# Patent

## Title
Metabolic engineering of xylose-fermenting eukaryotic cells

## Abstract

The present invention relates to genetic modifications in eukaryotic host cells that have been transformed to express a xylose isomerase that confers on the host cell the ability to isomerize xylose to xylulose. These genetic modifications are aimed at improving the efficiency of xylose metabolism and include. e.g., reduction of nonspecific aldose reductase activity, increased xylulose kinase activity and increased flux of the pentose phosphate pathway. The modified host cells of the invention are suitable for the production of a wide variety of fermentation products, including ethanol, in fermentation processes in which a source of xylose or a source of xylose and glucose are used as carbon source.

## Background
FIELD OF THE INVENTION
The present invention relates to further genetic modifications in eukaryotic host cells that have been transformed to express a xylose isomerase that confers the host cell the ability of isomerizing xylose to xylulose. The further genetic modifications are aimed at improving the efficiency of xylose metabolism and include e.g., reduction of nonspecific aldose reductase activity, increased xylulose kinase activity and increased flux of the pentose phosphate pathway. The modified host cells of the invention are suitable for the production of a wide variety of fermentation products in processes comprising xylose as carbon source.
BACKGROUND OF THE INVENTION
Economically viable ethanol production from the hemicellulose fraction of plant biomass requires the simultaneous conversion of both pentoses and hexoses at comparable rates and with high yields. Yeasts, in particular Saccharomyces spp., are the most appropriate candidates for this process since they can grow fast on hexoses, both aerobically and anaerobically. Furthermore they are much more resistant to the toxic environment of lignocellulose hydrolysates than (genetically modified) bacteria.
In previous studies evidence has been provided that metabolic engineering of S. cerevisiae for xylose utilization, should be based on the introduction of xylose isomerase (XI, EC 5.3.1.5) (Bruinenberg et al., 1983, Eur J. Appl. Microbiol. Biotechnol. 18:287-292). In contrast to strains that are based on xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9), strains expressing XI activity display high alcohol yields and hardly produce xylitol as has recently been demonstrated in WO 03/0624430 and Kuyper et al. 2004, FEMS Yeast Res. 4:655-664. From a theoretical point of view this is not surprising since the route via XR and XD leads to an obstruction in the NADH balance that in the absence of oxygen, can be relieved e.g., via xylitol formation.
WO03/0624430 discloses that the introduction of a functional Piromyces XI into S. cerevisiae allows slow metabolism of xylose via the endogenous xylulokinase (EC 2.7.1.17) encoded by XKS1 and the enzymes of the non-oxidative part of the pentose phosphate pathway and confers to the yeast transformants the ability to grow on xylose.
Kuyper et al. (supra) describe S. cerevisiae strains in which the Piromyces XI has been introduced and which are thereafter subjected to directed evolution in shake flasks show improved rates of xylose fermentation, but still required oxygen for growth. Further selection via a regime of extreme oxygen limitation under xylose excess, followed by anaerobic selection resulted in a laboratory strain (RWB202-AFX) which fulfils at least one of the prerequisites for hemicellulose utilization, namely an acceptable ethanol yield on xylose. However, the specific rate of ethanol production in this strain is still unacceptably low. In particular, the specific sugar consumption rate during growth on xylose (345 mg xylose/g biomass/h) is still ten-fold lower than on glucose. Attempts to further improve strain RWB202-AFX via evolutionary engineering have failed so far.
WO03/0624430 lists a number of alternative genetic modifications that may result in further improvement of the specific rates of ethanol production and/or sugar consumption on xylose in host cells expressing the Piromyces XI gene to a level that would be required for commercial hemicellulose utilization. These alternatives include: (a) increase transport of xylose into the host cell; (b) increased xylulose kinase activity; (c) increased flux of the pentose phosphate pathway; (d) decreased sensitivity to catabolite repression; (e) increased tolerance to ethanol, osmolarity or organic acids; and, (f) reduced production of by-products (such as e.g., xylitol, glycerol and/or acetic acid). More specifically, WO03/0624430 suggests to overexpress one or more of the genes encoding a hexose or pentose transporter, a xylulose kinase (such as the S. cerevisiae XKS1) an enzyme from the pentose phosphate pathway such as a transaldolase (TAL1) or a transketolase (TKL1) glycolytic enzymes, ethanologenic enzymes such as alcohol dehydrogenases, and/or to inactivate a hexose kinase gene, e.g., the S. cerevisiae HXK2 gene, the S. cerevisiae MIG1 or MIG2 genes, the (nonspecific) aldose reductase genes such as the S. cerevisiae GRE3 gene, or genes for enzymes involved in glycerol metabolism such as the S. cerevisiae glycerol-phosphate dehydrogenase 1 and/or 2 genes. WO03/0624430 however does not disclose which of these many alternatives actually does produce an improvement in the specific rates of ethanol production and/or xylose consumption in host cells carrying the Piromyces XI gene.
Karhumaa et al. (2004, “Development of a Xylose-growing Saccharomyces cerevisiae strain expressing bacterial xylose isomerase”, Poster presentation at 2nd Meeting on Physiology of Yeasts and Filamentous Fungi; Mar. 24-28, 2004 Anglet, France, p 43; and, “New Xylose-growing Saccharomyces cerevisiae strain for biofuel ethanol production”, Oral presentation at the 26th Symposium on Biotechnology for Fuels and Chemicals, May 9-12, 2004 Chattanooga Tenn., USA, p. 19) disclose a strain of S. cerevisiae expressing a bacterial XI from Thermus thermophilus. The strain further contains a number of the genetic modifications suggested in WO03/0624430: overexpression of xylulose kinase and all four enzymes of the non-oxidative pentose phosphate pathway as well as inactivation of the S. cerevisiae nonspecific aldose reductase gene (GRE3). However, despite these genetic modifications this strain is incapable of growth on xylose. Only after adaptation to aerobic growth on xylose a strain, TMB3050, was obtained that is capable of growth on xylose at a low rate (μ=0.04 h−1) and with a low specific xylose consumption rate of 4.3 mg xylose/g cells/h. Since undefined genetic modifications (accumulated during adaptation) are clearly required for growth on xylose in the first place, one cannot deduce from the work of Karhumaa et al. (supra) which, if any, of the defined genetic modifications (such as overexpression of xylulose kinase or any of the pentose phosphate pathway enzymes or inactivation of the aldose reductase gene) actually contribute to the ability of the adapted strain to grow on xylose.
It is therefore an object of the present invention to provide for eukaryotic host cells, such as fungal host cells, that are transformed with a XI gene that confers the ability to grow on xylose and which host cells have specific rates of xylose consumption and/or product (ethanol) formation that are compatible with commercial application of the host cells.
DESCRIPTION OF THE INVENTION
Definitions
Xylose Isomerase
The enzyme “xylose isomerase” (EC 5.3.1.5) is herein defined as an enzyme that catalyses the direct isomerization of D-xylose into D-xylulose and vice versa. The enzyme is also known as a D-xylose ketoisomerase. Some xylose isomerases are also capable of catalyzing the conversion between D-glucose and D-fructose and are therefore sometimes referred to as glucose isomerase. Xylose isomerases require bivalent cations like magnesium or manganese as cofactor. Xylose isomerases of the invention may be further defined by their amino acid sequence as herein described below. Likewise xylose isomerases may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a xylose isomerase as herein described below.
A unit (U) of xylose isomerase activity is herein defined as the amount of enzyme producing 1 nmol of xylulose per minute, under conditions as described by Kuyper et al. (2003, FEMS Yeast Res. 4: 69-78).
Xylulose Kinase
The enzyme “xylulose kinase” (EC 2.7.1.17) is herein defined as an enzyme that catalyses the reaction ATP+D-xylulose→ADP+D-xylulose 5-phosphate. The enzyme is also known as a phosphorylating xylulokinase, D-xylulokinase or ATP:D-xylulose 5-phosphotransferase. A xylulose kinase of the invention may be further defined by its amino acid sequence as herein described below. Likewise a xylulose kinase may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a xylulose kinase as herein described below. A unit of xylulokinase activity is defined in Example 1.13 herein.
Ribulose 5-phosphate Epimerase
The enzyme “ribulose 5-phosphate epimerase” (5.1.3.1) is herein defined as an enzyme that catalyses the epimerization of D-xylulose 5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme is also known as phosphoribulose epimerase; erythrose-4-phosphate isomerase; phosphoketopentose 3-epimerase; xylulose phosphate 3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate 3-epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphate epimerase; D-ribulose-5-P 3-epimerase; D-xylulose-5-phosphate 3-epimerase; pentose-5-phosphate 3-epimerase; or D-ribulose-5-phosphate 3-epimerase. A ribulose 5-phosphate epimerase of the invention may be further defined by its amino acid sequence as herein described below. Likewise a ribulose 5-phosphate epimerase may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a ribulose 5-phosphate epimerase as herein described below.
Ribulose 5-phosphate Isomerase
The enzyme “ribulose 5-phosphate isomerase” (EC 5.3.1.6) is herein defined as an enzyme that catalyses direct isomerization of D-ribose 5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme is also known as phosphopentosisomerase; phosphoriboisomerase; ribose phosphate isomerase; 5-phosphoribose isomerase; D-ribose 5-phosphate isomerase; D-ribose-5-phosphate ketol-isomerase; or D-ribose-5-phosphate aldose-ketose-isomerase. A ribulose 5-phosphate isomerase of the invention may be further defined by its amino acid sequence as herein described below. Likewise a ribulose 5-phosphate isomerase may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a ribulose 5-phosphate isomerase as herein described below.
Transketolase
The enzyme “transketolase” (EC 2.2.1.1) is herein defined as an enzyme that catalyses the reaction:

D-ribose 5-phosphate+D-xylulose 5-phosphatesedoheptulose 7-phosphate+D-glyceraldehyde 3-phosphate

and vice versa. The enzyme is also known as glycolaldehydetransferase or sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glycolaldehydetransferase. A transketolase of the invention may be further defined by its amino acid sequence as herein described below. Likewise a transketolase may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a transketolase as herein described below.

Transaldolase
The enzyme “transaldolase” (EC 2.2.1.2) is herein defined as an enzyme that catalyses the reaction:

sedoheptulose 7-phosphate+D-glyceraldehyde 3-phosphateD-erythrose 4-phosphate+D-fructose 6-phosphate

and vice versa. The enzyme is also known as dihydroxyacetone transferase; dihydroxyacetone synthase; formaldehyde transketolase; or sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate glyceronetransferase. A transaldolase of the invention may be further defined by its amino acid sequence as herein described below. Likewise a transaldolase may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a transaldolase as herein described below.

Aldose Reductase
The enzyme “aldose reductase” (EC 1.1.1.21) is herein defined as any enzyme that is capable of reducing xylose or xylulose to xylitol. In the context of the present invention an aldose reductase may be any nonspecific aldose reductase that is native (endogenous) to a host cell of the invention and that is capable of reducing xylose or xylulose to xylitol. Nonspecific aldose reductases catalyze the reaction:

aldose+NAD(P)H+H+alditol+NAD(P)+

The enzyme has a wide specificity and is also known as aldose reductase; polyol dehydrogenase (NADP+); alditol:NADP oxidoreductase; alditol:NADP+ 1-oxidoreductase; NADPH-aldopentose reductase; or NADPH-aldose reductase. A particular example of such an nonspecific aldose reductase that is endogenous to S. cerevisiae and that is encoded by the GRE3 gene (Träff et al., 2001, Appl. Environ. Microbiol. 67:5668-74). Thus, an aldose reductase of the invention may be further defined by its amino acid sequence as herein described below. Likewise an aldose reductase may be defined by the nucleotide sequences encoding the enzyme as well as by nucleotide sequences hybridizing to a reference nucleotide sequence encoding a aldose reductase as herein described below.
Sequence Identity and Similarity
Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).
Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g., the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.; Altschul, S., et al., supra. The well-known Smith Waterman algorithm may also be used to determine identity.
Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the “Ogap” program from Genetics Computer Group, located in Madison, Wis. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch (supra); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.
Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.
Hybridizing Nucleic Acid Sequences
Nucleotide sequences encoding the enzymes of the invention may also be defined by their capability to hybridize with the nucleotide sequences of SEQ ID NO.'s 9-16 and 18, respectively, under moderate, or preferably under stringent hybridization conditions. Stringent hybridization conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridize at a temperature of about 65° C. in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength, and washing at 65° C. in a solution comprising about 0.1 M salt, or less, preferably 0.2×SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e., at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having about 90% or more sequence identity.
Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridize at a temperature of about 45° C. in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6×SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e., at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridization conditions in order to specifically identify sequences varying in identity between 50% and 90%.
Operably Linked
As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
Promoter
As used herein, the term “promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
Homologous
The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically be operably linked to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later. Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.
Heterologous
The term “heterologous” when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations where at least two of the combined sequences are foreign with respect to each other.
In this document and in its claims, the verb “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to transformed eukaryotic host cells that have the ability of isomerizing xylose to xylulose as e.g., described in WO 03/0624430. The ability of isomerizing xylose to xylulose is conferred to the host cell by transformation of the host cell with a nucleic acid construct comprising a nucleotide sequence encoding a xylose isomerase. The transformed host cell's ability to isomerize xylose into xylulose is the direct isomerization of xylose to xylulose. This is understood to mean that xylose isomerized into xylulose in a single reaction catalyzed by a xylose isomerase, as opposed to the two step conversion of xylose into xylulose via a xylitol intermediate as catalyzed by xylose reductase and xylitol dehydrogenase, respectively.
The nucleotide sequence encodes a xylose isomerase that is preferably expressed in active form in the transformed host cell. Thus, expression of the nucleotide sequence in the host cell produces a xylose isomerase with a specific activity of at least 10 U xylose isomerase activity per mg protein at 30° C., preferably at least 20, 25, 30, 50, 100, 200, 300 or 500 U per mg at 30° C. The specific activity of the xylose isomerase expressed in the transformed host cell is herein defined as the amount of xylose isomerase activity units per mg protein of cell free lysate of the host cell, e.g., a yeast cell free lysate. Determination of the xylose isomerase activity, amount of protein and preparation of the cell free lysate are as described in Example 1.13. Accordingly, expression of the nucleotide sequence encoding the xylose isomerase in the host cell produces a xylose isomerase with a specific activity of at least 50 U xylose isomerase activity per mg protein at 30° C., preferably at least 100, 200, 500, 750 or 1000 U per mg at 30° C.
Preferably, expression of the nucleotide sequence encoding the xylose isomerase in the host cell produces a xylose isomerase with a Km for xylose that is less than 50, 40, 30 or 25 mM, more preferably, the Km for xylose is about 20 mM or less.
A preferred nucleotide sequence encoding the xylose isomerase may be selected from the group consisting of:

    
    
        (a) nucleotide sequences encoding a polypeptide comprising an amino acid sequence that has at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO. 1 and/or SEQ ID NO. 2;
        (b) nucleotide sequences comprising a nucleotide sequence that has at least 40, 50, 60, 70, 80, 90, 95, 97, 98, or 99% sequence identity with the nucleotide sequence of SEQ ID NO. 9 and/or SEQ ID NO. 10;
        (c) nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule sequence of (a) or (b);
        (d) nucleotide sequences the sequence of which differs from the sequence of a nucleic acid molecule of (c) due to the degeneracy of the genetic code.
    
    


The nucleotide sequence encoding the xylose isomerase may encode either a prokaryotic or an eukaryotic xylose isomerase, i.e., a xylose isomerase with an amino acid sequence that is identical to that of a xylose isomerase that naturally occurs in the prokaryotic or eukaryotic organism. The present inventors have found that the ability of a particular xylose isomerase to confer to a eukaryotic host cell the ability to isomerize xylose into xylulose does not depend so much on whether the isomerase is of prokaryotic or eukaryotic origin. Rather this depends on the relatedness of the isomerase's amino acid sequence to that of the Piromyces sequence (SEQ ID NO. 1). Surprisingly, the eukaryotic Piromyces isomerase is more related to prokaryotic isomerases than to other known eukaryotic isomerases. The Piromyces isomerase shares 61% amino acid identity with a Xanthomonas enzyme and 82% with a Bacteroides enzyme (SEQ ID NO. 2), whereas it only shares 49-52% identity with several plant xylose isomerases. No reports have issued of a plant xylose isomerase that is actively expressed in yeast. In contrast, in Example 3 herein we describe that a Bacteroides xylose isomerase confers to a eukaryotic host cell the ability to isomerase xylose into xylulose and to grow on xylose as sole carbon source. Therefore, a preferred nucleotide sequence encodes a xylose isomerase having an amino acid sequence that is related to the Piromyces sequence as defined above. A preferred nucleotide sequence encodes a fungal xylose isomerase (e.g., from a Basidiomycete), more preferably a xylose isomerase from an anaerobic fungus, e.g., a xylose isomerase from an anaerobic fungus that belongs to the families Neocallimastix, Caecomyces, Piromyces, Orpinomyces, or Ruminomyces. Alternatively, a preferred nucleotide sequence encodes a bacterial xylose isomerase, preferably a Gram-negative bacterium, more preferably an isomerase from the class Bacteroides, or from the genus Bacteroides, most preferably from B. thetaiotaomicron (SEQ ID NO. 2).
To increase the likelihood that the xylose isomerase is expressed in active form in a eukaryotic host cell such as yeast, the nucleotide sequence encoding the xylose isomerase may be adapted to optimize its codon usage to that of the eukaryotic host cell. The adaptiveness of a nucleotide sequence encoding the xylose isomerase (or other enzymes of the invention, see below) to the codon usage of the host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; also see: Jansen et al., 2003, Nucleic Acids Res. 31:2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7.
A host cell for transformation with the nucleotide sequence encoding the xylose isomerase as described above, preferably is a host capable of active or passive xylose transport into the cell. The host cell preferably contains active glycolysis. The host cell may further contain an endogenous pentose phosphate pathway and may contain endogenous xylulose kinase activity so that xylulose isomerized from xylose may be metabolized to pyruvate. The host further preferably contains enzymes for conversion of pyruvate to a desired fermentation product such as ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, β-lactam antibiotics and cephalosporins.
A preferred host cell is a host cell that is naturally capable of alcoholic fermentation, preferably, anaerobic alcoholic fermentation. The host cell further preferably has a high tolerance to ethanol, a high tolerance to low pH (i.e., capable of growth at a pH lower than 5, 4, 3, or 2,5) and towards organic acids like lactic acid, acetic acid or formic acid and sugar degradation products such as furfural and hydroxy-methylfurfural, and a high tolerance to elevated temperatures. Any of these characteristics or activities of the host cell may be naturally present in the host cell or may be introduced or modified by genetic modification. A suitable host cell is a eukaryotic microorganism like e.g., a fungus, however, most suitable as host cell are yeasts or filamentous fungi.
Yeasts are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York) that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism. Preferred yeasts as host cells belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia. Preferably the yeast is capable of anaerobic fermentation, more preferably anaerobic alcoholic fermentation.
Filamentous fungi are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism of most filamentous fungi is obligately aerobic. Preferred filamentous fungi as host cells belong to the genera Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, and Penicillium. 
Over the years suggestions have been made for the introduction of various organisms for the production of bio-ethanol from crop sugars. In practice, however, all major bio-ethanol production processes have continued to use the yeasts of the genus Saccharomyces as ethanol producer. This is due to the many attractive features of Saccharomyces species for industrial processes, i.e., a high acid-, ethanol- and osmo-tolerance, capability of anaerobic growth, and of course its high alcoholic fermentative capacity. Preferred yeast species as host cells include S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, K. fragilis. 
The host cell of the invention is thus a host cell that is transformed with a nucleic acid construct comprising the nucleotide sequence encoding the xylose isomerase as defined above. The nucleic acid construct comprising the xylose isomerase coding sequence preferably is capable of expression of the xylose isomerase in the host cell. To this end the nucleic acid construct may be constructed as described in e.g., WO 03/0624430. The host cell may comprise a single but preferably comprises multiple copies of the nucleic acid construct. The nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g., be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmids. Preferably, however, the nucleic acid construct is integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by illegitimate recombination but preferably nucleic acid construct is integrated into the host cell's genome by homologous recombination as is well known in the art of fungal molecular genetics (see e.g., WO 90/14423, EP-A-0 481 008, EP-A-0 635 574 and U.S. Pat. No. 6,265,186).
In a first aspect of the invention, the host cell of the invention comprises a genetic modification that increases the flux of the pentose phosphate pathway. In particular, the genetic modification causes an increased flux of the non-oxidative part pentose phosphate pathway. A genetic modification that causes an increased flux of the non-oxidative part of the pentose phosphate pathway is herein understood to mean a modification that increases the flux by at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to the flux in a strain which is genetically identical except for the genetic modification causing the increased flux. The flux of the non-oxidative part of the pentose phosphate pathway may be measured by growing the modified host on xylose as sole carbon source, determining the specific xylose consumption rate and subtracting the specific xylitol production rate from the specific xylose consumption rate, if any xylitol is produced. However, the flux of the non-oxidative part of the pentose phosphate pathway is proportional with the growth rate on xylose as sole carbon source, preferably with the anaerobic growth rate on xylose as sole carbon source. There is a linear relation between the growth rate on xylose as sole carbon source (μmax) and the flux of the non-oxidative part of the pentose phosphate pathway. The specific xylose consumption rate (Qs) is equal to the growth rate (μ) divided by the yield of biomass on sugar (Yxs) because the yield of biomass on sugar is constant (under a given set of conditions: anaerobic, growth medium, pH, genetic background of the strain, etc.; i.e., Qs=μ/Yxs). Therefore the increased flux of the non-oxidative part of the pentose phosphate pathway may be deduced from the increase in maximum growth rate under these conditions.
Genetic modifications that increase the flux of the pentose phosphate pathway may be introduced in the host cell in various ways. These including e.g., achieving higher steady state activity levels of xylulose kinase and/or one or more of the enzymes of the non-oxidative part pentose phosphate pathway and/or a reduced steady state level of nonspecific aldose reductase activity. These changes in steady state activity levels may be effected by selection of mutants (spontaneous or induced by chemicals or radiation) and/or by recombinant DNA technology e.g., by overexpression or inactivation, respectively, of genes encoding the enzymes or factors regulating these genes.
In a preferred host cell, the genetic modification comprises overexpression of at least one enzyme of the (non-oxidative part) pentose phosphate pathway. Preferably the enzyme is selected from the group consisting of the enzymes encoding for ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase. Various combinations of enzymes of the (non-oxidative part) pentose phosphate pathway may be overexpressed. E.g., the enzymes that are overexpressed may be at least the enzymes ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase; or at least the enzymes ribulose-5-phosphate isomerase and transketolase; or at least the enzymes ribulose-5-phosphate isomerase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase and transketolase; or at least the enzymes ribulose-5-phosphate epimerase and transaldolase; or at least the enzymes transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate epimerase, transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, transketolase and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transaldolase; or at least the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, and transketolase. In one embodiment of the invention each of the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase are overexpressed in the host cell. More preferred is a host cell in which the genetic modification comprises at least overexpression of both the enzymes transketolase and transaldolase as such a host cell is already capable of anaerobic growth on xylose. In fact, under some conditions we have found that host cells overexpressing only the transketolase and the transaldolase already have the same anaerobic growth rate on xylose as do host cells that overexpress all four of the enzymes, i.e., the ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase. Moreover, host cells overexpressing both of the enzymes ribulose-5-phosphate isomerase and ribulose-5-phosphate epimerase are preferred over host cells overexpressing only the isomerase or only the epimerase as overexpression of only one of these enzymes may produce metabolic imbalances.
There are various means available in the art for overexpression of enzymes in the host cells of the invention. In particular, an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the host cell, e.g., by integrating additional copies of the gene in the host cell's genome, by expressing the gene from an episomal multicopy expression vector or by introducing a episomal expression vector that comprises multiple copies of the gene.
Alternatively overexpression of enzymes in the host cells of the invention may be achieved by using a promoter that is not native to the sequence coding for the enzyme to be overexpressed, i.e., a promoter that is heterologous to the coding sequence to which it is operably linked. Although the promoter preferably is heterologous to the coding sequence to which it is operably linked, it is also preferred that the promoter is homologous, i.e., endogenous to the host cell. Preferably the heterologous promoter is capable of producing a higher steady state level of the transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e., mRNA molecules, per unit of time) than is the promoter that is native to the coding sequence, preferably under conditions where xylose or xylose and glucose are available as carbon sources, more preferably as major carbon sources (i.e., more than 50% of the available carbon source consists of xylose or xylose and glucose), most preferably as sole carbon sources. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters. A preferred promoter for use in the present invention will in addition be insensitive to catabolite (glucose) repression and/or will preferably not require xylose for induction.
Promoters having these characteristics are widely available and known to the skilled person. Suitable examples of such promoters include e.g., promoters from glycolytic genes, such as the phosphofructokinase (PPK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts or filamentous fungi; more details about such promoters from yeast may be found in (WO 93/03159). Other useful promoters are ribosomal protein encoding gene promoters, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADH1, ADH4, and the like), and the enolase promoter (ENO). Other promoters, both constitutive and inducible, and enhancers or upstream activating sequences will be known to those of skill in the art. The promoters used in the host cells of the invention may be modified, if desired, to affect their control characteristics.
The coding sequence used for overexpression of the enzymes preferably is homologous to the host cell of the invention. However, coding sequences that are heterologous to the host cell of the invention may likewise be applied.
A nucleotide sequence used for overexpression of ribulose-5-phosphate isomerase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with ribulose-5-phosphate isomerase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 4 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 12, under moderate conditions, preferably under stringent conditions.
A nucleotide sequence used for overexpression of ribulose-5-phosphate epimerase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with ribulose-5-phosphate epimerase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 5 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 13, under moderate conditions, preferably under stringent conditions.
A nucleotide sequence used for overexpression of transketolase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with transketolase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 6 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 14, under moderate conditions, preferably under stringent conditions.
A nucleotide sequence used for overexpression of transaldolase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with transaldolase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 7 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 15, under moderate conditions, preferably under stringent conditions.
Overexpression of an enzyme, when referring to the production of the enzyme in a genetically modified host cell, means that the enzyme is produced at a higher level of specific enzymatic activity as compared to the unmodified host cell under identical conditions. Usually this means that the enzymatically active protein (or proteins in case of multi-subunit enzymes) is produced in greater amounts, or rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Similarly this usually means that the mRNA coding for the enzymatically active protein is produced in greater amounts, or again rather at a higher steady state level as compared to the unmodified host cell under identical conditions. Overexpression of an enzyme is thus preferably determined by measuring the level of the enzyme's specific activity in the host cell using appropriate enzyme assays as described herein. Alternatively, overexpression of the enzyme may determined indirectly by quantifying the specific steady state level of enzyme protein, e.g., using antibodies specific for the enzyme, or by quantifying the specific steady level of the mRNA coding for the enzyme. The latter may particularly be suitable for enzymes of the pentose phosphate pathway for which enzymatic assays are not easily feasible as substrates for the enzymes are not commercially available. Preferably in the host cells of the invention, an enzyme to be overexpressed is overexpressed by at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
In a second aspect of the invention, the host cell of the invention comprises a genetic modification that increases the specific xylulose kinase activity. Preferably the genetic modification causes overexpression of a xylulose kinase, e.g., by overexpression of a nucleotide sequence encoding a xylulose kinase. The gene encoding the xylulose kinase may be endogenous to the host cell or may be a xylulose kinase that is heterologous to the host cell. A nucleotide sequence used for overexpression of xylulose kinase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with xylulose kinase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 3 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 11, under moderate conditions, preferably under stringent conditions.
A particularly preferred xylulose kinase is a xylose kinase that is related to the xylulose kinase from Piromyces (xylB; see WO 03/0624430). This Piromyces xylulose kinase is actually more related to prokaryotic kinase than to all of the known eukaryotic kinases such as the yeast kinase (SEQ ID NO. 3). The eukaryotic xylulose kinases have been indicated as non-specific sugar kinases, which have a broad substrate range that includes xylulose. In contrast, the prokaryotic xylulose kinases, to which the Piromyces kinase is most closely related, have been indicated to be more specific kinases for xylulose, i.e., having a narrower substrate range. Therefore, a more preferred nucleotide sequence for use in overexpression of xylulose kinase in the host cell of the invention is a nucleotide sequence encoding a polypeptide with xylulose kinase activity, whereby preferably the polypeptide has an amino acid sequence having at least 45, 50, 55, 60, 65, 70, 80, 90 or 95% identity with SEQ ID NO. 17 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 18, under moderate conditions, preferably under stringent conditions.
In the host cells of the invention, genetic modification that increases the specific xylulose kinase activity may be combined with any of the modifications increasing the flux of the pentose phosphate pathway as described above, but this combination is not essential for the invention. Thus, a host cell of the invention comprising only a genetic modification that increases the specific xylulose kinase activity is specifically included in the invention. The various means available in the art for achieving and analyzing overexpression of a xylulose kinase in the host cells of the invention are the same as described above for enzymes of the pentose phosphate pathway. Preferably in the host cells of the invention, a xylulose kinase to be overexpressed is overexpressed by at least a factor 1.1, 1.2, 1.5, 2, 5, 10 or 20 as compared to a strain which is genetically identical except for the genetic modification causing the overexpression. It is to be understood that these levels of overexpression may apply to the steady state level of the enzyme's activity, the steady state level of the enzyme's protein as well as to the steady state level of the transcript coding for the enzyme.
In a third aspect of the invention, the host cell of the invention comprises a genetic modification that reduces nonspecific aldose reductase activity in the host cell. Preferably, nonspecific aldose reductase activity is reduced in the host cell by one or more genetic modifications that reduce the expression of or inactivates a gene encoding an nonspecific aldose reductase. Preferably, the genetic modifications reduce or inactivate the expression of each endogenous copy of a gene encoding an nonspecific aldose reductase in the host cell. Host cells may comprise multiple copies of genes encoding nonspecific aldose reductases as a result of di-, poly- or aneuploidy, and/or the host cell may contain several different (iso)enzymes with aldose reductase activity that differ in amino acid sequence and that are each encoded by a different gene. Also in such instances preferably the expression of each gene that encodes an nonspecific aldose reductase is reduced or inactivated. Preferably, the gene is inactivated by deletion of at least part of the gene or by disruption of the gene, whereby in this context the term gene also includes any non-coding sequence up- or down-stream of the coding sequence, the (partial) deletion or inactivation of which results in a reduction of expression of nonspecific aldose reductase activity in the host cell. A nucleotide sequence encoding an aldose reductase whose activity is to be reduced in the host cell of the invention is a nucleotide sequence encoding a polypeptide with aldose reductase activity, whereby preferably the polypeptide has an amino acid sequence having at least 50, 60, 70, 80, 90 or 95% identity with SEQ ID NO. 8 or whereby the nucleotide sequence is capable of hybridizing with the nucleotide sequence of SEQ ID NO. 16 under moderate conditions, preferably under stringent conditions.
In the host cells of the invention, genetic modification that reduces nonspecific aldose reductase activity in the host cell may be combined with any of the modifications increasing the flux of the pentose phosphate pathway and/or with any of the modifications increasing the specific xylulose kinase activity in the host cells as described above, but these combinations are not essential for the invention. Thus, a host cell of the invention comprising only a genetic modification that reduces nonspecific aldose reductase activity in the host cell is specifically included in the invention.
In a further aspect the invention relates to modified host cells that are further adapted to xylose utilization by selection of mutants, either spontaneous or induced (e.g., by radiation or chemicals), for growth on xylose, preferably on xylose as sole carbon source, and more preferably under anaerobic conditions. Selection of mutants may be performed by serial passaging of cultures as e.g., described by Kuyper et al. (2004, FEMS Yeast Res. 4: 655-664) or by cultivation under selective pressure in a chemostat culture as is described in Example 4 herein.
In a preferred host cell of the invention at least one of the genetic modifications described above, including modifications obtained by selection of mutants, confer to the host cell the ability to grow on xylose as carbon source, preferably as sole carbon source, and preferably under anaerobic conditions. Preferably the modified host cell produce essentially no xylitol, e.g., the xylitol produced is below the detection limit or e.g., less than 5, 2, 1, 0.5, or 0.3% of the carbon consumed on a molar basis.
Preferably the modified host cell has the ability to grow on xylose as sole carbon source at a rate of at least 0.05, 0.1, 0.2, 0.25 or 0.3 h−1 under aerobic conditions, or, if applicable, at a rate of at least 0.03, 0.05, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15 or 0.2 h−1 under anaerobic conditions. Preferably the modified host cell has the ability to grow on a mixture of glucose and xylose (in a 1:1 weight ratio) as sole carbon source at a rate of at least 0.05, 0.1, 0.2, 0.25 or 0.3 h−1 under aerobic conditions, or, if applicable, at a rate of at least 0.03, 0.05, 0.1, 0.12, 0.15, or 0.2 h−1 under anaerobic conditions.
Preferably, the modified host cell has a specific xylose consumption rate of at least 346, 350, 400, 500, 600, 750, or 1000 mg xylose/g cells/h. Preferably, the modified host cell has a yield of fermentation product (such as ethanol) on xylose that is at least 55, 60, 70, 80, 85, 90, 95 or 98% of the host cell's yield of fermentation product (such as ethanol) on glucose. More preferably, the modified host cell's yield of fermentation product (such as ethanol) on xylose is equal to the host cell's yield of fermentation product (such as ethanol) on glucose. Likewise, the modified host cell's biomass yield on xylose is preferably at least 55, 60, 70, 80, 85, 90, 95 or 98% of the host cell's biomass yield on glucose. More preferably, the modified host cell's biomass yield on xylose is equal to the host cell's biomass yield on glucose. It is understood that in the comparison of yields on glucose and xylose both yields are compared under aerobic conditions or both under anaerobic conditions.
In a preferred aspect, the modified host cell of the invention is a host cell for the production of ethanol. In another aspect the invention relates to a transformed host cell for the production of fermentation products other than ethanol. Such non-ethanolic fermentation products include in principle any bulk or fine chemical that is producible by a eukaryotic microorganism such as a yeast or a filamentous fungus. Such fermentation products include e.g., lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, β-lactam antibiotics and cephalosporins. A preferred modified host cell of the invention for production of non-ethanolic fermentation products is a host cell that contains a genetic modification that results in decreased alcohol dehydrogenase activity.
In a further aspect the invention relates to fermentation processes in which the modified host cells of the invention are used for the fermentation of a carbon source comprising a source of xylose, such as xylose. In addition to a source of xylose the carbon source in the fermentation medium may also comprise a source of glucose. The source of xylose or glucose may be xylose or glucose as such or may be any carbohydrate oligo- or polymer comprising xylose or glucose units, such as e.g., lignocellulose, xylans, cellulose, starch and the like. For release of xylose or glucose units from such carbohydrates, appropriate carbohydrases (such as xylanases, glucanases, amylases and the like) may be added to the fermentation medium or may be produced by the modified host cell. In the latter case the modified host cell may be genetically engineered to produce and excrete such carbohydrases. An additional advantage of using oligo- or polymeric sources of glucose is that it enables to maintain a low(er) concentration of free glucose during the fermentation, e.g., by using rate-limiting amounts of the carbohydrases. This, in turn, will prevent repression of systems required for metabolism and transport of non-glucose sugars such as xylose. In a preferred process the modified host cell ferments both the xylose and glucose, preferably simultaneously in which case preferably a modified host cell is used which is insensitive to glucose repression to prevent diauxic growth. In addition to a source of xylose (and glucose) as carbon source, the fermentation medium will further comprise the appropriate ingredient required for growth of the modified host cell. Compositions of fermentation media for growth of microorganisms such as yeasts are well known in the art.
The fermentation process is a process for the production of a fermentation product such as e.g., ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, β-lactam antibiotics, such as Penicillin G or Penicillin V and fermentative derivatives thereof, and cephalosporins. The fermentation process may be an aerobic or an anaerobic fermentation process. An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e., oxygen consumption is not detectable), and wherein organic molecules serve as both electron donor and electron acceptors. In the absence of oxygen, NADH produced in glycolysis and biomass formation, cannot be oxidized by oxidative phosphorylation. To solve this problem many microorganisms use pyruvate or one of its derivatives as an electron and hydrogen acceptor thereby regenerating NAD+. Thus, in a preferred anaerobic fermentation process pyruvate is used as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, β-lactam antibiotics and cephalosporins.
The fermentation process is preferably run at a temperature that is optimal for the modified host cell. Thus, for most yeasts or fungal host cells, the fermentation process is performed at a temperature which is less than 42° C., preferably less than 38° C. For yeast or filamentous fungal host cells, the fermentation process is preferably performed at a temperature which is lower than 35, 33, 30 or 28° C. and at a temperature which is higher than 20, 22, or 25° C.
A preferred process is a process for the production of ethanol, whereby the process comprises the steps of: (a) fermenting a medium containing a source of xylose with a modified host cell as defined above, whereby the host cell ferments xylose to ethanol; and optionally, (b) recovery of the ethanol. The fermentation medium may also comprise a source of glucose that is also fermented to ethanol. In the process the volumetric ethanol productivity is preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per liter per hour. The ethanol yield on xylose and/or glucose in the process preferably is at least 50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as a percentage of the theoretical maximum yield, which, for glucose and xylose is 0.51 g. ethanol per g. glucose or xylose.
In a further aspect the invention relates to a process for producing a fermentation product selected from the group consisting of lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propane-diol, ethylene, glycerol, β-lactam antibiotics and cephalosporins. The process preferably comprises the steps of (a) fermenting a medium containing a source of xylose with a modified host cell as defined herein above, whereby the host cell ferments xylose to the fermentation product, and optionally, (b) recovery of the fermentation product. In a preferred process, the medium also contains a source of glucose.
Genetic Modifications
For overexpression of enzymes in the host cells of the inventions as described above, as well as for additional genetic modification of host cells, preferably yeasts, host cells are transformed with the various nucleic acid constructs of the invention by methods well known in the art. Such methods are e.g., known from standard handbooks, such as Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of fungal host cells are known from e.g., EP-A-0 635 574, WO98/46772, WO99/60102 and WO00/37671.
Promoters for use in the nucleic acid constructs for overexpression of enzymes in the host cells of the invention have been described above. In the nucleic acid constructs for overexpression, the 3′-end of the nucleotide acid sequence encoding the enzyme(s) preferably is operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice, such as e.g., the yeast species of choice. In any case the choice of the terminator is not critical; it may e.g., be from any yeast gene, although terminators may sometimes work if from a non-yeast, eukaryotic, gene. The transcription termination sequence further preferably comprises a polyadenylation signal.
Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term “marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Examples of suitable antibiotic resistance markers include e.g., dihydrofolate reductase, hygromycin-B-phosphotransferase, 3′-O-phosphotransferase II (kanamycin, neomycin and G418 resistance). Although the of antibiotic resistance markers may be most convenient for the transformation of polyploid host cells, preferably however, non-antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU2) or the S. pombe TPI gene (described by Russell P R, 1985, Gene 40: 125-130). In a preferred embodiment the host cells transformed with the nucleic acid constructs are marker gene free. Methods for constructing recombinant marker gene free microbial host cells are disclosed in EP-A-0 635 574 and are based on the use of bidirectional markers such as the A. nidulans amdS (acetamidase) gene or the yeast URA3 and LYS2 genes. Alternatively, a screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into the nucleic acid constructs of the invention allowing to screen for transformed cells.
Optional further elements that may be present in the nucleic acid constructs of the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromeres, telomeres and/or matrix attachment (MAR) sequences. The nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g., be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9:968-975) plasmids. Alternatively the nucleic acid construct may comprise sequences for integration, preferably by homologous recombination. Such sequences may thus be sequences homologous to the target site for integration in the host cell's genome. The nucleic acid constructs of the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and Russell (supra).
Methods for inactivation and gene disruption in yeast or filamentous fungi are well known in the art (see e.g., Fincham, 1989, Microbiol Rev. 53:148-70 and EP-A-0 635 574).



DESCRIPTION OF THE FIGURES
FIG. 1.
Typical graph of anaerobic growth of strain RWB 212 in fermentors on synthetic medium with 2% (w/v) xylose as the carbon source, duplicate experiments differed by less than 5%. Panel A: Xylose (●), ethanol (◯), glycerol (▪) and cumulative CO2 produced per liter as deduced from gas analysis (-). Panel B: dry weight (●), acetate (◯), xylitol (□), succinate (▴), lactate (Δ).
FIG. 2.
Typical graph of anaerobic growth of strain RWB 212 in fermenters on synthetic medium with 2% (w/v) glucose and 2% (w/v) xylose as the carbon source, duplicate experiments differed by less than 5%. Panel A: Glucose (●), xylose (◯), ethanol (▪), glycerol (□) and cumulative CO2 produced per liter as deduced from gas analysis (-). Panel B: dry weight (●), acetate (◯), xylitol (▪), lactate (□) succinate (▴).
FIG. 3.
Panel A: Residual xylose concentrations during anaerobic chemostat cultivation of RWB 212 on 30 g/l xylose as the carbon source. Data presented are the average of two independent chemostats and the experimental deviations.
Panel B: Culture dry weight during anaerobic chemostat cultivation of RWB 212 on 30 g/l xylose as the carbon source. Data presented are the average of two independent chemostats and the experimental deviations.
FIG. 4.
Carbon dioxide production profiles of three xylose metabolizing strains in anaerobic batch cultivations on glucose and xylose (20 g/l each). Exact experimental conditions varied so actual numeric values may not be compared.
FIG. 5.
Concentrations of glucose, xylose, ethanol, glycerol and CO2 measured during two independent anaerobic fermentor batches on 2% glucose and 2% xylose of selected strains originating from RWB 212.



EXAMPLES
1. Materials and Methods
1.1. Plasmid Construction
In order to integrate the TPI1 promoter in front of our target genes several plasmids were constructed. First the TPI1 promoter was cut as a XhoI-EcoRV fragment from pYX012-Aat (A. A. Winkler, derivative of pYX012 (R&D Systems, Minneapolis, Minn., USA)) and ligated to pUG6 [3] cut with SalI-PvuII. This gave us pUG6PTPI, which could then be used to PCR a Kanlox-PTPI integration cassette.
In cases were putative ORF's were located very close to the ATG of the target genes, we cloned those genes into pUG6PTPI. A 0.8 kb RPE1 fragment and a 2.3 kb TKL1 fragment were isolated from gel and cut with EcoRI and XhoI (present in the primers, see Table 3) and ligated into pUG6PTPI digested with EcoRI-SalI, resulting in pUG6PTPI-RPE1 and pUG6PTPI-TKL1.
In order to increase the activity of xylulokinase the XKS1 gene was amplified by PCR as a SpeI-SalI fragment (sites in the primers, see Table 3) and cloned into p415ADH [4] cut with XbaI-XhoI, resulting in p415ADHXKS.
Restriction endonucleases (New England Biolabs, Beverly, Mass., USA and Roche, Basel, Switzerland) and DNA ligase (Roche) were used according to the manufacturers' specifications. Plasmid isolation from E. coli was performed with the Qiaprep spin miniprep kit (Qiagen, Hilden, Germany). DNA fragments were separated on a 1% agarose (Sigma, St. Louis, Mo., USA) gel in 1×TBE [5]. Isolation of fragments from gel was carried out with the Qiaquick gel extraction kit (Quiagen). Amplification of RPE1, TKL1 and XKS1 was done with VentR DNA polymerase (New England Biolabs) according to the manufacturer's specification. The template was chromosomal DNA of CEN.PK113-7D (wt). The polymerase chain reaction (PCR) was performed in a Biometra TGradient Thermocycler (Biometra, Göttingen, Germany) with the following settings: 30 cycles of 1 min annealing at 60° C., 3 min extension at 75° C. and 1 min denaturing at 94° C.
1.2. Strains and Media
The Saccharomyces cerevisiae strain used in this study is RWB212 (MATA ura3-52 leu2-112 loxP-PTPI::(−266,−1)TAL1 gre3::hphMX pUGPTPI-TKL1 pUGPTPI-RPE1 KanloxP-PTPI::(−?,−1)RKI1), which is derived from CEN.PK102-3A (MATA ura3-52 leu2-112).
During construction strains were maintained on complex (YP: 10 g/l yeast extract (BD Difco), 20 g/l peptone (BD Difco)) or synthetic medium (MY) [6] supplemented with glucose (2%) as carbon source (YPD or MYD) and 1.5% agar in the case of plates After transformation integrants were selected by plating on YPD containing Geneticin® (G418) (Invitrogen/GIBCO) at 200 μg/ml or hygromycin B (Roche Diagnostics GmbH, Mannheim, Germany) at 300 μg/ml. After transformation with plasmids strains were plated on MYD. Transformations of yeast were done according to Gietz and Woods [7].
Plasmids were amplified in Escherichia coli strain XL-1 blue (Stratagene, La Jolla, Calif., USA). Transformation was performed according to Inoue et al. [8]. E. coli was grown on LB (Luria-Bertani) plates or in liquid TB (Terrific Broth) medium for the isolation of plasmids [5].
1.3. Strain Construction
For TAL1 and RKI1 integration of the TPI1 promoter 5′ of the open reading frame was done by amplifying a PCR fragment with the KanMX marker and the TPI1 promoter and directing it to the target location via homologous ends. The PCR was performed with Taq DNA polymerase (Amersham Pharmacia, Piscataway, USA) according to the manufacturer's specifications. The template was pUG6PTPI with PTALdisA and PTALdisB or PRKIdisA and PRKIdisB (Table 3) as primers.
In the case of TKL1 and RPE1, plasmids pUG6PTPI-TKL1 and pUG6PTPI-RPE1 were linearized with PvuII and SalI respectively and integrated into the genome. Correct integration of the constructs was verified colony PCR with TAL1 intern+KanA for TAL1 and PTPI primer+“intern” for TKL1, RPE1, and RPI1. The “intern” primers anneal downstream of the integrated constructs, while PTPI primer anneals at the 3′ end of the TPI1 promoter.
After integration of a construct the KanMX marker was removed with the cre recombinase. To this end strains were transformed with pSH47 [3]. Colonies with the plasmid were resuspended in YP with 1% galactose and incubated for 1 hour at 30° C. Then about 200 cells were plated on YPD. The resulting colonies were checked for loss of the KanMX marker and pSH47 (URA3).
In addition the GRE3 gene was replaced by the hphMX cassette from pAG32, conferring hygromycin resistance [9]. The hphMX cassette was amplified using oligo's 5′ gre3::Kanlox and 3′ gre3::Kanlox. Correct integration was verified using by PCR with 5′GRE3+KanA and 3′GRE3+KanB (Table 3). KanA and KanB anneal to the A. gossipi TEF promoter and terminator respectively, while the other primers anneal outside of the GRE3 open reading frame.
Colony PCR was done with Taq DNA polymerase (Amersham Pharmacia, Piscataway, USA) according to the manufacturer's specifications. As template cells were resuspended in 2.5 μl 0.02M NaOH to which the PCR reaction mixture was added. The PCR was performed in a Biometra TGradient Thermocycler (Biometra, Göttingen, Germany) with the following settings: 30 cycles of 1 min annealing at 60° C., 3 min extension at 72° C. and 1 min denaturing at 94° C.
The resulting strain, RWB212 (MATA ura3-52 leu2-112 loxP-PTPI::(−266,−1)TAL1 gre3::hphMX pUGPTPI-TKL1 pUGPTPI-RPE1 KanloxP-PTPI:: (−?,−1)RKI1), was then transformed with pAKX002, a multicopy vector containing the Piromyces sp. E2 XylA behind the TPI1 promoter, as well as p415ADHXKS. Which gave us RWB217 (MATA ura3-52 leu2-112 loxP-PTPI::(−266,−1)TAL1 gre3::hphMX pUGPTPI-TKL1 pUGPTPI-RPE1 KanloxP-PTPI::(−?,−1)RKI1 {pAKX002, p415ADHXKS}).
1.4. Strain Maintenance
Stock cultures were grown at 30° C. in shake flasks on synthetic medium [6] supplemented with 20 g of glucose/1. When stationary phase was reached, sterile glycerol was added to 30% (vol/vol), and 2-ml aliquots were stored in sterile vials at −80° C.
1.5. Cultivation and Media
Shake-flask cultivation was performed at 30° C. in a synthetic medium [6]. The pH of the medium was adjusted to 6.0 with 2 M KOH prior to sterilization. Precultures were prepared by inoculating 100 ml medium containing 20 g/l xylose in a 500 ml shake-flask with a frozen stock culture. After 24 to 48 h incubation at 30° C. in an orbital shaker (200 rpm), this culture was used to inoculate either shake-flask cultures or fermenter cultures. The synthetic medium for anaerobic cultivation was supplemented with 0.01 g/l ergosterol and 0.42 g/l Tween 80 dissolved in ethanol [10,11], this resulted in 11-13 mM ethanol in the medium.
1.6. Anaerobic Batch Cultivation in Fermenters
Anaerobic batch cultures were carried out in 2-liter laboratory fermenters (Applikon, Schiedam, The Netherlands) equipped with Norprene tubing, with a working volume of 1.5 liters, at 30° C. and at pH 5.0. Cultures were stirred at 800 rpm and sparged with 0.5 l/min of high-grade nitrogen (<5 ppm oxygen). The synthetic medium was supplemented with the anaerobic growth factors ergosterol and Tween 80 (0.01 and 0.42 g/l, respectively) as well as 100 μl/l of silicone antifoam (BDH, Poole, UK).
1.7. Determination of Culture Dry Weight
Culture samples (10.0 ml) were filtered over preweighed nitrocellulose filters (pore size 0.45 μm; Gelman Laboratory, Ann Arbor, USA). After removal of medium the filters were washed with demineralized water and dried in a microwave oven (Bosch, Stuttgart, Germany) for 20 min at 360 W and weighed. Duplicate determinations varied by less than 1%.
1.8. Gas Analysis
Exhaust gas was cooled in a condensor (2° C.) and dried with a Permapure dryer type MD-110-48P-4 (Permapure, Toms River, USA). O2 and CO2 concentrations were determined with a NGA 2000 analyzer (Rosemount Analytical, Orrville, USA). Exhaust gas-flow rate and specific oxygen-consumption and carbon-dioxide production rates were determined as described previously [12,13]. In calculating these biomass-specific rates, a correction was made for volume changes caused by withdrawing culture samples.
1.9. Metabolite Analysis
Glucose, xylose, xylitol, organic acids, glycerol and ethanol were detected by HPLC analysis on a Waters Alliance 2690 HPLC (Waters, Milford, USA) containing a Biorad HPX 87H column (Biorad, Hercules, USA). The column was eluted at 60° C. with 0.5 g/l H2SO4 at a flow rate of 0.6 ml/min. Detection was by means of a Waters 2410 refractive-index detector and a Waters 2487 UV detector. Xylulose was determined enzymatically in the following manner. The reaction mixture consisted of 100 mM Tris-HCl buffer (pH7.5) with 10 mM MgCl2, 0.30 mM NADH and an adequate amount of sample (1 ml total volume) the assay was started by the addition of 0.2 U sorbitol dehydrogenase (Sigma, St Louis, USA). The xylulose concentration was calculated using an absorption coefficient of 6.3 mM/cm for NADH.
1.10. Carbon Recoveries and Ethanol Evaporation
Carbon recoveries were calculated as carbon in products formed divided by the total amount of sugar carbon consumed and were based on a carbon content of biomass of 48%. To correct for ethanol evaporation during the fermentations, the amount of ethanol produced was assumed to be equal to the measured cumulative production of CO2 minus the CO2 production that occurred due to biomass synthesis (5.85 mmol CO2 per gram biomass [14]) and the CO2 associated with acetate formation as described previously [2].
1.11 Microarray Analysis
Sampling of cells from chemostats, probe preparation and hybridization to Affymetrix Genechip® microarrays were performed as described previously [15]. The results for each growth condition were derived from three independently cultured replicates.
1.12. Data Acquisition and Analysis
Acquisition and quantification of array images and data filtering were performed using the Affymetrix software packages: Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.
Before comparison, all arrays were globally scaled to a target value of 150 using the average signal from all gene features using Microarray Suite v5.0. From the 9,335 transcript features on the YG-S98 arrays a filter was applied to extract 6,383 yeast open reading frames of which there were 6,084 different genes. This discrepancy was due to several genes being represented more than once when sub-optimal probe sets were used in the array design.
To represent the variation in triplicate measurements, the coefficient of variation (C.V.; standard deviation divided by the mean) was calculated as previously described by Boer et al. [16].
For further statistical analyses Microsoft Excel running the Significant Analysis of Microarrays (SAM v1.12) add in was used [17] for possible pair wise comparisons of the eight data sets. Genes were considered as being changed in expression if they were called significantly changed using SAM (expected median false-discovery rate (FDR) of 1%) by at least 2-fold from each other condition. Hierarchical clustering of the obtained sets of significantly changed expression levels was subsequently performed by Genespring (Silicon Genetics).
1.13 Enzyme Assays
Xylose isomerase activity was assayed at 37° C. in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 10 mM xylose, 10 mM MgCl2 and a suitable amount of cell-free extract. The amount of xylulose formed was determined by the cysteine-carbazole method (9). Alternatively xylose isomerase activity was assayed at 30° C. enzyme assay as developed by Kersters-Hildersson et al. (Kinetic characterization of D-xylose isomerases by enzymatic assays using D-sorbitol dehydrogenase. Enz. Microb. Technol. 9 (1987) 145-148). The in vitro activity of xylose isomerase in the cell-free extracts of transformed S. cerevisiae strains is dependent on bivalent cations (Mg2+ or Co2+).
Xylulose kinase and xylose reductase activities were assayed as described by Witteveen et al. (28). One unit of activity is defined as the amount of enzyme producing 1 nmol of xylulose per min under the assay conditions. Xylulose formed was determined by the method of Dische and Borenfreund (Dische and Borenfreund, 1951, J. Biol. Chem. 192:583-587) or by HPLC using a Biorad HPX-87N Column operated at 80° C. and eluted at 0.6 ml/min using 0.01 M Na2HPO4 as the eluent. Xylose and xylulose were detected by a Refractive Index detector at an internal temperature of 60° C.
Specific activity is expressed as units per mg protein. Protein was determined with the Bio-Rad protein reagent (Bio-Rad Laboratories, Richmond, Calif., USA) with bovine γ-globulin as a standard.
2. Results
2.1 Overexpression of the Pentose Phosphate Pathway (PPP) Genes
Previously we have shown that expressing a fungal xylose isomerase in Saccharomyces cerevisiae is in principle enough to allow anaerobic growth of this yeast on xylose as the sole carbon source provided that sufficient selective pressure is applied [2]. The selected strain still however, did not perform up to industrial requirements (Table 1).
In order to investigate the possibility of rate limiting steps in pentose phosphate metabolism it was decided to construct a strain overproducing all the enzymes required to convert xylose into fructose-6-phosphate and glyceraldehyde-3-phosphate. The overexpressed enzymes were: xylose isomerase (XI), xylulokinase (XKS), ribulose-5-phosphate isomerase (R5PI), ribulose-5-phosphate epimerase (R5PE), transketolase (TKT) and transaldolase (TAL). Additionally the non-specific aldose reductase encoded by GRE3, which mediates unwanted production of xylitol [18] was deleted. Since some of the substrates of the enzymes in the PPP are not commercially available it was decided to check for overexpression via DNA arrays rather than via enzyme activity measurements. The results listed in Table 1 further confirmed that the transcription of the target genes was successfully modified in strain RWB 212.







TABLE 1







mRNA levels of structural genes encoding xylulokinase and pentose-phosphate-


pathway enzymes in the reference strain S. cerevisiae CEN.PK113-7D and in the engineered,


xylose-isomerase-expressing strain S. cerevisiae RWB212.












Systematic

Transcript level
Fold-change












Gene
name
Enzyme name
CEN.PK113-7D
RBW212
Mutant vs WT





XylA

xylose isomerase
n.d.
n.d.



XKS1
YGR194C
Xylulokinase
91 ± 7 
321 ± 54 
+3.5


TAL1
YLR354C
Transaldolase
574 ± 49 
959 ± 91 
+1.7


TKL1
YPR074C
transketolase 1
450 ± 37 
1982 ± 79 
+4.4


RPE1
YJL121C
D-ribulose-5-phosphate
299 ± 24 
2551 ± 385 
+8.5




3-epimerase





RKI1
YOR095C
D-ribose-5-phosphate
96 ± 8 
483 ± 64 
+5.0




ketol-isomerase





GRE3
YHR104w
aldose reductase
322 ± 6 
12 ± 0 
−26.8 


ACT1
YFL039C
Actin
408 ± 32 
366 ± 56 
 NCa


PDA1
YER178W
E1α subunit of pyruvate
2901 ± 142 
3217 ± 182 
NC




dehydrogenase complex





n.d. = not determined (not represented on Affymetrix microarrays);


aNC = not changed.






Both strains were grown in aerobic, glucose-limited chemostat cultures (D=0.10 h−1). Transcript levels were determined with Affymetrix GeneChip microarrays. Data are the average±average deviation of the mean of analyses on three independent cultures for each strain. ACT1 (Ng and Abelson, 1980, Proc. Nat. Acad. Sci. USA. 77:3912-3916) and PDA1 (Wenzel et al., 1995, Nucleic. Acids Res. 23:883-884) are included as internal standards.

2.2 Physiological Characterization of the Engineered Strain

One of the striking properties of the engineered strain was its ability to grow anaerobically on xylose (FIG. 1) without any selective pressure being required. Anaerobic growth on xylose in mineral medium proceeded with a growth rate as high as 0.09 h−1. Xylulose was not accumulated but xylitol formation, though extremely small, was detectable (FIG. 1) biomass, ethanol and glycerol yields of stain RWB 212 on xylose were comparable to those of RWB 202-AFX which was obtained via evolutionary engineering (Table 2). From Table 2 a specific xylose consumption rate of more than 1.0 g xylose/g biomass/h can be calculated (Qs=0.09/0.085=1,059 gram Xyl/gram X/h), compared to 345 mg xylose/g biomass/h for RWB 202-AFX while a yield at least similar to the yield on glucose was obtained.
2.3 Mixed Substrate Utilization
As pointed out in the introduction: economic conversion of hemicellulose hydrolysates to ethanol requires the fermentation of both glucose and xylose, preferably simultaneously. In order to verify the properties of strain RWB 212 with respect to mixed sugar utilization, the yeast was grown in a mixture of glucose and xylose (20 g/l each). The results depicted in FIG. 2 show that both sugars were completely consumed but glucose was the preferred substrate. Xylose consumption commenced after approximately 80% of the glucose was consumed.
3. Functional Expression of the B. thetaiotaomicron Xylose Isomerase in Yeast
The nucleotide sequence encoding the B. thetaiotaomicron VPI-5482 xylose isomerase (Acc. No.'s AA075900 or NP 809706; SEQ ID NO. 10) was cloned into a multicopy yeast expression vector to give p426GPDBtXI. This plasmid was used to transform RWB215 (MATα ura3-52 leu2-112loxP-PTPI::(−266,−1)TAL1 gre3::hphMX pUGPTPI-TKL1 pUGPTPI-RPE1 KanloxP-PTPI::(−?,−1)RKI1), which was further transformed with p415ADHXKS for overexpression of xylulokinase. Two independent transformants were picked and both were able to grow on minimal medium with xylose as sole carbon source and in lysates of the transformants a specific xylose isomerase activity of 140+/−20 U per mg protein was measured, compared to about 1300 U per mg protein for the strains expressing the Piromyces xylose isomerase.
4. Selection of RWB 212
The strain RWB 212 (see above) was placed under selective pressure by cultivation in anaerobic chemostat cultures (duplo) with 30 g/l xylose as the carbon source with an estimated growth rate of 0.06 h−1. The selection process was monitored by determination of culture dry weight and residual xylose concentration. The initial residual xylose concentration was around 30 mM but already after 26 generations (300 hours) residual xylose concentrations had decreased to less than 15 mM (FIG. 3A) and a corresponding increase in biomass concentration was also observed (FIG. 3B).
From these chemostat cultures samples were taken at 530 hours and these were plated on mineral medium agar plates supplemented with 2% glucose. After 62 hours at 30° C. single colonies from these plates were restreaked on fresh glucose plates. After another 72 hours incubation at 30° C., two colonies were selected (one colony originating from each separate chemostat) and used to inoculate precultures (aerobic shake flasks, mineral medium with glucose) for anaerobic fermentor batches on 20 g/l glucose and 20 g/l xylose.
From the CO2 off-gas signals in FIG. 4 it is evident that these cultures have superior xylose utilization characteristics compared to the parental strain RWB 212 and the other selection strain RWB 202-AFX. The “new” selection strain displays an increase in CO2 production rate when consuming xylose, which is not observed in the parental strain. FIG. 5 depicts the measured concentrations of carbon source and products in supernatants of these two independent batches, mainly during the xylose consumption phase. The overall (i.e., glucose+xylose phase) volumetric ethanol production rate of both experiments is higher than 0.5 g/L/hr and the first batch also demonstrates a volumetric productivity in the xylose consumption phase of higher than 0.5 g/L/hr.
5. Testing of Strains RWB 204, 206, 208 and 210
The strains tested have been constructed as has been described in the patent text as well as in Kuyper et al., 2005, FEMSYR 5:399-409. The modified genes are listed in the Table below:
Listing of the Genes Overexpressed and Deleted in the Used Strains












Strain
Overexpression
Deletion







RWB 204
TAL1



RWB 206
TAL1
gre3


RWB 208
TAL1, TKL1
gre3


RWB 210
TAL1, TKL1, RPE1
gre3


RWB 212
TAL1, TKL1, RPE1, RKI1
gre3









After the introduction of the modifications listed in the above Table all strains were transformed with two plasmids; pAKX002 expressing the Piromyces xylose isomerase and p415ADHXKS a second plasmid expressing the endogenous xylulokinase. In the above article RWB 212 transformed with the two plasmids was given a separate number: RWB 217. A repeat of the transformation of RWB 212 with the two plasmids resulted in RWB 223.
After transformation single colonies were streaked on synthetic medium agar plates with glucose. From these plates shake flask cultures with synthetic medium and 20 g/l xylose were inoculated and incubated at 30′C for 48 hours. Each shake flask culture was used to inoculate an anaerobic fermentor with synthetic medium and 20 g/l xylose.
After 48 hours incubation the shake flask inoculated with RWB 204 had not grown as determined by visual inspection. All four flasks were used to inoculate one fermentor. The growth in the fermentors was monitored by measuring the CO2 concentrations in off gas.
For reference purposes the CO2 profiles of RWB 217 and RWB 223 (both with TAI, TKL, RPE and RKI overexpression) are also given in FIG. 1. Over a period of 100 hours no significant CO2 production could be measured in the off gas of the batches with RWB 204 and RWB 206. The growth rate determined from these CO2 production graphs is 0.12 h−1 for RWB 208, 217 and 223, for RWB 210 it was determined at 0.08 h−1. From these result follows that overexpression of both transaldolase and transketolase are required for anaerobic growth on xylose. Furthermore, the additional overexpression of ribose 5-phosphate epimerase in RWB 210 decreases the growth rate on xylose. The overexpression of RPE1 probably disturbs the equilibrium between xylulose-5P, ribulose-5P and ribose-5P, hindering the activity of transketolase. Under these experimental conditions the additional overexpression of the R5P-epimerase and -isomerase does not further improve the performance of anaerobic xylose fermentation.







TABLE 2







Growth parameters, sugar consumption and product formation by the wild-type


CEN.PK 113-7D, the selected strain RWB 202-AFX and the engineered strain RWB 212 during


anaerobic batch cultivation in fermenters.













CEN.PK 113-7D
RWB 202-AFX
RWB 202-AFX
RWB 212
RWB 212





Carbon source (w/v)
Glucose (2%)
Glucose (2%)
Xylose (2%)
Xylose (2%)
Glucose (2%) +







Xylose (2%)


Specific growth rate (hr−1)
0.34 ± 0.00
0.24 ± 0.00
0.03 ± 0.00
0.09 ± 0.00
 0.25 ± 0.00a


Biomass yield (g/g)
0.099 ± 0.003
0.079 ± 0.000
0.088 ± 0.004
0.085 ± 0.002
0.074 ± 0.001


Ethanol yieldb (g/g)
0.40 ± 0.01
0.40 ± 0.00
0.42 ± 0.00
0.43 ± 0.00
0.43 ± 0.00


Carbon recoveryb (%)
104.0 ± 1.1 
103.7 ± 0.8 
105.5 ± 0.0 
105.9 ± ??  
103.2 ± ??  


Sugar consumed (mM)
116.1 ± 0.3 
114.9 ± 0.4 
137.4 ± 0.2 
133.9 ± 0.1 
108.5 ± 0.2 +







136.0 ± 0.3







Products:












Biomass (g/l
2.07 ± 0.06
1.64 ± 0.01
1.81 ± 0.08
1.70 ± 0.04
2.97 ± 0.04


CO2 (mmoles/l)
197.1 ± 3.4 
196.9 ± 1.3 
199.7 ± 1.5 
199.9 ± 1.5 
391.6 ± 0.6 


Ethanolc (mM)
181.6 ± 3.9 
180.3 ± 1.4 
186.8 ± 2.2 
188.5 ± 1.3 
370.7 ± 0.4 


Xylitol (mM)
<0.01
<0.01
2.76 ± 0.03
0.38 ± 0.04
0.78 ± 0.00


Xylulose (mM)d
<0.01
<0.01
7.98 ± 0.09
<0.01
<0.01


Glycerol (mM)
22.9 ± 0.2 
24.2 ± 0.1 
18.3 ± 0.3 
17.8 ± 0.2 
32.7 ± 0.3 


Acetate (mM)
3.42 ± 0.11
6.93 ± 0.02
2.26 ± 0.16
1.40 ± 0.07
3.54 ± 0.02


Succinate (mM)
0.26 ± 0.01
0.27 ± 0.02
0.75 ± 0.00
0.39 ± 0.02
0.96 ± 0.00


Lactate (mM)
1.70 ± 0.02
1.49 ± 0.02
0.95 ± 0.02
1.46 ± 0.01
2.78 ± 0.03





Values are presented as the average and experimental deviation of two independent batch cultivations.


adetermined from the glucose consumption phase.


bcalculation based on the ethanol concentrations deduced from the CO2 production, see Section 1.10.


cdeduced from the CO2 production, see Section 1.10.


dtransient accumulation. This value represents the highest concentration during the mid-log phase. At the end of growth all xylulose had been reconsumed, in all other cultures the xylulose concentration remained below the detection limit.













TABLE 3







Primers used








Oligo name
Sequence





PTALdisA
CCTTTCCAACGAATCGTATATACTAACATGCGCGCGCTTCCTATGCAT



AGGCCACTAGTGGATCTG


 


PTALdisB
AGAGAGTTGTTAGCAACCTTTTGTTTCTTTTGAGCTGGTTCAGACATG



GTGAATTCCTGTATGTG


 


5′TAL1
CTGACTGAGCCATATTGAGG


 


TAL1 intern
CACCAGTGTCGGCAACAACG


 


PRKIdisA
TCTTGTAGAAAATTAACAACATCGTGTTACATAAACTTGGTTACGCAT



AGGCCACTAGTGGATCTG


 


PRKIdisB
TTGCCCAAAGATTCTAACGCATCAATTTTTGGGACACCGGCAGCCATG



GTGAATTCCTGTATGTG


 


RKI1intern
CAGCTCTCTTGGCATCCTCC


 


EcoRI-5′TKL1
GGAATTCATGACTCAATTCACTGACATTG


 


3′TKL1-XhoI
GGCCTCGAGCTTGAATGGTGTGATTCTCT


 


TKL1intern
CCGCCATTGGTGATCTACAG


 


EcoRI-5′RPEI
GGAATTCATGGTCAAACCAATTATAGC


 


3′RPEI-XhoI
CCGCTCGAGTTAGGCACTTACGTATCTTG


 


RPE1intern
GGAAGCCTTATGGAGTGTCA


 


PTPI1primer
TGGCATGTGAGATTCTCCGA


 


KanA
CGCACGTCAAGACTGTCAAG


 


KanB
TCGTATGTGAATGCTGGTCG


 


5′gre3::Kanlox
AAAATACTGTAATATAAATCGTAAAGGAAAATTGGAAATTTTTTCAGC



TGAAGCTTCGTACGC


 


3′gre3::Kanlox
TGGATTTTACTGGCTGGATCAGGCAAAAGTGGGGAATTTACCGCATAG



GCCACTAGTGGATCTG


 


5′GRE3
CCTGGTGGAACATCCTAGAA


 


3′GRE3
GGATGACACCACAGGCAGAA


 


SpeI-5′XKS1
GACTAGTATGTTGTGTTCAGTAATTCAG


 


3′XKS1-SalI
TGCAGTCGACATTTTAGATGAGAGTCTTTTCC
















TABLE 4





Plasmids used

















pUG6
loxP-KanMX-loxP cassette
Guldener et al. [3]


pUG6PTPI1
pUG6 with the TPI1 promoter
present document


pUG6TPI1-RPE1
pUG6 with RPE1 behind theTPI1 promoter
present document


pUG6TPI1-TKL1
pUG6 with TKL1 behind the TPI1 promoter
present document


pAG32
loxP-hphMX-loxP cassette
Goldstein et al. [9]


PAKX002
2μ, URA3, Piromyces XylA behind the TPI1 promoter
Kuyper et al. [20]


P415ADH
CEN, LEU2, ADH1 promoter
Mumberg et al. [21]


p415ADHXKS1
CEN, LEU2, PADH1-XKS1
present document


PSH47
CEN, URA3, Cre recombinase behind PGAL1
Guldener et al..[3]









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## Claims

1. A yeast or filamentous fungal host cell transformed with a nucleic acid construct comprising a nucleotide sequence that encodes a xylose isomerase enzyme comprising an amino acid sequence that has at least 85% sequence identity with the amino acid sequence SEQ ID NO:2, which construct, upon transformation of the host cell, confers on the host cell the ability to directly isomerize xylose to xylulose, and wherein the host cell comprises a genetic modification that
(a) increases the flux of the pentose phosphate pathway, and
(b) results in overexpression of at least one gene of the non-oxidative branch of the pentose phosphate pathway.

2. The host cell according to claim 1, wherein the nucleotide sequence encodes a xylose isomerase enzyme that is obtainable from a bacterium of the class Bacteroides.

3. The host cell according to claim 1, wherein the nucleotide sequence is adapted to optimize its codon usage to that of the host cell.

4. The host cell according to claim 1, wherein the gene of the non-oxidative branch of the pentose phosphate pathway is selected from the group consisting of the gene encoding
(i) ribulose-5-phosphate isomerase,
(ii) ribulose-5-phosphate epimerase,
(iii) transketolase and
(iv) transaldolase.

5. The host cell according to claim 4, wherein the at least one gene that is overexpressed encodes a transketolase or a transaldolase.

6. The host cell according to claim 1, wherein the at least one gene that is overexpressed is endogenous to the host cell.

7. The host cell according to claim 1, wherein the host cell further comprises a genetic modification that increases specific xylulose kinase enzymatic activity.

8. The host cell according to claim 7, wherein the further genetic modification comprises overexpression of the gene encoding said xylulose kinase.

9. The host cell according to claim 8, wherein the overexpressed xylulose kinase gene is endogenous to the host cell.

10. A host cell according to claim 1, wherein the host cell further comprises a genetic modification that reduces nonspecific aldose reductase activity in the cell.

11. The host cell according to claim 10, wherein the reduced activity results from reduced expression of or inactivation of a gene encoding nonspecific aldose reductase.

12. The host cell according to claim 11, wherein the nonspecific aldose reductase gene is inactivated by (i) deletion of at least part of the gene or (ii) disruption of the gene.

13. The host cell according to claim 11, wherein (i) expression of each gene in the host cell that encodes a nonspecific aldose reductase is reduced or (ii) each gene that encodes a nonspecific aldose reductase is inactivated.

14. The host cell according to claim 1 that is a yeast cell.

15. The yeast cell according to claim 14 that is a member of one of the following genera: Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, or Yarrowia.

16. The yeast cell according to claim 14 that is a member of one of the following species: S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, or K. fragilis.

17. The host cell according to claim 1 that is a filamentous fungus.

18. The filamentous fungus according to claim 17 that is a member of one of the following genera: Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, or Penicillium.

19. The host cell according to claim 1, that expresses one or more enzymes that confers on the host cell the ability to produce at least one fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and a cephalosporin.

20. A process for producing ethanol, comprising the steps of:
(a) fermenting a medium containing a source of xylose with the modified host cell according to claim 1 that ferments xylose to ethanol, and, optionally,
(b) recovering of the ethanol from the medium.

21. The process according to claim 20, wherein the medium also contains a source of glucose.

22. The process according to claim 20, wherein volumetric ethanol productivity is at least 0.5 g ethanol per liter per hour.

23. The process according to claim 20, wherein ethanol yield is at least 50%.

24. A fermentation process for producing one of the following products: lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and a cephalosporin, which process comprises the steps of:
(a) fermenting a medium containing a source of xylose with the modified host cell according to claim 1 that ferments xylose to the product, and, optionally,
(b) recovering the product from the medium.

25. The process according to claim 24, wherein the medium also contains a source of glucose.