5 while preventing the rejection of plasmid pLOI297. Plasmid pTS3 was constructed by inserting the Vitreoscilla hemoglobin gene, vgb, into plasmid pKT230. Plasmid pKT230 has two origins of replication each of which is in a different incompatibility group than ColE1 plasmids. Bagdasarian M., et al. “Specific-purpose plasmid cloning vectors: II. Broad host range, high copy number, RSF1010-derived vectors, and a host vector system for gene cloning in Pseudomonas.” Gene 16 (1981): 237-247. pKT230 is composed of plasmid pACYC177 ligated with plasmid RSF1010. RSF1010 belongs to the IncQ incompatibility group and pACYC177 contains the replication system of miniplasmid P15A. Chang, A. C. Y. and Cohen, S. N., “Construction and Characterization of Amplifiable Multicopy DNA Cloning Vehicles Derived from P15A Cryptic Miniplasmid.” Journal of Bacteriology 134.3 (1978): 1141-1156.
Strain FBR5/pTS4 was developed by introduction of novel plasmid pTS4 (FIG. 2) into strain FBR5 together with pLOI297. Plasmid pTS4 was constructed by insertion of vgb into plasmid pBBR1MCS-5. Plasmid pBBR1MCS-5 has an origin of replication that is compatible with ColE1 plasmids. Kovach, M. E., et al. “Four new derivatives of the broad-host-range cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes.” Gene 166 (1995): 175-176.
The third novel E. coli strain was developed by combining all three genes of interest, pdc, adhb and vgb, into one novel plasmid (pTS5), curing FBR5 of the pLOI297 plasmid to produce strain NZN111 and introducing pTS5 into strain NZN111.
After the three novel ethanol producing strains were developed: (1) FBR5/pTS3, (2) FBR5/pTS4 and (3) NZN111/pTS5, the cell physiology of these strains was studied. Data collection focused on Vitreoscilla hemoglobin (VHb) production and ethanol production under different growth conditions.
The VHb expression level of FBR5/pTS3, the lowest VHb expression of the novel strains, was approximately twice the normal induced level in Vitreoscilla. Geckil, H., et al. “Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogens carrying Vitreoscilla hemoglobin gene.” Bioprocess and Biosystem Engineering 26 (2004): 325-330. Of the three novel strains, only NZN111/pTS5 expressed VHb at levels as high as those commonly seen for plasmids in E. coli with the VHb gene, vgb, controlled by the native promoter and FBR5/pTS4 expressed VHb at about half of commonly seen levels. Dikshit, K. L., D. A. Webster. “Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli.” Gene 70 (1988): 377-386. Fish, P. A, et al., “Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo.” Journal of Molecular Catalysis B: Enzymatic 9 (2000): 75-82.
In the study by Tsai et al. 1996, where VHb expression in E. coli from an IPTG inducible plasmid under microaerobic conditions was found to reduce ethanol production monotonically with increasing VHb concentrations including the lowest VHb concentration tested. The lowest VHb expression level tested was 500 nmoles/gram dry cell weight or approximately 125 nmoles/gram wet cell weight. Tsai, P. S., et al. “Effect of Vitreoscilla Hemoglobin Dosage on Microaerobic Escherichia coli Carbon and Energy Metabolism.” Biotechnology and Bioengineering 49 (1996): 139-150. This expression level was comparable to the expression level of FBR5/pTS4 under microaerobic conditions while the expression level of FBR5/pTS3 under microaerobic conditions was approximately half of the lowest level of VHb expression tested by Tsai et al. (FIG. 8).
Thus, the study of Tsai et al. teaches away from VHb expression for increased ethanol production, particularly in E. coli which produce ethanol with the wild-type ethanol pathway. However, it has been surprisingly found that lower levels of VHb expression than those tested by Tsai et al. actually increase ethanol production.
The results indicate that a relatively low level of VHb expression is beneficial to ethanol production, even under the selective pressure of additional antibiotics and even at the metabolic cost of maintaining additional plasmid DNA. For example, FBR5/pTS3 produced 15% higher ethanol concentration (v/v) on buffered LB enriched with 8% (w/v) glucose under microaerobic conditions with antibiotics and 119-138% higher ethanol concentration (v/v) on buffered LB enriched with 8% (w/v) xylose under microaerobic conditions without antibiotics.
EXAMPLES
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention as defined in the appended claims.
Example 1
Development of Novel Strains Expressing a Pyruvate Decarboxylase and a Alcohol Dehydrogenase from a Bacteria of the Genus Zymomonas; a Xylose Isomerase Enzyme; and a Hemoglobin Gene from a Bacteria of the Genus Vitreoscilla 
A. Cloning Methods Utilized
Polymerase chain reactions were separately performed using two polymerase mixtures. For cloning purposes, TcR (tetracycline resistance) was PCR amplified from plasmid pBR322 using JumpStart ReadyMix Taq (Sigma-Aldrich Catalog #P2893). Taq ReadyMix was also used for diagnostic purposes to detect for the presence of particular plasmid or gene in cells. All other PCR amplification for cloning was performed with the Phusion High-Fidelity PCR Kit (New England BioLabs Catalog #F-5535).
All primers for PCR amplification were obtained from Integrated DNA Technologies (Coralville, Iowa) and the following primers were used for each amplification: (1) vgb with PstI ends was PCR amplified from pUC8:16 Primer 1—(SEQ ID NO: 11) 5′-AAA CTG CAG GTT AAA AGT ATT TGA GTT TTG ATG TGG A-3′ and Primer 2—(SEQ ID NO: 12) 5′-CCA ATG CAT TGG TTC TGC AGG TGT AAA TAT CAG ACG TAA AAA GAC CA-3′; (2) TcR with EcoRI ends was PCR amplified from pBR322 using Primer 1—(SEQ ID NO: 13) 5′-AAA ACT GCA GAA AAC CCG GGC TCT TCC TTT TTC AAT ATT ATT GAA GCA-3′ and Primer 2—(SEQ ID NO: 14) 5′-TGC ATT GGC TGC AGT TTC CCG GGT TTT TGA ATT CAT ATG TTC TGC CAA GGG TTG GTT TG-3′; (3) TcR with HindIII ends and point mutation was PCR amplified from pBR322 using Primer 1—(SEQ ID NO: 15) 5′-CCC AAG CTT TTG ACA GCT TAT CAT CGA TAA GCT ATA ATG CGG TAG TTT ATC AC-3′ and Primer 2—(SEQ ID NO: 16) 5′-CCC AAG CTT ATA TGT TCT GCC AAG GGT TGG TTT G-3′; (4) vgb+TcR cassette with EcoRI ends was PCR amplified from pTS2 using Primer 1—(SEQ ID NO: 17) 5′-GGC GAA TTC CTG CAA GGC GAT TAA GTT GG-3′ and Primer 2—(SEQ ID NO: 18) 5′-GGC GAA TTC CAA GGC ACA CCT GAA GAC G-3′; (5) pdc+adhb cassette with BamHI ends was PCR amplified from pLOI297 using Primer 1—(SEQ ID NO: 19) 5′-AAA GGA TCC GCG CAA CGT AAT TAA TGT GAG TT-3′ and Primer 2—(SEQ ID NO: 20) 5′-TTT GGA TCC CCA AAT GGC AAA TTA TT-3′; and (6) vgb+TcR cassette with XhoI ends was PCR amplified from pTS2 using Primer 1—(SEQ ID NO: 21) 5′-GGC CTC GAG CTG CAA GGC GAT TAA GTT GG-3′ and Primer 2—(SEQ ID NO: 22) 5′-GGC CTC GAG CAA GGC ACA CCT GAA GAC G-3′.
PCR amplification cycles were the following: (1) vgb with PstI ends PCR amplified from pUC8:16 [step 1—94° C. for 5 minutes, step 2—94° C. for 30 seconds, step 3—59° C. for 30 seconds, step 4—72° C. for 1 minute and 15 seconds, step 5—72° C. for 5 minutes and step 6—held at 4° C.; amplification cycle (steps 2-4) repeated 30 times]; (2) TcR with EcoRI ends was PCR amplified from pBR322 using the same cycle as for vgb with PstI ends, including annealing temperature, except elongation step 4—72° C. for 2 minutes; (3) TcR with HindIII ends and point mutation was PCR amplified from pBR322 again using the same cycle for vgb with PstI ends, except a temperature gradient for annealing temperatures was used between 57.7° C. and 61.6° C. where all temperatures worked and elongation step 4—72° C. for 2 minutes (4) vgb+TcR cassette with EcoRI ends was PCR amplified from pTS2 using Phusion polymerase [step 1—98° C. for 30 seconds, step 2—98° C. for 10 seconds, step 3—55° C. for 30 seconds, step 4—72° C. for 1 minute and 30 seconds, step 5—72° C. for 5 minutes and step 6—held at 4° C.; amplification cycle (steps 2-4) repeated 35 times]; (5) pdc+adhb cassette with BamHI ends was PCR amplified from pLOI297 using Phusion polymerase and the same cycle as amplicon 4 except a temperature gradient for annealing temperatures was used between 50° C. and 70° C. where all temperatures worked and elongation step 4—72° C. for 2 minutes; and (6) vgb+TcR cassette with XhoI ends was PCR amplified from pTS2 using Phusion polymerase and same cycle as for the vgb+TcR cassette with EcoRI ends.
All PCR reactions were run in a MJ Research PTC-200 Peltier Thermal Cycler. Excluding use of the PCR-Script Amp system, before enzymatic digestion, all PCR products were cleaned with QIAquick PCR Purification kit (Qiagen Catalog #28104).
DNA gel electrophoresis was done using 1% agarose gels (35 ml of either 1×TAE or 0.5×TBE buffer prepared as described in Sambrook 2001 (Sambrook, J., and D. W. Russell. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001) and 0.35 g of electrophoresis grade agarose (Amresco Catalog #0710-500G) with 1:10,000 dilution of gel stain GelRed (Biotium Catalog #41003). Agarose weights were measured using a Denver Instruments APX-60 balance. Gels were run in a horizontal gel box (similar to an Owl Separation Systems Model B1) with a power supply (VWR Scientific Model VWR 105) set at 130 V. All ladders used were HindIII restriction endonuclease digestions of λ-DNA.
All endonuclease digestions were performed using New England BioLabs enzymes and buffers. Reactions were incubated in an incubator (VWR Scientific Model VWR1530) at 37° C. for a minimum of 1 hour to as long as overnight.
For all ligation reactions, except the cloning of pTS1 and pTS3, all digested DNA was extracted from gels before preparation of ligation reactions. For pTS3, the insert was gel extracted, but pKT230 was too large (12 kb) to gel extract because it could not be removed from beads due to excessively tight binding. All gel extractions were performed using the Qiaex II system (Qiagen Catalog #20021). The sole deviation from the standard protocol was that DNA was heated to 60° C. for ten minutes in order to elute from beads.
All ligations, other than PCR-Script Amp ligations, were incubated overnight at between 4° C. and 16° C. using T4 DNA ligase from New England BioLabs (Catalog #M0202S). Total concentration of DNA in ligations (vector+insert) was approximately 10 ng/μl, generally 100 ng in 10 μl ligation reactions. Insert to plasmid molar ratios were approximately 7:1.
Plasmids were prepped according to three primary protocols (1) Qiagen QlAprep Spin Miniprep Kit (Catalog #27104), (2) Promega PureYield Plasmid Midiprep System (Catalog #A2492) and (3) alkaline lysis (Sambrook 2001).
For pTS3, pTS4 and pTS5, PCR products were prepared for insertion by ligation into intermediate plasmid PCR-Script Amp. The Stratagene PCR-Script Amp cloning kit (Catalog #211188) was used. All reactions were performed at half volume to extend kit life. Also the competent cells provided by the kit were not used because they were found to exhibit a high degree of tetracycline antibiotic resistance.
Chemical competent and electro-competent cells were prepared according to the protocol of Sambrook 2001. It was found that electrocompetent cells had a very short shelf-life at −80° C. NEB 5-alpha electrocompetent E. coli were used for several transformations and found to have a much longer shelf life at −80° C. (New England Biolabs Catalog #C2989K).
All heat shock transformations were carried out as described in Sambrook 2001. When tetracycline selection was used, heat shock was the necessary procedure because electroporation has low efficiency. Steele, C., S. Zhang, and E. J. Shillitoe. “Effect of Different Antibiotics on Efficiency of Transformation of Bacteria by Electroporation.” BioTechniques 17.2 (1994): 360-365. Electroporation was done using a BTX Electro Square Porator ECM830 and BTX 1 mm gap cuvettes. The settings used were 500 V and pulse length of 17 ms.
Working antibiotic concentrations for ampicillin (Amp), kanamycin (Km), streptomycin (Sm), gentamicin (Gm) and tetracycline (Tc) were 100 μg/ml, 50 μg/ml, 50 μg/ml, 5 μg/ml and 25 μg/ml, respectively, throughout the experiments. Stock solutions were prepared in sterile dH2O of (1) Amp-sodium salt of 25 mg/ml, (2) Km 10 mg/ml, (3) Sm-sulfate 10 mg/ml, and Gm 10 mg/ml. The stock solution for Tc was 5 mg/ml in 50% dH2O and 50% EtOH.
All plates used for FBR5 and derivative strains had LB, appropriate antibiotics and were supplemented with 8% (w/v) xylose (D-xylose Sigma-Aldrich X1500). Selection and maintenance plates were used as follows: FBR5 on LB/Amp, FBR5/pTS3 on LB/Amp/Sm, FBR5/pTS4 on LB/Amp/Gm, NZN111 on LB/Km, pTS5/NZN111 on LB/Amp and both DH5α/pTS1 and DH5α/pTS2 on LB/Tc.
Blue white screening for β-galactosidase activity was done on LB/antibiotic plates to which 100 μl pool of LB had been added and into which 100 μl of 2% X-gal (in dimethylformamide) and 100 μl of 10 mM IPTG (in sterile dH2O) were added. This mixture was distributed evenly across the surface of the plate and allowed to soak into plates for 30 minutes.
B. Development of Novel Plasmids and Novel Strains
A strategy was developed to clone TcR (tetracycline resistance gene) from plasmid pBR322 into the HindIII site of vgb containing plasmid pUC8:16 (HindIII sites lies adjacent to vgb). The primary challenge encountered was that a HindIII site existed in the −10 sequence of the promoter of TcR of pBR322. In response, primers were designed to amplify TcR with a one base pair mismatch creating a point mutation to knockout the HindIII site and enhance the −10 sequence of the promoter by changing it from TTTAAT to the consensus sequence TATAAT. The point mutation by primer mismatch PCR was successful. The insert was ligated into pUC8:16 to create pTS2. Construction of pTS2 was confirmed by HindIII digestion, by EcoRI digestion, and PCR amplification of the insert.
A PCR strategy was developed to amplify the region of pTS2 containing vgb and TcR as one amplicon. The vgb+TcR cassette was successfully amplified with the Phusion polymerase. The vgb+TcR cassette was then successfully ligated into the intermediate cloning vector PCR-Script Amp.
The vgb+TcR cassette was digested from plasmid PCR-Script Amp+vgb+TcR and inserted into the EcoRI site of pKT230 to create pTS3 (FIG. 1), and similarly inserted into the EcoRI site of pBBR1-MCS-5 to create pTS4 (FIG. 2). Construction of plasmids pTS3 and pTS4 was confirmed by EcoRI digestion, and further confirmed by PCR amplification of the insert.
Next, pTS3 and pTS4 were separately introduced into strain FBR5 by electroporation to generate strains FBR5/pTS3 and FBR5/pTS4. After electroporation, FBR5/pTS3 transformants were selected on plates of xylose enriched LB media with ampicillin and streptomycin. The presence of the two plasmids within the cells allowed for growth because pLOI297 alone conferred antibiotic resistance to ampicillin and pTS3 alone conferred resistance to streptomycin. After electroporation, FBR5/pTS4 transformants were selected on plates of xylose enriched LB media with ampicillin and gentamicin. The presence of the two plasmids within the cells allowed for growth because pLOI297 alone conferred antibiotic resistance to ampicillin and pTS4 alone conferred resistance to gentamicin.
Development of the novel strains (FBR5/pTS3 and FBR5/pTS4) was confirmed by PCR amplification of pdc+adhb from pLOI297 in one reaction and vgb+TcR on pTS3 or pTS4 in another reaction (FIGS. 4 and 5).
In order to potentially increase ethanol production efficiency by combining all three genes of interest, vgb, pdc and adhb, on one high-copy-number plasmid and to simplify growth conditions by reducing the number of antibiotics required for plasmid stabilization, pTS5 was constructed (FIG. 3).
The first step in the construction of pTS5 was use of a strategy to PCR amplify pdc and adhb together as a cassette including the lac promoter. The pdc+adhb cassette was successfully amplified with the Phusion polymerase. The pdc+adhb cassette (3.2 kb) was then successfully ligated into cloning vector PCR-Script Amp (3 kb) using blue-white screening. Previous use of the vgb+TcR cassette with EcoRI ends was not applicable for construction of pTS5 because an EcoRI restriction site was present between the lac promoter and pdc and adhb. There was no restriction site for XhoI present in the pdc+adhb cassette or within the vgb+TcR cassette, but a unique XhoI site was present on the plasmid PCR-Script Amp. Therefore, the vgb+TcR cassette was PCR amplified with XhoI ends.
Insertion of the vgb+TcR cassette with XhoI ends into cloning vector PCR-Script Amp was complicated by the occurrence of an internal deletion in TcR. It remains unknown why the vgb+TcR cassette with XhoI ends exhibited internal deletion when the vgb+TcR cassette with EcoRI ends did not. Yet, by use of blue-white screening construction of PCR-Script Amp+insert of partial vgb+TcR cassette was confirmed by digestion with restriction endonuclease XhoI, and by PCR amplification of the insert using primers specific to the vgb+TcR cassette. For clones #3 and #6, it was determined by PCR amplification with primers specific to vgb that the internal deletion in the vgb+TcR cassette was within the region of TcR and that vgb including its native oxygen sensitive promoter was intact.
Subsequently, vgb with XhoI ends was released from PCR-Script Amp vgb by restriction endonuclease digestion with XhoI and inserted into the unique XhoI site of PCR-Script Amp pdc+adhb to create novel plasmid pTS5. Selection for successful construction was difficult because the blue-white screen had been expended in generation of PCR-Script Amp pdc+adhb and the insert carried no antibiotic resistance. Ligation was performed with a molar ratio between insert and plasmid much higher that 7:1 used in previous ligations, risking multiple inserts while reducing the probably of plasmid self ligation. The ligation mixture was electroporated into NEB 5-alpha cells and plated to LB/ampicillin. All colonies were picked and used to inoculate 2 ml LB/ampicillin cultures grown overnight. 1.5 ml of each culture was pelleted and pellets were visually examined to distinguish pink color that might be attributed to VHb. Promising pellets were mini-prepped and tested for presence of PCR-Script Amp pdc+adhb+vgb (pTS5) with single cut restriction endonuclease digestion with PstI and vgb insert releasing digestion with XhoI, and further with BamHI. Construction of pTS5 was further verified by PCR amplification of pdc+adhb in one reaction and vgb in another reaction.
In order to generate novel E. coli strain NZN111/pTS5, FBR5 had to first be cured of plasmid pLOI297. FBR5 was repeatedly restreaked on xylose enriched LB/kanamycin plates because the kanamycin resistance gene was present on the genomic DNA where it had been used to knock out the lactate dehydrogenase gene. After repeated restreaks, FBR5 was found to have lost ampicillin resistance indicating that it had been cured of plasmid pLOI297 generating the underlying parent strain of FBR5, NZN111. NZN111 was cultured and prepared for electroporation and pTS5 was introduced by electroporation. NZN111/pTS5 transformants were screened on xylose enriched LB/ampicillin plates. Generation of novel E. coli strain NZN111/pTS5 was verified by restriction endonuclease digestion with XhoI, BamHI, and PstI as well as PCR amplification with primers to pdc+adhb in one reaction and primers to vgb in another reaction (FIG. 6).
Example 2
VHb Expression and Ethanol Production Studies of Novel Strains
A. Methods for Measurement of Ethanol Production, Methods for Measurement of VHb Expression, Strain Maintenance, and Statistical Tests
Fermentation studies were performed with the following media: phosphate buffered LB enriched with 8% (w/v) of one of D-xylose or D-glucose. All sugar percentages herein are in percent (w/v) unless otherwise specified. Carbon monoxide difference spectra, measuring VHb expression, were collected for FBR5, FBR5/pTS3, FBR5/pTS4 and NZN111/pTS5 cultured in phosphate buffered LB enriched with 8% (w/v) xylose or 8% (w/v) glucose.
For making buffered LB the following stock solutions were prepared: (1) 40% (w/v) sugar solution in dH2O of D-xylose or D-glucose (the sugar was autoclaved separately); (2) 2×LB with no NaCl made by dissolving 10 g tryptone and 5 g yeast extract in 500 ml dH2O and autoclaving; (3) phosphate buffer, pH 7.0, by dissolving 10.8 g sodium phosphate monobasic, monohydrate and 17.3 g of sodium phosphate dibasic in 200 ml dH2O and autoclaving, and (4) sodium acetate 10% (w/v) in dH2O (also autoclaved).
The buffered and enriched LB was prepared as phosphate buffered: phosphate buffered included 50 ml 2×LB, 20 ml phosphate buffer, 20 ml of sugar stock, 9 ml dH2O and 1 ml sodium acetate.
Stock solutions of antibiotics were prepared in sterile dH2O of (1) ampicillin (Amp), 25 mg/ml, (2) kanamycin (Km), 10 mg/ml, (3) streptomycin (Sm), 10 mg/ml, and gentamicin (Gm), 10 mg/ml. Working antibiotic concentrations were 100 μg/ml Amp for FBR5 cultures, 100 μg/ml Amp and 50 μg/ml Sm for FBR5/pTS3 cultures, 100 μg/ml Amp and 5 μg/ml Gm for FBR5/pTS4 cultures and 100 μg/ml Amp for NZN111/pTS5 cultures.
All strains were maintained on LB plates with the appropriate antibiotic(s) enriched with 8% xylose. The plates were made by substituting 20% of the volume of dH2O with xylose in dH2O (40% w/v). All liquid cultures were started from single colonies as small pre-cultures of approximately 2 ml in LB with the appropriate antibiotic(s) enriched with 8% xylose. Pre-cultures were grown to stationary phase and optical densities were measured. Larger cultures were started with approximately 500 μl of pre-culture, but inoculation volumes were adjusted, taking into account optical density, to equalize biomass of inoculums across cultures.
Each aerobic culture (for VHb expression measurements) was grown as 50 ml of culture in a 250 ml Erlenmeyer flask. The media composition of the aerobic cultures was identical to the media composition of the microaerobic cultures, but all the volumes were halved. The flasks were clamped onto the base plate of a platform shaker (Lab-Line Incubator Shaker) at 37° C. and approximately 180 rpm.
All microaerobic cultures were grown as 100 ml of culture in a 125 ml Erlenmeyer flask. The flask was capped with a rubber stopper pierced with a 22 gauge needle for CO2 exhaust. Similarly, the flasks were clamped onto the base plate of a platform shaker at approximately 180 rpm and 37° C.
All cultures were run as sets which included the control strain growing in the same shaker's incubation conditions, batch of media, and at the same times as each of the other strains being tested. For ethanol and optical density measurements at the first time point, one ml was removed for assays from each flask and placed in a capped 1.5 ml micro-centrifuge tube and cultures were quickly returned to the shaker in order to minimize disruption of growth conditions.
All optical density (OD) measurements throughout the cell physiology studies were done on a ThemoSpectronic Genesys 10uv spectrophotometer at 600 nm. The cultures were diluted 1:10 with the appropriate medium (LB or LB enriched with hydrolysate) in order to maintain the linearity of the OD measurements and read against blanks of the corresponding media. All OD measurements as reported below are the original 1:10 dilution measurements multiplied by 10 to reflect full OD.
Carbon monoxide difference spectra were taken as previously described. Dikshit, K. L., D. A. Webster. “Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli.” Gene 70 (1988): 377-386. The majority of carbon monoxide (CO) difference measurements were made under aerobic culture conditions at early stationary phase. All cultures were closely monitored by measurement of OD as cultures approached stationary phase. As OD readings began to level off, 40 ml of the cultures were harvested by centrifugation in a Sorvall Instruments RC5C at 4000 rpm for 10 minutes. Cultures grown under microaerobic conditions were similarly harvested for CO difference measurements immediately after data were collected at the second time point (44-47 hours of growth).
Centrifuge tubes used to spin down cells were weighed before cell culture was added and after cells had been pelleted in order to determine the wet weight of cells. If CO difference was not measured immediately, pellets were stored at −20° C. For CO difference measurement, cells were resuspended at a concentration of 40 mg wet weight of cells per ml in 0.10 M sodium phosphate buffer pH 7.5. Once cells were evenly resuspended, 3 ml of cell suspension was placed into each of two quartz cuvettes. To each cuvette was further added a match head worth of the reducing agent sodium dithionite and cuvettes were mixed by gentle inversion. Both cuvettes were placed into the duel beam spectrophotometer (Varian Cary 300 Scan UV-Visible), the SBW parameter was set to 4.0 and baseline absorbance was measured starting at 600 nm and ending at 400 nm with a scan rate of 200 nm/minute. Then the cuvette positioned toward the front of the machine was removed and bubbled with CO at a rate of approximately one bubble per second for a period of 2 minutes. The CO bubbled cuvette was replaced into the spectrophotometer and sample data was overlaid over the baseline trace with the same scan parameters as the baseline.
Once a CO difference trace had been collected, VHb concentration was determined by measuring absorbance at 419 nm (a positive value) and the absolute value of the absorbance at 436 nm (a negative value) and adding the two values to get AA. Concentration of VHb in nanomoles/ml was determined by ΔA/274*1000. Concentration of VHb in nanomoles/gram cell wet weight was determined by converting nanomoles/ml by multiplying by 1 ml/0.04 g. All VHb concentrations reported herein are in nmoles VHb/g cell wet weight.
All ethanol measurements were made using BioAssay Systems EnzyChrom Ethanol Assay Kit (ECET-100). This enzymatic assay is based on alcohol dehydrogenase catalyzed oxidation of ethanol, in which the NADH formed is coupled to formazan (MTT)/phenazine methosulfate. The intensity of the color produced, measured at 565 nm, is proportionate to the ethanol concentration of the sample. Since this is an enzymatic assay, it requires time to proceed and color is allowed to develop over 5 minutes. Standard curves were prepared for each kit purchased and all ethanol concentrations were determined from the standard curve for the kit used to take measurements. The ECET-100 protocol was followed and reagents were mixed fresh for each sample. OD values at 5 minutes were adjusted by the OD at 5 minutes of the blank (with no ethanol) to determine ethanol concentration, rather than determining the DOD (OD at 5 minutes minus OD at time 0) as described in the protocol. This deviation was due to the relatively large variability in OD at time zero for a sample depending on the time taken to get the cuvette into the spectrophotometer whereas OD changed more slowly around the 5 minute time point. A ThermoSpectronic Genesys 10uv and Brand Ultramicro Cuvettes (Catalog #759200) were used for all ethanol OD measurements. All ethanol measurements reported below are in percentage v/v, unless otherwise specified.
For ethanol measurements, 1 ml was aliquoted from cultures and placed into a 1.5 ml micro-centrifuge tube. The cultures were diluted 100-200 fold, 50 μl in 5 ml dH2O or 50 μl in 10 ml dH2O for ethanol measurement. A blank reaction with all reagents added to a sample of dH2O was prepared for each set of ethanol measurements.
Statistical analysis was done on the data using t-tests to determine the probably that the means of the compared data sets were the same. All probability values provided herein are for two-tailed t-tests. When the number of samples in compared data sets was the same, a paired t-test was used. When the number of samples in compared data sets was different, a t-test with the assumption of homoscedasticity was used because this provided a more conservative probability estimate and there was no reason to believe the variances of the compared data sets were different.
B. VHb Expression Measurements
Once the novel strains of ethanol producing E. coli were developed, each strain was grown in phosphate buffered LB media enriched with sugar under aerobic conditions to measure the concentration of VHb production. Mainly aerobic culture conditions were used for the measurement of VHb production by the novel strains because expression of VHb is commonly measured with 50 ml of culture in a 250 ml flask and cells harvested at early stationary phase. The three novel strains were found to produce distinctly different concentrations of VHb on fermentation media (FIG. 7); this allowed examination of the VHb dosage effect on ethanol fermentation. The control strain FBR5 tested negative for VHb expression.
Fermentation Under Microaerobic Conditions with Antibiotics
Fermentation studies were performed with phosphate buffered LB enriched with glucose under microaerobic conditions with antibiotics for the control strain, FBR5, and all three novel strains. FIG. 9 shows that the concentration of ethanol produced by FBR5/pTS3 exceeded the concentration of ethanol produced by FBR5 in phosphate buffered LB with 8% glucose in microaerobic conditions with antibiotics at the 44-47 hour time point by 15%. A t-test provided greater than 99% confidence for this finding of higher ethanol production by FBR5/pTS3 at the 44-47 hour time point.
The ratio of ethanol concentration to cell biomass was 31% higher for FBR5/pTS3 than FBR5 in phosphate buffered LB with 8% glucose in microaerobic conditions with antibiotics at the 44-47 hour time point (FIG. 12). A t-test provided greater than 90% confidence for this finding. This finding indicates more efficient production of ethanol on a cell mass basis (i.e. a gram of cells with vgb expression produce more ethanol than a gram of control cells).
Since previous measures of VHb expression had been made for aerobic cultures, two sets of measures were made for VHb expression under microaerobic conditions with antibiotics for FBR5/pTS3 and FBR5/pTS4 (FIG. 8). Also, FBR5/pTS3 maintained similar VHb expression levels under microaerobic conditions without antibiotics: on phosphate buffered LB with 8% xylose, 37.38 nmoles VHb/g wet weight of cells, standard deviation 19.12, n equals 3; on phosphate buffered LB with 8% glucose, 54.17 nmoles VHb/g wet weight of cells, standard deviation 13.47, n equals 3.
Fermentation Under Microaerobic Conditions without Antibiotics
Additional fermentation studies were performed with phosphate buffered LB enriched with xylose under microaerobic conditions without antibiotics for the control strain, FBR5, and only FBR5/pTS3, because it appeared that the VHb expression level of FBR5/pTS3 was the most beneficial to ethanol production. FIG. 10 shows that the concentration of ethanol produced by FBR5/pTS3 exceeded the concentration of ethanol produced by FBR5 in phosphate buffered LB with 8% xylose in microaerobic conditions without antibiotics at the 18-22 hour time point by 138%. A t-test provided greater than 98% confidence for this finding of higher ethanol production by FBR5/pTS3.
FIG. 11 shows that the concentration of ethanol produced by FBR5/pTS3 exceeded the concentration of ethanol produced by FBR5 in phosphate buffered LB with 8% xylose in microaerobic conditions without antibiotics at the 44-47 hour time point by 119%. A t-test provided greater than 98% confidence for this finding of higher ethanol production by FBR5/pTS3.
The ratio of ethanol concentration to cell biomass was 44% higher for FBR5/pTS3 than FBR5 in phosphate buffered LB with 8% xylose in microaerobic conditions without antibiotics at the 44-47 hour time point (FIG. 13). A t-test provided greater than 97% confidence for this finding. This finding indicates more efficient production of ethanol on a cell mass basis (i.e. a gram of cells with vgb expression produce more ethanol than a gram of control cells).
A number of patents, patent application publications, and scientific publications are cited throughout and/or listed at the end of the description. Each of these is incorporated herein by reference in their entirety. Likewise, all publications mentioned in an incorporated publication are incorporated by reference in their entirety.
Examples in cited publications and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the cited publications will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
The words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively.

## Claims

1. A method for increasing production of ethanol on a cell-mass basis comprising:
(a) providing a microorganism of the genus Saccharomyces which utilizes a carbon source to produce ethanol wherein said microorganism expresses a xylose isomerase gene;
(b) genetically modifying said microorganism, wherein said modifying comprises at least one genetic modification which provides for expression of a hemoglobin gene from a bacterium of the genus Vitreoscilla, and wherein the expression of the hemoglobin gene from the bacterium of the genus Vitreoscilla produces a concentration of intracellular hemoglobin greater than 0 and less than 125 nmoles per gram wet weight of cells; and
(c) contacting the modified hemoglobin expressing microorganism of step (b) with at least one carbon source comprising xylose under substantially anaerobic conditions,
wherein production of ethanol is greater on a cell-mass basis after said microorganism is genetically modified to provide for expression of said hemoglobin than before said microorganism is genetically modified to provide for expression of said hemoglobin.

2. The method of claim 1, wherein the expression of the hemoglobin gene from the bacterium of the genus Vitreoscilla produces a concentration of intracellular hemoglobin greater than 0 and less than 100 nmoles per gram wet weight of cells.

3. The method of claim 1, wherein the expression of the hemoglobin gene from the bacterium of the genus Vitreoscilla produces a concentration of intracellular hemoglobin greater than 0 and less than 75 nmoles per gram wet weight of cells.

4. The method of claim 1, wherein the xylose isomerase gene is an endogenously or heterologously gene.

5. The method of claim 4, wherein the xylose isomerase gene is a wild-type gene, a mutated gene or a purposefully modified gene.

6. The method of claim 4, wherein the xylose isomerase gene is a heterologously gene and wherein said xylose isomerase gene has been inserted into a chromosome of the microorganism.

7. The method of claim 4, wherein the xylose isomerase gene is a heterologously gene and wherein said xylose isomerase gene is in a plasmid or vector.

8. The method of claim 1, wherein the carbon source is derived from cellulosic biomass.