Xylose isomerases that confer efficient xylose fermentation capacity in yeast.

Abstract

The present invention relates to novel nucleic acid sequences encoding bacterial xylose isomerases which, upon transformation of a eukaryotic microbial host cell, such as yeast, confer upon the host cell the ability to isomerize xylose into xylulose. The nucleic acid sequences encode xylose isomerases originating from bacteria such as Eubacterium sp., Clostridium cellulosii, and others. The invention further relates to fermentation processes in which the transformed host cells ferment a medium containing xylose to produce ethanol or other fermentation products.

Description

1 / 86 Descriptive Report of the Invention Patent for: "XYLOSE ISOMERASES THAT CONFER EFFICIENT FERMENTATION CAPACITY OF XYLOSE IN YEAST" Field of invention

[001] The present invention relates to the fields of microbiology and fermentation technology. In particular, the invention relates to nucleic acid sequences encoding xylose isomerases which, upon transformation of a eukaryotic microbial host cell, such as yeast, confer upon the host cell the ability to isomerize xylose into xylulose. The invention further relates to fermentation processes in which the transformed host cells ferment a medium containing pentose to produce ethanol or other fermentation products. Antecedent Technique

[002] The yeast Saccharomyces cerevisiae has been the main organism of choice in industrial fermentation processes, including alcoholic beverages and bioethanol production. The dominance of this organism in these industries is due to its superior properties, such as high productivity and ethanol yield, high tolerance to ethanol and other inhibitors, and its excellent maintenance of viability during production, storage, and transport. Furthermore, being one of the most intensively studied microorganisms, numerous molecular tools are available to it. Petition 870190079230, dated 08 / 15 / 2019, page 9 / 101 2 / 86 available for your genetic and physiological manipulation (1).

[003] On the other hand, natural strains of S. cerevisiae are not useful in lignocellulosic ethanol industries. This is mainly due to their inability to metabolize pentose sugars, particularly xylose. Xylose is the second most abundant sugar in nature. It accounts for one third of the total sugar present in lignocellulosic biomass, such as agricultural and forestry residues, and municipal solid waste. Thus, the efficient use of xylose is crucial for the production of lignocellulosic (second generation) bioethanol (2).

[004] There are several microorganisms capable of naturally fermenting xylose. However, unlike S. cerevisiae, these organisms do not possess sufficient inherent robustness to cope with the adverse environments found in industrial fermentations. Compared to S. cerevisiae, they are less tolerant to ethanol and several growth and fermentation inhibitors, such as organic acids, furan derivatives, and phenolic compounds that are present in lignocellulosic hydrolysates (3). For this reason, much effort is being made to engineer S. cerevisiae for efficient xylose fermentation, rather than conferring industrial robustness to natural xylose using microorganisms. Petition 870190079230, dated 08 / 15 / 2019, page 10 / 101 3 / 86

[005] Two different xylose utilization pathways have been designed in yeast. The first pathway, called the fungal pathway or the redox pathway, works by a two-step enzymatic conversion of xylose to xylulose. In the first step, the NADPH-dependent Xylose Reductase (XR) enzyme reduces xylose to xylitol. Xylitol is subsequently oxidized to xylulose by NAD-dependent Xylitol Dehydrogenase (XDH). Xylulose can then be phosphorylated to Xylulose-5-Phosphate by native Xylulokinase. Although yeast strains expressing the fungal redox pathway can efficiently ferment xylose, they generally produce less ethanol per gram of biomass due to the accumulation of xylitol as a byproduct (4). The low ethanol production and high xylitol accumulation are due to the cofactor imbalance generated by the heterologous XR / XDH enzymes. Several strategies have been applied to solve the cofactor imbalance problem.This includes modifying the specificity of the XR and XDH cofactor, and the expression of heterologous transhydrogenases that catalyze the transfer of H+ between NADPH and NAD+ (5-7). Balancing cofactor use in yeast expressing XR / XDH has shown good potential, but so far has not been able to eliminate xylitol production as a byproduct. The ethanol yield per unit of sugar consumed by these strains remains very low.

[006] The second route works with a conversion into a Petition 870190079230, dated 08 / 15 / 2019, page 11 / 101 4 / 86 xylose to xylulose step using Xylose Isomerase (XI). This pathway alleviates the cofactor imbalance associated with the fungal redox pathway. The XI pathway is predominantly found in bacteria, but also in some fungi. Many previous attempts to express bacterial XI in yeast failed or resulted in very low expression. The first functionally active bacterial XI expressed in yeast was encoded by the XylA gene from the thermophilic bacterium Thermus thermophiles (8). However, optimal enzymatic activity was observed at 85 °C, well above the ideal temperature at which yeasts can grow. However, the recombinant strain was able to grow very slowly with xylose as the sole carbon source. Subsequently, the expression of an enzymatically active fungal XI from Piromyces sp. became a major success story (9). Subsequently, other XIs from various species of bacteria or fungi were actively expressed in S. cerevisiae (10).However, the activity of these enzymes in yeast remains lower compared to the XI of Piromyces sp. The first bacterial XI that showed very good enzymatic activity when expressed in yeast was the XI of the bacterial species Clostridium phytofermentans. This enzyme was less inhibited by xylitol than by the xylose isomerases of other bacterial species (11). However, despite the high in vitro enzymatic activities of these XIs reported until now. Petition 870190079230, dated 08 / 15 / 2019, page 12 / 101 5 / 86 now, recombinant strains expressing these enzymes exhibited only slow growth and xylose fermentation capacity. Further improvement by mutagenesis or adaptive evolution of recombinant yeast is needed to obtain an acceptable xylose fermentation capacity (12).

[007] To date, hundreds of XylA sequences are available in the NCBI sequence databases. These sequences are a great tool for searching for functionally active XIs originating from various species. Despite the vast sequence information, only a few XIs originating from various bacterial species have been functionally expressed in yeast. It has recently been reported that most XIs actively expressed in yeast originate from the Bacteroidetes group that lives in the mammalian intestine (10). A disadvantage of XIs from the Bacteroidetes group is their strong inhibition by xylitol (11). However, many bacterial XIs other than those originating from the Bacteroidetes group cannot be functionally expressed in yeast, and we cannot yet predict in advance whether a specific XI will be functionally expressed in yeast or not.

[008] There is therefore still a need in the technique for nucleotide sequences encoding other xylose isomerases that can be used to transform cells. Petition 870190079230, dated 08 / 15 / 2019, page 13 / 101 6 / 86 host cells such as S. cerevisiae to give them the ability to isomerize xylose into xylulose, so as to allow the use of the transformed host cell in processes for the production of ethanol or other fermentation products by fermenting feedstock containing pentose. Summary of the invention

[009] In a first aspect, the invention relates to a eukaryotic microbial cell comprising a nucleotide sequence, the expression of which confers or enhances in the cell the ability to directly isomerize xylose to xylulose, wherein the nucleotide sequence encodes a polypeptide with xylose isomerase activity, which polypeptide comprises an amino acid sequence that has at least 68% sequence identity with the amino acid sequence of SEQ ID NO. 7. Preferably, the nucleotide sequence encodes an amino acid sequence that is obtained from a bacterium of the genus Eubacterium, more preferably a bacterium of the species Eubacterium sp. CAG_180.A preferred cell according to the invention further comprises a second nucleotide sequence, the expression of which confers or enhances in the cell the ability to directly isomerize xylose to xylulose, wherein the nucleotide sequence encodes a polypeptide with xylose isomerase activity, said polypeptide being... Petition 870190079230, dated 08 / 15 / 2019, page 14 / 101 7 / 86 comprises an amino acid sequence that has at least 71% sequence identity with the amino acid sequence of SEQ ID No. 10. Preferably, the second nucleotide sequence encodes an amino acid sequence that is obtained from a bacterium of the genus Clostridium, more preferably a bacterium of the species Clostridium cellulosii.

[0010] The eukaryotic microbial cell according to the invention is preferably a yeast or a filamentous fungus of a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Naumovia, Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium and Penicillium.

[0011] In one embodiment, the eukaryotic microbial cell is preferably a yeast capable of anaerobic alcoholic fermentation. Preferably, the yeast belongs to a Saccharomyces species selected from the group consisting of S. cerevisiae, S. bayanus, S. bulderi, S. cervazzii, S. cariocanus, S. castellii, S. dairenensis, S. exiguus, S. kluyveri, S. kudriazevii, S. mikatae, S. paradoxus, S. pastorianus, S. turicensis, and S. unisporus.

[0012] In a eukaryotic microbial cell according to the invention, the nucleotide sequence encoding the polypeptide with xylose isomerase activity is Petition 870190079230, dated 08 / 15 / 2019, page 15 / 101 8 / 86 preferably operatively linked to a promoter that is insensitive to catabolic suppression and / or that does not require xylose for induction.

[0013] The eukaryotic microbial cell according to the invention preferably comprises at least one genetic modification selected from: a) a genetic modification that increases the specific activity of xylulose kinase; b) a genetic modification that increases the flux of the pentose phosphate pathway; and, c) a genetic modification that reduces the non-specific activity of aldose reductase in the cell. The cell further preferably comprises at least one genetic modification that results in a characteristic selected from the group consisting of: a) increased tolerance to ethanol; b) increased tolerance to acetic acid; c) reduced glycerol production; d) increased rate of xylose fermentation to ethanol; and, e) increased heat tolerance.More preferably in the cell: a) the genetic modification is a modification that introduces an allele of one or more of the genes ADE1, KIN3, MKT1, VPS70, SWS2 and APJ1 that confers increased tolerance to ethanol as described in WO 2012 / 175552 and WO 2014 / 170330; b) the genetic modification is a modification that introduces an allele of one or more of the genes GLO1, DOT5, CUP2 and HAA1 that confers greater tolerance to acetic acid, as described in documents WO 2015 / 181169 and WO 2016 / 083397; c) the modification. Petition 870190079230, dated 08 / 15 / 2019, page 16 / 101 9 / 86 genetic modification is a modification that introduces a mutant SSK1 gene that encodes a truncated ssk1 protein as described in WO 2014 / 048863; d) the genetic modification is a modification that introduces an allele of the NNK1 gene that confers an increased rate of xylose fermentation to ethanol, as described in WO 2015 / 086805; and, e) the genetic modification is the overexpression of at least one of a gene encoding the Prp42 protein and a gene encoding the Smd2 protein.

[0014] In a preferred eukaryotic microbial cell according to the invention, the nucleotide sequence encoding the polypeptide with xylose isomerase activity is integrated into the cell genome.

[0015] A eukaryotic microbial cell according to the invention is preferably a cell of an industrial yeast strain or derived from an industrial yeast strain. The cell may be a diploid, aneuploid or polyploid cell.

[0016] In one embodiment, a eukaryotic microbial cell according to the invention is a cell that is improved in at least one industrially relevant phenotype by evolutionary engineering, wherein, preferably, the industrially relevant phenotype is the xylose utilization rate.

[0017] A eukaryotic microbial cell according to the invention has even more preferably the ability to Petition 870190079230, dated 08 / 15 / 2019, page 17 / 101 10 / 86 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, amino acids, 1,3-propanediol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics and cephalosporins.

[0018] In a second aspect, the invention relates to a process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propanediol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics and cephalosporins. The process preferably comprises the steps of: (a) fermenting a medium containing a xylose source and, optionally, a glucose source, with a eukaryotic microbial cell according to the invention, whereby the cell ferments the xylose and, optionally, the glucose, to the fermentation product and, optionally, (b) recovering the fermentation product.

[0019] In a third aspect, the invention relates to the use of a eukaryotic microbial cell according to the first aspect in a process according to the second aspect. Petition 870190079230, dated 08 / 15 / 2019, p. 18 / 101 11 / 86 aspect. Description of the invention Definitions

[0020] The enzyme xylose isomerase (EC 5.3.1.5) is defined herein as an enzyme that catalyzes the direct isomerization of D-xylose to D-xylulose and vice versa. The enzyme is also known as 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 magnesium as a cofactor. The xylose isomerases of the invention can be further defined by their amino acid sequence as described below. Similarly, xylose isomerases can be defined by the nucleotide sequences that encode the enzyme, as well as by the nucleotide sequences that hybridize with a reference nucleotide sequence that encodes a xylose isomerase, as described below.One unit (U) of xylose isomerase activity is defined here as the amount of enzyme producing 1 nmol of xylulose per minute in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 10 mM xylose, and 10 mM MgCl2 at 37 °C. The xylulose formed was determined by the method of Dische and Borenfreund (1951, J. Biol. Chem. 192: 583-587) or by HPLC as it is known in the art.

[0021] The terms homology, “sequence identity Petition 870190079230, dated 08 / 15 / 2019, page 19 / 101 12 / 86 and similar terms are used interchangeably herein. Sequence identity is defined herein as a relationship between two or more amino acid sequences (polypeptide or protein) or two or more nucleic acid sequences (polynucleotide), as determined by comparing the sequences. In the art, identity also means the degree of relationship between amino acid or nucleic acid sequences, as the case may be, determined by the correspondence between the chains of such sequences. The similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide with the sequence of a second polypeptide. Identity and similarity can be readily calculated by known methods.

[0022] “Sequence identity” and “sequence similarity” can be determined by aligning two peptides or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences along their entire length, while sequences of substantially different lengths are preferably aligned Petition 870190079230, dated 08 / 15 / 2019, page 20 / 101 13 / 86 using a local alignment algorithm (e.g., Smith-Waterman). Sequences can then be referred to as substantially identical or essentially similar when they (when optimally aligned by, for example, the GAP or BESTFIT programs using default parameters) share at least a certain minimum percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences along their entire length (total length), maximizing the number of matches and minimizing the number of gaps. A global alignment is appropriately used to determine sequence identity when the two sequences have similar lengths. Generally, the default GAP parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and a gap extension penalty = 3 (nucleotides) / 2 (proteins).For nucleotides, the standard scoring matrix used is nwsgapdna, and for proteins, the standard scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and percent sequence identity scores can be determined using computer programs such as the Wisconsin GCG Package, Version 10.3, available from Accelrys Inc., Scranton Road 9685, San Diego, CA 92121-3752 USA, or using open-source software, such as... Petition 870190079230, dated 08 / 15 / 2019, page 21 / 101 14 / 86 as the “needle” program (using the global Needleman Wunsch algorithm) or “water” program (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for the GAP above, or using the default settings (both for 'needle' and for 'water' and both for protein and for DNA alignments, the default gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have substantially different global lengths, local alignments are preferred, such as those using the Smith Waterman algorithm.

[0023] Alternatively, percent similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx (version 2.0) programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403–10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the molecules of Petition 870190079230, dated 08 / 15 / 2019, page 22 / 101 15 / 86 nucleic acid oxidoreductase of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain interval alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / .

[0024] Optionally, in determining the degree of amino acid similarity, the specialist may also take into account conservative amino acid substitutions, as will become clear to the specialist. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of amino acid residue classes for conservative substitutions are given in the Tables below. Acidic residues Asp (D) and Glu (E) Basic residues Lys (K), Arg (R), and His (H) Uncharged hydrophilic residues Ser (S), Thr (T), Asn (N), and Gln (Q) Petition 870190079230, dated 08 / 15 / 2019, p. 23 / 101 16 / 86 Non-aliphatic charged residues Gly (G), Leu (L), and Ile (I) Ala (A), Val (V), Non-polar uncharged residues Cys (C), Met (M), and Pro (P) Aromatic residues Phe (F), Tyr (Y), and Trp (W)

[0025] Alternative classes of conservative amino acid residue substitution. 1 AST 2 OF 3 NQ 4 RK 5 ILM 6 FYW

[0026] Alternative Physical and Functional Classifications of Amino Acid Residues. Residues containing alcohol groups S and T Aliphatic residues I, L, V, and M Residues associated with cycloalkenyl F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Petition 870190079230, dated 08 / 15 / 2019, p. 24 / 101 17 / 86 Small waste A, C, D, G, N, P, S, T, and V; Very small waste A, G, and S; Waste involved in shift work A, C, D, E, G, H, K, N, Q, R, S, P, and T; Flexible waste Q, T, K, S, G, P, D, E, and R

[0027] The nucleotide sequences encoding the xylose isomerases of the invention can also be defined by their ability to hybridize with the nucleotide sequences encoding xylose isomerases as exemplified herein, under moderate or preferably stringent hybridization conditions. Strict hybridization conditions are defined here as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50, 75 or 100, and more preferably about 200 or more nucleotides, to hybridize at a temperature of about 65 °C in a solution comprising about 1 M of salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at 65 °C in a solution comprising about 0.1 M of salt, or less, preferably 0.2 x SSC or any other solution having a comparable ionic strength.Ideally, hybridization should be carried out overnight, i.e., for at least 10 hours, and preferably washing should be carried out for at least one hour with at least two changes of the washing solution. These conditions... Petition 870190079230, dated 08 / 15 / 2019, p. 25 / 101 18 / 86 typically allow sequence-specific hybridization with approximately 90% or greater sequence identity.

[0028] Moderate conditions are defined here as conditions that allow a nucleic acid sequence 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 6x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6x SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is carried out overnight, i.e., for at least 10 hours, and preferably the washing is carried out for at least one hour with at least two changes of the washing solution. These conditions usually allow specific hybridization of sequences with up to 50% sequence identity.The expert in the technique will be able to modify these hybridization conditions in order to specifically identify sequences that vary in identity between 50% and 90%.

[0029] A “nucleic acid construct” or “nucleic acid vector” is understood here to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term nucleic acid construct Petition 870190079230, dated 08 / 15 / 2019, page 26 / 101 19 / 86 nucleic acid does not, therefore, include naturally occurring nucleic acid molecules, although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms expression vector or expression construct refer to nucleotide sequences that are capable of affecting the expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least appropriate transcriptional regulatory sequences and, optionally, 3' transcription termination signals. Additional factors necessary or useful for effecting expression may also be present, such as expression-enhancing elements. The expression vector will be introduced into a suitable host cell and will be able to effect expression of the coding sequence in an in vitro cell culture of the host cell.The expression vector will be suitable for replication in the host cell or organism of the invention.

[0030] As used herein, the term promoter or transcription regulatory sequence refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences and is located upstream of the transcription direction from the transcription initiation site of the coding sequence, and is structurally identified by the presence of a site Petition 870190079230, dated 08 / 15 / 2019, page 27 / 101 20 / 86 binding sites for DNA-dependent RNA polymerase, transcription initiation sites, and other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to a person skilled in the art to act directly or indirectly in regulating the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An inducible promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer.

[0031] The term selectable marker is a term familiar to a person skilled in the art and is used here to describe any genetic entity that, when expressed, can be used to select a cell or cells containing the selectable marker. The term “reporter” can be used interchangeably with marker, although it is primarily used to refer to visible markers such as green fluorescent protein (GFP). Selectable markers can be dominant or recessive or bidirectional.

[0032] As used herein, the term “operationally linked” refers to a linking of elements of Petition 870190079230, dated 08 / 15 / 2019, p. 28 / 101 21 / 86 polynucleotides in a functional relationship. A nucleic acid is operatively linked when it is placed in a functional relationship with another nucleic acid sequence. For example, a transcription regulatory sequence is operatively linked to a coding sequence if it affects the transcription of the coding sequence. Operationally linked means that the linked DNA sequences are typically contiguous and, when necessary, to join two protein-coding regions, contiguous and in the reading frame.

[0033] The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, three-dimensional structure, or origin.

[0034] Fungi (singular fungus) are understood here as heterotrophic eukaryotic microorganisms that digest their food externally, absorbing nutrient molecules within their cells. Fungi are a separate kingdom of eukaryotic organisms and include yeasts, molds, and mushrooms. The terms fungi, fungi, and fungi, as used herein, expressly include yeasts as well as filamentous fungi.

[0035] The term gene means a fragment of DNA comprising a region (transcribed region), which is Petition 870190079230, dated 08 / 15 / 2019, page 29 / 101 22 / 86 transcribed into an RNA molecule (e.g., mRNA) in a cell, operatively linked to appropriate regulatory regions (e.g., a promoter). A gene will typically comprise several operatively linked fragments such as a promoter, a 5' command sequence, a coding region, and a 3' untranslated sequence (3' end) comprising a polyadenylation site. Gene expression refers to the process by which a region of DNA that is operatively linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA that is biologically active, i.e., capable of being translated into a biologically active protein or peptide.

[0036] The term homologous, when used to indicate the relationship between a given recombinant nucleic acid or polypeptide molecule and a given host organism or host cell, means 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 (but not necessarily) be operationally linked to a different promoter sequence (heterologous) and, if applicable, a different secretion signal sequence (heterologous) and / or termination sequence than in its natural environment. It is understood that the sequences Petition 870190079230, dated 08 / 15 / 2019, p. 30 / 101 23 / 86 Regulatory sequences, signaling sequences, termination sequences, etc., can also be homologous to the host cell. In this context, the use of only “homologous” sequence elements allows the construction of “self-cloned” genetically modified organisms (GMOs) (self-cloning is defined here as in European Directive 98 / 81 / EC, Annex II). When used to indicate the relationship of two nucleic acid sequences, the term homologous means that a single-stranded nucleic acid sequence can hybridize with a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on several factors, including the amount of identity between the sequences and the hybridization conditions, such as temperature and salt concentration, as discussed later.

[0037] The term heterologous, when used in relation 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 they are introduced, but have been obtained from another cell by recombinant production or Petition 870190079230, dated 08 / 15 / 2019, p. 31 / 101 24 / 86 synthetically. Generally, though not necessarily, these nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are 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 a person skilled in the art would recognize as heterologous or foreign to the cell in which it is expressed is here encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to unnatural combinations of nucleic acid or amino acid sequences, that is, combinations in which at least two of the combined sequences are foreign to each other. Description of the modalities

[0038] To date, a vast number of xylose isomerase amino acid sequences are publicly available in GenBank and other sequence databases. Among them are some xylose isomerase amino acid sequences known for their functional expression in yeast, including, for example, xylose isomerases from anaerobic fungi such as Piromyces, from the Bacteroidetes group that lives in the intestines of mammals, as well as bacterial xylose isomerases from the Clostridium species. Petition 870190079230, dated 08 / 15 / 2019, page 32 / 101 25 / 86 phytofermentans. The present inventors have surprisingly discovered xylose isomerase amino acid sequences that are not related to Piromyces, Bacteroidetes, and C. phytofermentans enzymes—in the sense that most of them share less than 70% amino acid sequence identity with the amino acid sequences of Piromyces (PiXI; SEQ ID NO: 18) and C. phytofermentans (CpXI; SEQ ID NO: 17) enzymes (see Table 1)—and yet possess the capacity for functional (i.e., active) expression in yeast.

[0039] The functional expression of a xylose isomerase in a yeast is here understood as the expression of an optimized codon-coding sequence for the xylose isomerase from a glycolytic promoter on a 2μ-based plasmid in a host strain of S. cerevisiae, whose expression allows detectable growth of the yeast on xylose as the sole carbon source, preferably under anaerobic conditions with ethanol production at the expense of xylose, more preferably with at least one of a growth rate, biomass and ethanol yield that is at least 10, 20, 50 or 80% of that achieved with a codon-optimized sequence encoding for the Piromyces xylose isomerase (with the amino acid sequence of SEQ ID NO: 18) under identical conditions. The S. cerevisiae host strain is preferably a modified host strain for Petition 870190079230, dated 08 / 15 / 2019, page 33 / 101 26 / 86 growth in xylose by overexpression of xylulose kinase (XKS1) and all genes of the pentose phosphate pathway (PPP), such as, for example, the M315CpXIΔ / CpXIΔ strain (see Examples). Preferably, functional expression is expression that allows detectable growth of the host strain in xylose as the sole carbon source at temperatures below 35, 33, 30 or 28°C and above 20, 22 or 25°C.

[0040] Table 1. Sequence identity of xylose isomerase amino acid sequences compared with the amino acid sequence of Piromyces sp. XI (PiXI) and C. phytofermentans XI (CpXI). Source of identity of xylose isomerase % for PiXI % identity for CpXI Functional expression in yeast SEQ ID NO. Code Lachnoclostridium phytofermentans 54.99 96.12 + 1 Lp1XI Clostridium algidicarnis 53.83 72.60 + 2 Ca2XI Mageeibacillus indolicus 53.02 69.35 + 3 Mi3XI Ruminococcus sp, NK3A76 52.19 68.64 - 4 Rs4XI Epulopiscium sp, 'N,t, morphotype B 52.94 67.28 + 5 Es5XI Petition 870190079230, dated 08 / 15 / 2019, p. 34 / 101 27 / 86 Alkaliphilus metalliredigens 52.76 65.53 + 6 Am6XI Eubacterium sp, CAG_180 54.38 65.44 + 7 Es7XI Clostridium saccharoperbuty lacetonicum 53.23 64.61 + 8 Cs8XI Fusobacterium mortiferum 51.96 65, 67 + 9 Fm9X1 54.50 62.70 - 13 Cd13XI Caldicellulosir uptor acetigenus 50.35 61.75 - 14 Ca14XI Agrobacterium tumefaciens 49.89 52.50 — 15 At15XI Burkholderia cenocepacia 49.32 51.70 — 16 Bc16XI

[0041] In a first aspect, the invention relates to a transformed host cell that has the ability to isomerize xylose to xylulose. The ability to isomerize xylose to xylulose is conferred on the host cell by Petition 870190079230, dated 08 / 15 / 2019, page 35 / 101 28 / 86 transformation of the host cell with a nucleic acid construct comprising a nucleotide sequence encoding a xylose isomerase. The ability of the transformed host cell to isomerize xylose to xylulose is understood to mean the direct isomerization of xylose, in a single reaction catalyzed by xylose isomerase, to xylulose, as opposed to the two-step conversion of xylose to xylulose via a xylitol intermediate as catalyzed by xylose reductase and xylitol dehydrogenase, respectively.

[0042] In one embodiment, the nucleotide sequence encoding xylose isomerase is selected from the group consisting of:

[0043] (a) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 65.5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 7 (Eubacterium sp. CAG_180);

[0044] (b) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 64, 9, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, Petition 870190079230, dated 08 / 15 / 2019, p. 36 / 101 29 / 86 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 10 ([Clostridium] cellulosi);

[0045] (c) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence that has at least 64,7, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 8 (Clostridium saccharoperbutylacetonicum);

[0046] (d) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence that has at least 64, 6, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 11 (Cellulosilyticum lentocellum);

[0047] (e) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 67, 3, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, Petition 870190079230, dated 08 / 15 / 2019, p. 37 / 101 30 / 86 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence SEQ ID NO. 5 (Epulopiscium sp. 'Nt morphotype B);

[0048] (f) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 96.2, 96.5, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO. 1 (Lachnoclostridium phytofermentans);

[0049] (g) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 65, 6, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 6 (Alkaliphilus metalliredigens);

[0050] (h) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 69.4, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 3 (Mageeibacillus indolicus); Petition 870190079230, dated 08 / 15 / 2019, page 38 / 101 31 / 86

[0051] (i) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence that has at least 72, 7, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 2 (Clostridium algidicarnis);

[0052] (j) a nucleotide sequence encoding a polypeptide with xylose isomerase activity, wherein the polypeptide comprises an amino acid sequence having at least 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence of SEQ ID NO. 12 (Peptoclostridium difficile);

[0053] (k) a complementary nucleotide sequence that hybridizes with a nucleotide sequence of one of (a) - (j); and,

[0054] (l) a nucleotide sequence whose sequence differs from the sequence of a nucleotide sequence of (k) due to genetic code degeneracy.

[0055] The nucleotide sequences of the invention encode a new class of xylose isomerases that can be functionally expressed in eukaryotic microbial host cells of the invention, as defined below. The Petition 870190079230, dated 08 / 15 / 2019, page 39 / 101 32 / 86 nucleotide sequences of the invention preferentially encode xylose isomerases that occur naturally in the parent organism, for example, the parent bacterium.

[0056] A preferred nucleotide sequence of the invention thus encodes a xylose isomerase with an amino acid sequence that is identical to that of a xylose isomerase obtainable from (or occurring naturally in) a bacterium of the family Clostridiaceae, more preferably a bacterium of the genus Clostridium, for example, Clostridium algidicarnis, but more preferably is Clostridium saccharoperbutylacetonicum and most preferably is [Clostridium] cellulosi.

[0057] Another preferred nucleotide sequence of the invention encodes a xylose isomerase with an amino acid sequence that is identical to that of a xylose isomerase that can be obtained from (or occurs naturally in) a bacterium of the family Eubacteriaceae, more preferably a bacterium of the genus Eubacterium, of which the species Eubacterium sp. CAG_180 is the most preferred.

[0058] Alternatively, the nucleotide sequence of the invention encodes a xylose isomerase with an amino acid sequence that is identical to that of a xylose isomerase obtainable from (or occurring naturally in) a bacterium of a genus selected from the group consisting of Cellulosilyticum, Epulopiscium, Petition 870190079230, dated 08 / 15 / 2019, p. 40 / 101 33 / 86 Lachnoclostridium, Alkaliphilus, Mageeibacillus and Peptoclostridium, more preferably a bacterium of a species selected from the group consisting of Cellulosilyticum ladocellum, Epulopiscium sp. 'Nt morphotype B, Lachnoclostridium phytofermentans, Metaloidigens Alkaliphilus, Mageeibacillus indolicus and Peptoclostridium difficile.

[0059] It is understood, however, that nucleotide sequences encoding manipulated forms of any of the xylose isomerases defined above and comprising one or more amino acid substitutions, insertions and / or deletions compared to the corresponding naturally occurring xylose isomerases, but which are within the ranges of identity or similarity as defined herein, are expressly included in the invention. Therefore, in one embodiment, the nucleotide sequence of the invention encodes a xylose isomerase amino acid sequence comprising a xylose isomerase signature sequence as defined by Meaden et al. (1994, Gene, 141: 97-101): VXW [GP] GREG [YSTA] (present at positions 187-195) and [LIVM] EPKPX [EQ] P (present at positions 232239), where X can be any amino acid and where the amino acids in parentheses indicate that one of the amino acids in parentheses may be present at that position in the signature sequence.The amino acid sequence of xylose. Petition 870190079230, dated 08 / 15 / 2019, p. 41 / 101 The invention's 34 / 86 isomerase further preferably comprises the conserved amino acid residues His-102, Asp-105, and Asp-340, which constitute a triad directly involved in catalysis; Lys-235 plays a structural as well as a functional catalytic role; and Glu-233 is involved in magnesium binding (Vangrysperre et al., 1990, Biochem J. 265: 699-705; Henrick et al., J. Mol Biol 208: 129-157; Bhosale et al., 1996 Microbiol). The amino acid positions of the above signature sequences and conserved residues refer to the positions in the reference amino acid sequence of Piromyces xylose isomerase of SEQ ID NO: 18.In amino acid sequences of the present invention other than SEQ ID NO: 18, preferably, the amino acid positions of the above signature sequences and conserved residues are present in the amino acid positions corresponding to the positions of the signature sequences and conserved residues in SEQ ID NO: 18, preferably in a ClustalW (1,83 or 1,81) sequence alignment using standard settings. A person skilled in the art will know how to identify corresponding amino acid positions in xylose isomerase amino acid sequences other than SEQ ID NO: 18 using amino acid sequence alignment algorithms as defined above. An example of such alignment is shown in Table 2.

[0060] In some modalities, therefore, the sequence Petition 870190079230, dated 08 / 15 / 2019, p. 42 / 101 The nucleotide sequence of the invention encodes a xylose isomerase, the amino acid sequence of which, at least, comprises, in each of the invariant positions (which are indicated in Table 2 with a *), the amino acid present in the invariant position. Preferably, the amino acid sequence also comprises, in the strongly conserved positions (which are indicated in Table 2 with a :), one of the amino acids present in a strongly conserved position. More preferably, the amino acid sequence also comprises, in the less strongly conserved positions (which are indicated in Table 2 with a ).) one of the amino acids present in a less conserved position. It is unlikely that amino acid substitutions from these invariant and conserved positions will affect the activity of xylose isomerase. Furthermore, to date, a vast number of amino acid sequences of xylose isomerases are known in the art, and new ones are continually being added. The sequence alignments of SEQ ID NO: 18 and the xylose isomerase sequences of the invention with these. Petition 870190079230, dated 08 / 15 / 2019, page 43 / 101 36 / 86 amino acid sequences of xylose and isomerase will indicate more conserved regions and amino acid positions, whose conservation is important for enzymatic structure and activity.

[0061] The nucleotide sequence encodes a xylose isomerase that is preferentially expressed in its active form in the host cell. Thus, expression of the nucleotide sequence in the host cell produces a xylose isomerase with a specific activity of at least 10 xylose isomerase activity units per mg of protein at 25°C, preferably at least 20, 25, 30, 50, 100, 200, or 300 U per mg at 25°C. The specific activity of the xylose isomerase expressed in the host cell is defined here as the amount of xylose isomerase activity units per mg of protein in the cell-free lysate of the host cell, for example, a cell-free yeast lysate. The determination of xylose isomerase activity, amount of protein, and preparation of the cell-free lysate is as described in the Examples.Preferably, the expression of the nucleotide sequence 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.

[0062] The nucleotide sequence encodes a xylose isomerase that preferentially has reduced sensitivity to Petition 870190079230, dated 08 / 15 / 2019, page 44 / 101 37 / 86 inhibition by xylitol. Preferably, the xylose isomerase shows less inhibition by xylitol than the Piromyces isomerase (SEQ ID NO: 18), more preferably the xylose isomerase shows less inhibition by xylitol than the C. phytofermentans isomerase (SEQ ID NO: 17). The nucleotide sequence thus encodes a xylose isomerase in a preferred manner that has an apparent inhibition constant Ki that is greater than 4,6, xylitol 5, 10, 14, 51, 15 mM. The sensitivity to inhibition by xylitol and the apparent inhibition constant Ki for xylitol can be determined as described in (11).

[0063] The nucleotide sequences of the invention, which encode polypeptides with xylose isomerase activity, can be obtained from genomic and / or cDNA of a bacterium belonging to a phylum, class, order, family or genus as described above, using the method for isolating nucleotide sequences that are well known in the art per se (see for example Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York). The nucleotide sequences of the invention are, for example, obtained in a process where a) degenerate PCR primers (such as those in SEQ ID Nos. 19 and 20) are used on genomic and / or cDNA of a suitable organism (for example, a bacterium as indicated above) to generate Petition 870190079230, dated 08 / 15 / 2019, page 45 / 101 38 / 86 a PCR fragment comprising part of the nucleotide sequences encoding polypeptides with xylose isomerase activity, b) the PCR fragment obtained in a) is used as a probe to search for cDNA and / or genomic library of the organism and c) production of cDNA or genomic normal DNA comprising the nucleotide sequence encoding a polypeptide with xylose isomerase activity.

[0064] To increase the probability of xylose isomerase being expressed at sufficient levels and in its active form in the host cells of the invention, the nucleotide sequence encoding these enzymes, as well as other enzymes of the invention (see below), are preferably adapted to optimize their codon usage to that of the host cell in question. The adaptability of a nucleotide sequence encoding an enzyme for codon utilization in a host cell can be expressed as the codon adaptation index (CAI). The codon adaptation index is defined here as a measure of the relative adaptability of a gene's codon utilization to the codon utilization of genes highly expressed in a particular host cell or organism. The relative adaptability (w) of each codon is the ratio between the use of each codon and the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptability values.Non-synonymous codons and codons of. Petition 870190079230, dated 08 / 15 / 2019, p. 46 / 101 39 / 86 termination (dependent on the genetic code) are excluded. CAI values ​​range from 0 to 1, with higher values ​​indicating a greater proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; see also: Jansen et al., 2003, Nucleic Acids Res 31 (8): 2242-51). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9. The most preferred sequences are those that have been encoded for expression in S. cerevisiae cells, as listed in SEQ ID NOs: 21-34, of which SEQ ID NOs: 27, 28, and 30 are preferred, and SEQ ID NO: 28 is most preferred.

[0065] The host cell to be transformed with a nucleic acid construct comprising a nucleotide sequence encoding a xylose isomerase of the invention is preferably a eukaryotic microbial host, more preferably a fungal host cell, such as a yeast or a filamentous fungal host cell. Preferably, the host cell is a cultured cell. The host cell of the invention is preferably a host capable of active or passive pentose (xylose and preferably also arabinose) transport into the cell. The host cell preferably contains active glycolysis. The host cell may also preferably contain an endogenous pentose phosphate pathway and may Petition 870190079230, dated 08 / 15 / 2019, page 47 / 101 40 / 86 contain endogenous xylulose kinase activity so that xylulose isomerized from xylose can be metabolized to pyruvate. The host also preferentially contains enzymes for converting a pentose (preferably via pyruvate) into a desired fermentation product such as ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propanediol, ethylene, glycerol, β-lactam antibiotics, and cephalosporins. A particularly preferred host cell is one that is naturally capable of alcoholic fermentation, preferably anaerobic alcoholic fermentation.The host cell preferably also has a high tolerance to ethanol, a high tolerance to low pH (i.e., capable of growing at pH less than 5, 4, or 3) and to organic acids such as lactic acid, acetic acid, or formic acid and sugar degradation products such as furfural and hydroxymethylfurfural, 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, preferably by self-cloning or by the methods of the invention described below. A suitable cell is a cultured cell, a cell that can be cultured in the process of... Petition 870190079230, dated 08 / 15 / 2019, page 48 / 101 41 / 86 fermentation, for example, in submerged or solid-state fermentation. Particularly suitable cells are eukaryotic microorganisms such as, for example, fungi; however, yeasts or filamentous fungi are more suitable for use in the present invention.

[0066] Yeasts are defined here as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Yeasts: Characteristics and Identification, J.A. Barnett, R.W. Payne, D. Yarrow, 2000, 3rd ed., Cambridge University Press, Cambridge, UK; and, Yeasts, a Taxonomic Study, C.P. Kurtzman and J.W. Fell (eds) 1998, 4th ed., Elsevier Science Publ. BV, Amsterdam, Netherlands) that grow predominantly in unicellular form. Yeasts can grow by budding from a unicellular thallus or can grow by fission of the organism. The preferred yeasts as host cells belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania, and Naumovia. Preferred yeast species as host cells include S. cerevisiae, S. exigus, S. bayanus, K. lactis, K. marxianus, and Schizosaccharomyces pombe.

[0067] Preferably, the yeast cell of the invention is a yeast cell that is naturally capable of anoxic fermentation, more preferably fermentation Petition 870190079230, dated 08 / 15 / 2019, page 49 / 101 42 / 86 alcoholic and more preferably anoxic alcoholic fermentation. Over the years, suggestions have been made for the introduction of various organisms for the production of bioethanol from crop sugars. In practice, however, all major bioethanol production processes have continued to use yeasts of the genus Saccharomyces as ethanol producers. This is due to the many attractive characteristics of Saccharomyces species for industrial processes, namely, a high anaerobic growth capacity, acid tolerance, ethanol and osmotolerance and, of course, their high alcoholic fermentative capacity. More preferably, therefore, a yeast host cell of the invention belongs to a species selected from the group consisting of Saccharomyces cerevisiae, S. bayanus, S. bulderi, S. cervazzii, S. cariocanus, S. castellii, S. dairenensis, S. exiguus. , S. kluyveri, S. kudriazevii, S. mikatae, S. paradoxus, S. pastorianus, S. turicensis and S.unisporus (Kurtzman, 2003, supra; and JA Barnett, RW Payne, D. Yarrow, 2000, supra). Preferably, the yeast cell of the present invention is an industrial yeast strain or a yeast strain derived from an industrial yeast strain. Industrial yeast strains are generally diploid, polyploid, or aneuploid and have proven suitability for large-scale industrial fermentation applications. The strains of... Petition 870190079230, dated 08 / 15 / 2019, pages 50 / 101 43 / 86 Suitable industrial yeasts include, for example, the commercial yeast strains Gert Strand Turbo, Alltech SuperStart™, Fermiol Super HA™, Thermosacc™ and Ethanol Red™. Yeast cells derived from any of these strains by modifications as described herein are also suitable.

[0068] Filamentous fungi are defined herein 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 in most filamentous fungi is obligately aerobic. Preferred filamentous fungi as host cells belong to the genera Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, and Penicillium.

[0069] In a host cell of the invention, the nucleotide sequence encoding xylose isomerase as defined above is preferably operationally linked to a promoter that causes sufficient expression of the nucleotide sequences in the cell to confer upon the cell the ability to convert xylose to xylulose. More preferably, the Petition 870190079230, dated 08 / 15 / 2019, pp. 51 / 101 The 44 / 86 promoter causes sufficient expression of nucleotide sequences to confer on the cell the ability to grow on xylose as the sole source of carbon and / or energy, most preferentially under anaerobic conditions. Suitable promoters for nucleotide sequence expression, as defined above, include promoters that are insensitive to catabolite (glucose) repression and / or that do not require xylose for induction. Promoters with these characteristics are widely available and known to the specialist. Suitable examples of such promoters include, for example, promoters of glycolic genes such as phosphofructokinase (PPK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase (PGK), glucose-6-phosphate isomerase promoters (PGI1) of yeasts or filamentous fungi; More details about such yeast promoters can be found in (WO 93 / 03159).Other useful promoters are those of genes encoding ribosomal protein, the lactase gene promoter (LAC4), alcohol dehydrogenase promoters (ADH1, ADH4 and similar), the enolase promoter (ENO), the hexose (glucose) transporter promoter (HXT7), and the cytochrome c1 promoter (CYC1). Other promoters, both constitutive and inducible, enhancers and / or upstream activating sequences will be known to those skilled in the art. In one way. Petition 870190079230, dated 08 / 15 / 2019, pp. 52-101 45 / 86 preferred, the promoter that is operationally linked to the nucleotide sequence as defined above is homologous to the host cell.

[0070] In a host cell of the invention, the nucleotide sequence encoding xylose isomerase as defined above is preferably expressed from an expression construct in which the coding sequence is operatively linked to a promoter as defined above. An expression construct in a host cell of the invention may be present in a plasmid, preferably a multicopy plasmid. However, more preferably the expression construct is integrated into the host cell genome. Preferably, the host cell comprises multiple copies of the expression construct integrated into its genome. More preferably, the multiple copies (e.g., 2, 3, 4, 5, 6, 8, 10 or more copies) of the expression construct are integrated into more than one, for example, at least two different genomic or chromosomal locations in the host cell genome.A preferred chromosomal location for integrating an expression construct into the genome of a host cell of the invention is an intergenic region, e.g., the intergenic region downstream of TYE7 and upstream of the tRNA gene tP (UGG) O3 on chromosome XV. In one embodiment, the host cell is a diploid, polyploid, or aneuploid host cell. Preferably... Petition 870190079230, dated 08 / 15 / 2019, page 53 / 101 46 / 86 In the diploid, polyploid, or aneuploid host cell, the expression construct is present at a chromosomal location that is present in at least two copies in the cell's genome. Optionally, more than one tandem copy, for example, two copies, of the expression construct is integrated at a genomic or chromosomal location.

[0071] In one embodiment, a host cell of the invention comprises more than one different type of nucleotide sequence encoding, for example, at least two different xylose isomerases as defined above, or for example, encoding xylose isomerases as defined above in combination with any other xylose isomerase, for example, a xylose isomerase already known in the art.

[0072] The host cell of the invention further preferably comprises xylulose kinase activity, such that the xylulose isomerized from xylose can be metabolized to pyruvate. Preferably, the cell contains endogenous xylulose kinase activity. More preferably, a cell of the invention comprises a genetic modification that increases the specific activity of xylulose kinase. Preferably, the genetic modification causes the overexpression of a xylulose kinase, for example by overexpression of a nucleotide sequence encoding a xylulose kinase. The gene encoding the xylulose kinase may be endogenous to the cell or may be a xylulose kinase that is heterologous to the Petition 870190079230, dated 08 / 15 / 2019, pp. 54 / 101 47 / 86 cell. A nucleotide sequence that can be used for the overexpression of xylulose kinase in the cells of the invention is, for example, the S. cerevisiae xylulose kinase gene (XKS1) as described by Deng and Ho (1990, Appl. Biochem. Biotechnol. 24-25: 193-199). Another preferred xylulose kinase is a xylose kinase that is related to Piromyces xylulose kinase (xylB; see document WO 03 / 0624430). This Piromyces xylulose kinase is, in fact, more closely related to prokaryotic kinase than to any known eukaryotic kinase, such as yeast kinase. Eukaryotic xylulose kinases have been identified as non-specific sugar kinases, which have a wide range of substrates that includes xylulose.In contrast, prokaryotic xylulose kinases, to which Piromyces kinase is most closely related, have been indicated as more specific kinases for xylulose, i.e., having a narrower substrate range. In the cells of the invention, a xylulose kinase to be overexpressed is overexpressed by at least a factor of 1.1, 1.2, 1.5, 2, 5, 10, or 20 compared to a strain that is genetically identical except for the genetic modification that causes the overexpression. It is to be understood that these levels of overexpression may apply to the steady-state level of enzyme activity, the steady-state level of the enzyme protein, as well as the steady-state level of... Petition 870190079230, dated 08 / 15 / 2019, pp. 55 / 101 48 / 86 transcript that codes for the enzyme.

[0073] A cell of the invention further preferably comprises a genetic modification that increases the flux of the pentose phosphate pathway as described in WO 06 / 009434. In particular, the genetic modification causes an increase in the flux of the non-oxidative pentose phosphate pathway. A genetic modification that causes an increased flux of the non-oxidative part of the pentose phosphate pathway is understood here as meaning a modification that increases the flux by at least a factor of 1.1, 1.2, 1.5, 2, 5, 10 or 20 when compared to the flux in a strain that is genetically identical except for the genetic modification that causes the increased flux. The flux of the non-oxidative part of the pentose phosphate pathway can be measured as described in WO 06 / 009434.

[0074] Genetic modifications that increase the flux of the pentose phosphate pathway can be introduced into the cells of the invention in various ways. These include, for example, achieving higher steady-state activity levels of xylulose kinase and / or one or more of the enzymes of the non-oxidative pentose phosphate pathway and / or a reduced steady-state level of nonspecific aldose reductase activity. These changes in steady-state activity levels can be effected by selection of spontaneous mutants (or induced by chemicals or Petition 870190079230, dated 08 / 15 / 2019, pp. 56 / 101 49 / 86 radiation) and / or by recombinant DNA technology, for example, by overexpression or inactivation, respectively, of genes that encode the enzymes or factors that regulate these genes.

[0075] In a preferred cell of the invention, the genetic modification comprises the overexpression of at least one enzyme of the (non-oxidative) pentose phosphate pathway. Preferably, the enzyme is selected from the group consisting of enzymes encoding ribulose-5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, transketolase, and transaldolase. Various combinations of enzymes of the (non-oxidative) pentose phosphate pathway may be overexpressed. In one embodiment of the invention, each of the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, transketolase, and transaldolase is overexpressed in the cell of the invention.

[0076] There are several means available in the art for overexpression of enzymes in the cells of the invention. In particular, an enzyme can be overexpressed by increasing the number of copies of the gene encoding the enzyme in the cell, by integrating additional copies of the gene into the cell's genome, by expressing the gene from an expression vector of multiple episomal copies, or by introducing an episomal expression vector comprising multiple copies of the gene. The sequence of Petition 870190079230, dated 08 / 15 / 2019, pp. 57 / 101 The 50 / 86 coding used for enzyme overexpression is preferably homologous to the host cell of the invention. However, coding sequences that are heterologous to the host cell of the invention may also be applied.

[0077] Alternatively, the overexpression of enzymes in the cells of the invention can be achieved by using a promoter that is not native to the coding sequence of the enzyme to be overexpressed, i.e., a promoter that is heterologous to the coding sequence to which it is operatively bound. Although the promoter is preferably heterologous to the coding sequence to which it is operatively bound, it is also preferred that the promoter be homologous, i.e., endogenous to the cell of the invention.Preferably, the heterologous promoter is capable of producing a higher stationary level of transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e., mRNA molecules, per unit time) than 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 primary carbon sources (i.e., more than 50% of the available carbon source consists of xylose or xylose and glucose), more preferably as the sole carbon sources. Petition 870190079230, dated 08 / 15 / 2019, pages 58 / 101 51 / 86 Suitable promoters in this context include promoters as described above for the expression of nucleotide sequences encoding xylose isomerases as defined above.

[0078] Another preferred cell of the invention comprises a genetic modification that reduces the nonspecific aldose reductase activity in the cell. Preferably, the nonspecific aldose reductase activity is reduced in the host cell through one or more gene modifications that reduce the expression of or inactivate a gene encoding a nonspecific aldose reductase. Preferably, the genetic modifications reduce or inactivate the expression of each endogenous copy of a gene encoding a nonspecific aldose reductase capable of reducing an aldopentose, including xylose, xylulose, and arabinose, in the cell's genome. A given cell may comprise multiple copies of the genes encoding nonspecific aldose reductase as a result of di-, poly-, or aneuploidy, and / or a cell may contain several different (iso) enzymes with aldose reductase activity that differ in amino acid sequence and are each encoded by a different gene.Also in such cases, the expression of each gene encoding a non-specific aldose reductase is preferentially reduced or inactivated. Preferably, the gene is inactivated by deletion of at least part of the gene or by disruption of the gene, so that, in this context, the term gene also includes... Petition 870190079230, dated 08 / 15 / 2019, pp. 59 / 101 52 / 86 any non-coding sequence upstream or downstream of the coding sequence, the (partial) deletion or inactivation of which results in a reduction of the 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 cell, and amino acid sequences of such aldose reductases are described in WO 06 / 009434 and include, for example, the (nonspecific) aldose reductase genes of the GRE3 gene of S. cerevisiae (Trâff et al., 2001, Appl. Environm. Microbiol. 67: 5668-5674) and their orthologs in other species.

[0079] A host cell of the invention further preferably comprises at least one genetic modification resulting in a selected group characteristic consisting of: a) increased tolerance to ethanol; b) increased tolerance to acetic acid; c) reduced glycerol production; d) increased xylose for the rate of alcoholic fermentation; ee) increased heat tolerance.

[0080] The genetic modification that results in increased ethanol tolerance is preferably a modification such as those described in WO 2012 / 175552 and WO 2014 / 170330, such as, for example, a modification that introduces alleles of one or more ADE1, KIN3, MKT1 and VPS70 that confer greater ethanol tolerance, and / or a modification Petition 870190079230, dated 08 / 15 / 2019, pages 60 / 101 53 / 86 that overexpresses a wild-type SWS2 gene and / or inactivates the APJ1 gene, which also confers increased ethanol tolerance.

[0081] The genetic modification that results in increased acetic acid tolerance is preferably a modification such as described in WO 2015 / 181169 and WO 2016 / 083397, such as, for example, a modification that introduces an allele of one or more of the GLO1, DOT5, CUP2 and HAA1 genes that confers greater tolerance to acetic acid.

[0082] The genetic modification that results in reduced glycerol production is preferably a modification such as that described in WO 2014 / 048863, such as, for example, a modification that introduces a mutant SSK1 gene encoding a truncated ssk1 protein.

[0083] The genetic modification that results in an increased rate of xylose fermentation to ethanol is preferably a modification such as described in WO 2015 / 086805, such as, for example, a modification that introduces an allele of the NNK1 gene that confers an increased xylose to ethanol fermentation rate.

[0084] The genetic modification that results in increased heat tolerance is preferably a modification such as, for example, described in WO 2014 / 090930, such as, for example, a modification that introduces the overexpression of at least one gene encoding the Petition 870190079230, dated 08 / 15 / 2019, pages 61 / 101 54 / 86 protein Prp42 and a gene that codes for the Smd2 protein.

[0085] A preferred host cell of the invention is a host cell that is improved in at least one industrially relevant phenotype by evolutionary engineering. Evolutionary engineering is a process in which the industrially relevant phenotypes of a microorganism, here yeast, can be coupled to specific growth rate and / or affinity for a nutrient, by a rational configuration natural selection process. Evolutionary engineering is, for example, described in detail in Çakar et al. (2011, FEMS Yeast Research 12: 171-182). Preferably, the D-xylose utilization rate of the host cell is improved by evolutionary engineering. The improvement of the D-xylose utilization rate of yeast host cells by evolutionary engineering is described in detail by Demeke et al. (12, 15 and 16).

[0086] In a preferred host cell according to the invention, the nucleic acid construct confers upon the host cell the ability to grow on xylose as a carbon / energy source, preferably as the sole carbon / energy source and preferably under anaerobic conditions, i.e., conditions as defined herein below for the anaerobic fermentation process. Preferably, when grown on xylose as a carbon / energy source, the transformed host essentially does not produce xylitol, therefore Petition 870190079230, dated 08 / 15 / 2019, pages 62 / 101 55 / 86 For example, the xylitol produced is below the detection limit or, for example, less than 5, 2, 1, 0.5 or 0.3% of the carbon consumed on a molar basis.

[0087] A host cell of the invention preferably has the ability to grow on xylose as the sole carbon / energy source at a rate of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3 h-1 under aerobic conditions, or, more preferably, at a rate of at least 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h-1 under anaerobic conditions. A cell of the invention preferably has the ability to grow on a mixture of glucose and xylose (in a weight ratio of 1:1) as the sole carbon / energy source at a rate of at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.25 or 0.3 h-1 under aerobic conditions, or, more preferably, at a rate of at least 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.12, 0.15 or 0.2 h-1 under anaerobic conditions.Thus, in a preferred host cell according to the invention, the nucleic acid construct confers upon the host cell the ability to anaerobically ferment xylose as the sole carbon source in a process where, ultimately, pyruvate is utilized as an electron (and hydrogen acceptor) and is reduced to fermentation products such as ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propanediol. Petition 870190079230, dated 08 / 15 / 2019, pages 63 / 101 56 / 86 ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics and cephalosporins.

[0088] Preferably, a cell of the invention has a specific xylose consumption rate of at least 200, 300, 400, 600, 700, 800, 900 or 1000 mg h-1 (g dry weight) -1. Preferably, a cell of the invention has a fermentation product yield (such as ethanol) in xylose that is at least 20, 40, 50, 60, 80, 90, 95 or 98% of the fermentation product yield (such as ethanol) in glucose. More preferably, the modified host cell yield of the fermentation product (such as ethanol) in xylose is equal to the host cell yield of the fermentation product (such as ethanol) in glucose. Similarly, the biomass yield of the xylose-modified host cell is preferably at least 55, 60, 70, 80, 85, 90, 95, or 98% of the biomass yield of the glucose host cell.More preferably, the biomass yield of the xylose-modified host cell is equal to the biomass yield of the glucose host cell. It is understood that, in comparing glucose and xylose yields, both yields are compared under aerobic conditions or both under anaerobic conditions.

[0089] In another aspect, the invention relates to a process for the production of a fermentation product. Petition 870190079230, dated 08 / 15 / 2019, pp. 64 / 101 57 / 86 selected from the group consisting of ethanol, lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propanediol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics and cephalosporins. The process preferably comprises the steps of: a) fermenting a medium containing a xylose source with a cell as defined above, wherein the cell ferments xylose into the fermentation product and, optionally, b) recovery of the fermentation product.

[0090] In addition to a xylose source, the carbon source in the fermentation medium may also comprise a glucose source. The expert will further appreciate that the fermentation medium may also comprise other types of carbohydrates, such as, for example, in particular an arabinose source. The xylose and glucose sources may be xylose and glucose as such (i.e., as monomeric sugars) or may be in the form of any oligo- or carbohydrate polymer comprising xylose and / or glucose units, such as, for example, lignocellulose, xylans, cellulose, starch and the like. To release xylose and / or glucose units from these carbohydrates, appropriate carbohydrases may be added (such as xylanases, glucanases, amylases, cellulases, Petition 870190079230, dated 08 / 15 / 2019, pages 65 / 101 58 / 86 glucanases and similar substances) can be added to the fermentation medium or produced by the modified host cell. In the latter case, the modified host cell can be genetically modified to produce and excrete these carbohydrases. An additional advantage of using oligo- or polymeric glucose sources is that it allows maintaining a low concentration of free glucose during fermentation, and using rate-limiting amounts of carbohydrases preferably during fermentation. This, in turn, will prevent the repression of systems necessary for the metabolism and transport of non-carbohydrate sugars, such as xylose. In a preferred process, the modified host cell ferments xylose and glucose, preferably simultaneously, in which case a modified host cell that is insensitive to glucose repression is preferably used to prevent diauxic growth.In addition to a source of xylose (and glucose) as a carbon source, the fermentation medium will also comprise the appropriate ingredient necessary for the growth of the modified host cell. Compositions of fermentation media for the growth of eukaryotic microorganisms such as yeasts and filamentous fungi are well known in the art.

[0091] The fermentation process can be an aerobic or anaerobic fermentation process. A process of Petition 870190079230, dated 08 / 15 / 2019, pages 66 / 101 59 / 86 Anaerobic fermentation is defined here as a fermentation process carried out 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 (i.e., oxygen consumption is not detectable) and in which organic molecules serve as electron acceptors and electron donors. In the absence of oxygen, the 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, thus 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, as well as non-ethanol fermentation products such as lactic acid, 3-hydroxypropionic acid, acrylic acid, acetic acid, succinic acid, citric acid, amino acids, 1,3-propanediol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, β-lactam antibiotics, and cephalosporins. The anaerobic processes of the invention are preferred to aerobic processes because anaerobic processes do not require investment and energy for aeration and, moreover, anaerobic processes produce higher product yields than aerobic processes. Petition 870190079230, dated 08 / 15 / 2019, pages 67 / 101 60 / 86 aerobic processes. Alternatively, the fermentation process of the invention can be carried out under limited aerobic oxygen conditions. Preferably, in an aerobic process under limited oxygen conditions, the oxygen consumption rate is at least 5.5, more preferably at least 6, and even more preferably at least 7 mmol / L / h.

[0092] The fermentation process is preferably carried out at a temperature that is optimum for the modified cells of the invention. Thus, for most yeasts or fungal cells, the fermentation process is carried out at a temperature below 42 °C, preferably below 38 °C. For filamentous yeasts or fungal cells, the fermentation process is preferably carried out at a temperature below 35, 33, 30 or 28 °C and at a temperature above 20, 22 or 25 °C. For some species, such as Kluyveromyces marxianus, and manipulated Saccharomyces cerevisiae strains, the fermentation process can be carried out at considerably higher temperatures, i.e., at 42 °C, 43 °C, or preferably between 45 and 50 °C, or in rare cases between 50 and 55 °C.

[0093] Preferably, in the fermentation processes of the invention, the cells stably maintain the nucleic acid constructs that confer on the cell the ability to isomerize xylose into xylulose and, optionally, convert Petition 870190079230, dated 08 / 15 / 2019, pages 68 / 101 61 / 86 arabinose to D-xylulose 5-phosphate. Preferably, in the process, at least 10, 20, 50 or 75% of the cells retain the capacity to isomerize xylose to xylulose and, optionally, convert arabinose to D-xylulose 5-phosphate after 50 generations of growth, preferably under industrial fermentation conditions.

[0094] A preferred fermentation process according to the invention is a process for the production of ethanol, wherein the process comprises the steps of: a) fermenting a medium containing a xylose source with a cell as defined above, wherein the cell ferments xylose and optionally b) recovering the ethanol. The fermentation medium may also be made as described above. In the process, the volumetric productivity of ethanol is preferably at least 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g of ethanol per liter per hour. The ethanol yield from xylose and / or glucose in the process is preferably at least 50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is defined herein as a percentage of the maximum theoretical yield, which for xylose and glucose is 0.51 g ethanol per g xylose or glucose.

[0095] In this document and its claims, the verb "to understand" and its conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Furthermore, the reference to an element by the article Petition 870190079230, dated 08 / 15 / 2019, pp. 69 / 101 62 / 86 The indefinite article a or an does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article a or an normally means at least one.

[0096] All patent and literature references cited in this specification are incorporated herein by reference in their entirety.

[0097] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Brief description of the drawings

[0098] Figure 1. Xylose fermentation performance of the M315CpXIΔ / CpXIΔ strain expressing a plasmid containing one of the first 7 XylA genes. The code indicating the bacterial origin of the XylA genes is explained in Table 1. Fermentation was performed in duplicate using two independent transformants at an initial cell density of 1 g DW / L in 50 mL of YP medium containing 4% xylose at 35 °C. The average value is shown in the graph. CO2 production was estimated by measuring weight loss during fermentation.

[0099] Figure 2. Xylose fermentation performance of the M315CpXIΔ / CpXIΔ strain expressing a plasmid with one of the 11 XylA genes. The code indicating the bacterial origin of the XylA genes is explained in Table 1. The fermentation was carried out Petition 870190079230, dated 08 / 15 / 2019, pages 70 / 101 63 / 86 using an initial cell density of 1 g DW / L in 45 mL of YP medium containing 4% xylose at 35 °C. CO2 production was estimated by measuring weight loss during fermentation.

[00100] Figure 3. Integration of XylA genes into the genome.

[00101] A) Integration method using the CRISPR / Cas9 methodology on chromosome XV between TYE7 and tp(UGG)O3. The arrows indicated by g1 and g2 are gRNA sites where Cas9 performs a double-strand break in the chromosomes, guided by two gRNA cleavage sites on a single guide RNA plasmid. A plasmid-based donor DNA (pDonor) contained two Xyl sequences, XI1 and XI2, flanked by the H1 and H2 sequences, which are homologous to the integration site to support homologous recombination.

[00102] B) PCR gel electrophoresis image performed to verify proper insertion of XylA genes into the genome using two primers flanking homologous sequences H1 and H2 [shown as prFw (GY94) and prRv (GY95)] at the bottom of panel A. Insertion of a single copy of XylA into both chromosome alleles produced a PCR product of approximately 3kb (e.g., Lane CpXI 1, 2, and 5), while insertion of two copies into both alleles resulted in a PCR product of 5kb (e.g., Lane CpXI 3 and 6). The absence of an XylA insertion is expected to produce a PCR band of approximately 1.6 Kb, which is the size of the PCR band obtained for the control strain T18. Petition 870190079230, dated 08 / 15 / 2019, pp. 71 / 101 64 / 86

[00103] Figure 4. Xylose fermentation performance of GSE16-T18CpXIΔ / CpXIΔ-based strains after genetic integration of different XylA genes. The code indicating the bacterial origin of the XylA genes is explained in Table 1. For each XylA gene, two strains containing 2 or 4 copies were selected. Fermentation was performed using an initial cell density of 1 g DW / L in 50 mL of YP medium containing 4% xylose at 35 °C. CO2 production was estimated by measuring weight loss during fermentation.

[00104] Figure 5. A) Xylose fermentation performance of MDS130-based strains with genomic integration of different XylA genes as indicated. The code indicating the bacterial origin of the XylA genes is explained in Table 1. B) Direct comparison of the xylose fermentation performance of the MDS130 strain with the MDC5 strain. Examples Example 1 Introduction

[00105] Despite the vast amount of information on xylose isomerase sequences in public sequence databases, only a few have been functionally expressed in yeast. A bottleneck may be due to differences in the regulatory mechanisms of protein synthesis between prokaryotes and eukaryotes. Bacterial protein synthesis in yeast may not be adequately regulated, which could be the Petition 870190079230, dated 08 / 15 / 2019, pp. 72-101 65 / 86 reason for the frequent occurrence of inactive or insoluble proteins. Studies have shown that adequate gene expression does not necessarily correlate with adequate enzymatic activity (13). In most strains expressing XylA, high XI enzymatic activity and adequate xylose fermentation capacity were observed only after evolutionary adaptation of the host strain, indicating that there are other regulatory mechanisms necessary for the proper functioning of XI enzymes and their proper connection with other enzymes in the yeast fermentation pathway (11,12). Although the regulatory mechanisms are not well understood, certain genetic alterations are required by the recombinant host for adequate XI activity. This lack of a suitable yeast strain as a host for functional expression, in turn, makes it difficult to screen potential XIs that may be active in an appropriate host but not in a regular yeast strain.

[00106] To overcome the bottleneck of the absence of a suitable host strain for screening active bacterial XI genes, we developed two yeast strains that are able to grow directly and efficiently ferment xylose after the expression of a bacterial XI. These strains have the same industrial yeast lineage as the strain that successfully expressed the Clostridium phytofermentans XylA (CpXI) gene (12,15,16). The first strain M315_CpXIΔ / CpXIΔ was Petition 870190079230, dated 08 / 15 / 2019, pp. 73 / 101 66 / 86 was developed by deleting both copies of CpXI from the genome of the M315 strain. This M315 progenitor strain was developed by random mutagenesis of a recombinant industrial strain, Ethanol Red, which contained two copies of CpXI and xylulokinase (XKS1), and all genes of the pentose phosphate pathway (PPP) were overexpressed on the chromosome. The second platform strain, GSE16-T18CpXI Δ / CpXIΔ, was developed by deleting all copies of the CpXI gene from the chromosome of the xylose fermentation industrial strain GSE16-T18, which contained 16 to 18 copies of CpXI. GSE16-T18 was developed from the M315 lineage through a series of evolutionary adaptive rounds in synthetic media and lignocellulose hydrolysate. Eliminating all CpXI copies of the strain completely abolished xylose fermentation performance. Reintroducing xylose isomerase into these platform strains restored the efficiency of xylose fermentation capacity.Therefore, these two strains provided us with a useful tool for screening potential XylA genes from different sources for functional expression in yeast. Using these platform strains, we were able to select several bacterial XylA genes for rapid xylose fermentation capability, which resulted in the identification of several genes expressing xylose isomerase with superior performance. Materials and methods Petition 870190079230, dated 08 / 15 / 2019, pp. 74 / 101 67 / 86 Construction of multi-copy plasmids carrying XylA genes.

[00107] Each of the 14 bacterial XylA genes was synthesized in two blocks of approximately 700 bp with 30 bp of overlap. The two gblock gene fragments were PCR-linked using a pair of primers, each with a 30 bp tail sequence to create overlap at the 5' and 3' ends of a linearized p426tef1 vector. The p426tef1 vector (Mumberg et al., 1995, Yeast vectors for controlled heterologous protein expression in different gene pools. Gene, 156; 119-122) was linearized using PstI and HindIII restriction enzymes between the tef1 promoter and the cyc1 terminator. The PCR fragment and the linearized vector were assembled using the Gibson Assemblies cloning kit (New England BioLabs, USA), and transformed into chemically competent Top10 E. coli strain (Invitrogen). Plasmids were subsequently isolated from E. coli using the NucleoSpin® Plasmid EasyPure kit (MACHEREY-NEGEL GMBH & CO. KG, Germany).The isolated plasmids were transformed into the host yeast strain using the standard LiAc / PEG method (18). CpXI suppression of GSE16-T18

[00108] The GSE16-T18 strain carried between 16 and 18 copies of the CpXI gene, which was originally inserted in two copies, replacing part of the PYK2 gene on chromosome XV. The gene Petition 870190079230, dated 08 / 15 / 2019, pp. 75 / 101 68 / 86 CpXI was amplified at the chromosomal locus in multiple tandem repeats during an evolutionary engineering step (15).

[00109] Multiple copies of the XylA genes were eliminated using a CRISPR / Cas9-based methodology. First, a single plasmid carrying gRNA with two target gRNA sequences from both ends of the amplified xylA genes and a hygromycin resistance gene hph was constructed. Next, two donor DNA fragments were produced by PCR amplification of two selection marker genes, the kanamycin resistance gene kan and the nourseotricin resistance marker nat. Each marker gene was flanked by sequences homologous to the upstream and downstream gRNA target sequences in the genome. After that, the GSE16-T18 strain was transformed with a Cas9 plasmid having a selection marker of ble. The GSE16-T18-Cas9 strain expressing Cas9 was subsequently transformed with the gRNA plasmid and the two donor DNA fragments. Transformants were selected only for the hph marker in the gRNA.Positive transformants expressing the resistance marker hph were evaluated for effective replacement of multiple copies of the XyIA gene with the two markers kan and NAT, both phenotypically and by PCR. A strain that replaced all Xyla copies with one kan and one nat marker was selected, and the markers were... Petition 870190079230, dated 08 / 15 / 2019, pp. 76 / 101 69 / 86 was subsequently removed by another CRISPR / Cas9 step using a gRNA plasmid that targets each of the markers and kan nat. A complete PYK2 sequence flanked by sequences upstream and downstream of the genome-widely inserted markers was used as donor DNA to cure the partially deleted PYK2 gene. The final strain, devoid of any CpXI gene and carrying the entire PYK2, was referred to as GSE16-T18CpXIΔ / CpXIΔ. Optimization of the CRISPR / Cas9 method for genomic integration of XylA genes.

[00110] Genomic integration of 2 to 4 copies of each of the XylA genes was performed using an optimized CRISPR / Cas9 system. First, donor DNA was constructed into a multicopy plasmid containing two XylA sequences flanked by sequences homologous to sequences upstream and downstream of the integration site to trigger homologous recombination. The donor plasmid DNA (pDonor) was transformed into the yeast strain GSE16-T18CpXIA / CpXIA and directly selected on plates containing xylose as a carbon source. Strains carrying the pDonor plasmid were then transformed with a gRNA plasmid with the hph marker and a Cas9 plasmid with the kan marker and selected for geneticin and hygromycin YPD+. Transformants that grow in the presence of both antibiotic resistance markers were transferred to a Petition 870190079230, dated 08 / 15 / 2019, pp. 77 / 101 70 / 86 new YPD plates were used to assess correct integration of donor DNA into the genome. This was performed by PCR using a pair of primers upstream and downstream of the insertion site. Once insertion was confirmed, the strains were depleted by culturing them in YPD medium for 5 days and then serially transferring the strains to a new YPD plate every 24h. After 5 days, a sample was spread to single colonies and several colonies were evaluated for loss of gRNA and Cas9 plasmids containing the hph and kan markers, respectively. Colonies that lost both plasmids were checked by PCR to assess loss of the donor plasmid once the donor plasmid was devoid of selection marker. Small-scale fermentations

[00111] Small-scale fermentations were carried out essentially according to the protocol described previously (12). Briefly, cells were pre-cultured in 5 ml of YPD for 24 h. Subsequently, 1 ml of culture was transferred to 50 mL of YPD in a 300 mL Erlenmeyer flask. After 24 h of growth, the cells were harvested and a quantity of 1 g DW / L of cells was inoculated into 50 mL of YP medium containing 4% w / v xylose as a carbon source, in cylindrical tubes with rubber stoppers and glass tubes. The cultures were continuously stirred with a magnetic stirrer at 120 rpm and incubated at 35 °C. The Petition 870190079230, dated 08 / 15 / 2019, pages 78 / 101 71 / 86 progress of fermentations was followed by measuring the weight due to CO2 loss during fermentation. Results Screening for XI sequences that support the growth of S. cerevisiae on xylose as the sole carbon source.

[00112] Expression in yeast of XIs originating from various bacterial species has been reported in the last decade. Most enzymes did not show reasonable enzymatic activity in S. cerevisiae. Only a limited number of XIs with good enzymatic activity are available so far. As there are a large number of sequences in public databases, such as NCBI, we explored sequence databases to search for XylA genes originating from diverse environments. We selected 16 sequences encoding XI from 16 bacterial species. The sequences ranged from 62% to 96% identity with the C. phytofermentans XylA sequence (Table 1) and between 50 and 55% with the Piromyces sp E2 XylA sequence at the amino acid level.

[00113] Each sequence was encoded and synthesized by IDT (Integrated DNA Technologies, Heverlee, Belgium). The genes with optimized codons were subsequently cloned into a yeast p426-TEF1 expression vector, under the control of the TEF1 promoter and Cyc1 terminator. For comparison, we also constructed a plasmid with the CpXI gene under the control of the same promoter and terminator. The plasmids Petition 870190079230, dated 08 / 15 / 2019, pp. 79 / 101 72 / 86 constructed were subsequently transformed into the M315CpXIΔ / CpXIΔ platform strain.

[00114] Transformants were selected in synthetic medium containing xylose as a carbon source (SCX plate). After 5 days at 30 °C, 7 of the 14 transformants were able to grow on the SCX plate. Later, 4 additional transformants grew in smaller colonies after 7 days, indicating that the genes of these 4 additional transformants only supported the slow growth of xylose. However, a total of 11 of the 16 genes tested were able to support growth in medium with xylose as the sole carbon source. Confirmation of correct gene expression.

[00115] In order to confirm the presence of the gene expressed in the host strain, polymerase chain reaction (PCR) was performed using primers that specifically amplify each gene. As expected, a positive PCR result was obtained with the expected molecular weight of 1.2 kb for all strains tested (data not shown). The negative control strains M315CpXIΔ / CpXIΔ and M315CpXI failed to show a PCR band, confirming the specificity of the PCR product. Fermentation in a medium with xylose

[00116] Since growth and fermentation are different characteristics and often do not coincide Petition 870190079230, dated 08 / 15 / 2019, pages 80 / 101 73 / 86 correlate well with each other, we evaluated all 11 XylA transformants for fermentation performance in YP medium containing xylose as the sole carbon source. The first 7 XylA transformants were tested in an initial batch of fermentation assays. Interestingly, all 7 XylA transformants showed rapid xylose fermentation capability in YP medium containing 4% xylose (Figure 1). A control strain with the CpXI gene was also evaluated for comparison. Two of the newly isolated genes (Es7XI and Cc10XI) maintained xylose fermentation performance similar to that of CpXI.

[00117] Subsequently, we repeated the fermentation test and included the four slow-growing XylA transformants. As shown in Figure 2, all 11 transformants were able to ferment xylose very well. The 7 strains showing fast fermentation in the first fermentation test also showed the same fast fermentation profile. In addition, two strains from the second slow-growing batch (Es5XI and Cl11XI) showed a fermentation profile similar to the first 7 fast-fermenting strains. Therefore, 9 of the 11 transformants were able to support the fast fermentation capacity of xylose in an industrial yeast background.

[00118] To confirm that the XylA transformants did not actually carry any more CpXI, which is capable of supporting Petition 870190079230, dated 08 / 15 / 2019, pages 81 / 101 74 / 86 due to its high xylose fermentation capacity, we tested all cultures at the end of fermentation by PCR using specific primers unique to the CpXI sequence. As expected, none of the cultures were positive for the CpXI gene, while the control strain carrying the CpXI plasmid was positive for the expected molecular weight band (data not shown). Integration of XylA genes into the genome

[00119] Since the number of plasmid copies varies greatly in vivo during growth or fermentation, selecting the most active gene based on the fermentation performance of plasmid-carrying strains can create a strong bias. Furthermore, plasmids are unstable and not an ideal gene expression system for industrial application. Thus, we performed genomic integration of 3 of the 8 genes that support the best xylose fermentation capacity, and also the CpXI gene for comparison. The integration was performed in the genome of a robust industrial platform yeast strain GSE16T18CpXIΔ / CpXIΔ, using a modified CRISPR / Cas9 system that we optimized for a single transformation step and for efficient integration of foreign genes in 2 to 4 copies, as described in the methods section. Using this methodology, we were able to stably integrate 2 to 4 copies of each of the genes into an intergenic region. Petition 870190079230, dated 08 / 15 / 2019, pages 82 / 101 75 / 86 downstream of TYE7 and upstream of the tRNA tP (UGG) 03 gene on chromosome XV. Proper integration of the genes into the genome was confirmed by PCR (Figure 3). Fermentation performance after XylA genomic integration

[00120] The fermentation performance of strains carrying 2 to 4 copies of each XylA gene was evaluated in YP medium with xylose as the sole carbon source. As shown in Figure 4, strains carrying the Es7XI, Cs8XI, or Fm9XI gene in their genome showed high xylose fermentation capacity. Strains carrying Cs8XI and Fm9XI showed a xylose fermentation rate comparable to that of the strain carrying CpXI from the beginning, while the strain carrying Es7XI showed a delay in the onset of fermentation but later recovered a high xylose fermentation rate. Although the strain with two copies of Cs8XI showed a slightly slower xylose fermentation rate than the strain with two copies of CpXI, it showed the highest fermentation rate during the exponential phase of fermentation (Figure 4). Furthermore, the strains with 4 copies of Cs8XI fermented at a higher rate than the strain with the same number of copies of CpXI. Conclusion

[00121] Eleven of the 16 recently identified xylA genes confer very good xylose fermentation performance in an industrial yeast strain when expressed from Petition 870190079230, dated 08 / 15 / 2019, pages 83 / 101 76 / 86 a multi-copy plasmid, under the control of the TEF1 promoter and Cyc1 terminator. Except for the XylA gene obtained from L. phytofermentans, which has 96% sequence identity with that of CpXI, all functionally expressed XylA genes lack significant sequence identity with any of the xylose isomerases that have been actively expressed to date. The bacterial species from which these XylA genes were obtained are isolated from diverse environments. Although most species inhabit environments rich in plant matter, which explains their cellulolytic capacity, the bacterium M. indolicus is a non-cellulolytic organism that was isolated from the female genital tract (17). From an evolutionary point of view, this would indicate that there is no correlation with the functionality of Xylose Isomerase, since there is no need for XI to remain active in an environment without hemicellulose. On the other hand, it cannot be excluded that the bacterium M.Indolicus also lives in environments where the use of xylose is important for its survival, but not for lignocellulolytic capacity.

[00122] Three of the 11 XylA genes were studied after their integration into the genome. The Cs8XI gene was among the best for conferring xylose fermentation capability to the industrial strain of the platform when integrated in 2 or 4 copies. This gene is derived from an acetone-butanol producing bacterial species, C. saccharoperbutylacetonicum. Although if Petition 870190079230, dated 08 / 15 / 2019, pages 84 / 101 77 / 86 know that the bacterium uses xylose, the XI gene of this organism has never been expressed in the yeast S. cerevisiae. On the other hand, the Fm9XI gene has been previously expressed in yeast (WO 2010 / 074577). Interestingly, the Cs8XI and Fm9XI XylA genes have only 68% sequence identity at the amino acid level. The low sequence identity of the two XylA genes is not surprising, since the two originating organisms are not related. Cs8XI is therefore a novel gene that confers excellent xylose fermentation capacity in yeast with chromosomal integration of only 2 to 4 copies. Integration of additional copies of the gene may further improve xylose fermentation capacity. Furthermore, the integration of the other genes identified in this work into the genome of the platform strain is important for stable gene expression and may also result in high xylose fermentation capacity. Example 2 Performance of Es7XI and Cc10XI in the MDS130 lineage

[00123] We further improved the GSE16-T18 strain for enhanced xylose fermentation and inhibitor tolerance through genomic rearrangement and evolutionary adaptation. The MDS130 strain was thus selected, showing highly improved xylose fermentation capacity in inhibitor-rich hydrolysates. Subsequently, we completely eliminated the CpXI genes from the MDS130 genome using the CRISPR / Cas9 technique. Petition 870190079230, dated 08 / 15 / 2019, pages 85 / 101 78 / 86 as described above in the section “CpXI deletion from GSE16-T18. As expected, the knockout strain MDS130CpXIΔ / CpXIΔ was unable to utilize xylose (Figure 5A).

[00124] Next, we introduced the two new high-performing XI genes, Es7XI and Cc10XI, into the MDS130CpXIA / CpXIA genome downstream of the TYE7 gene on chromosome XV. With only two copies of each gene introduced, the deletion strain was able to utilize xylose, but at a slower rate compared to the original MDS130 strain which carried approximately 18 copies of CpXI. In order to assess whether the combination of the two genes improved xylose fermentation performance, we introduced 4 additional copies of Cc10XI into strains containing two copies of Es7X. This resulted in a significant improvement in the fermentation rate, approaching the performance of the MDS130 strain (Figure 5A).

[00125] It has been previously shown that a gene of interest adjacent to an ARS sequence is frequently amplified when cells grow under selective pressure requiring high expression of the gene of interest (WO2016026954). For this reason, we introduced Ex7XI approximately 2000 nucleotides upstream of ARS1529 in two copies and evolved it in YP + 4% xylose to induce chromosome amplification. After 3 weeks, isolates from individual cells were evaluated and the MDC5 strain that best Petition 870190079230, dated 08 / 15 / 2019, pages 86 / 101 The 79 / 86 strain developed from the tested single-cell isolates was selected. Analysis of gene copy number by qPCR showed that this strain contained approximately 12 copies of Es7XI. The performance of the MDC5 strain with 12 copies of Es7XI was similar to that of MDS130, which carried approximately 18 copies of CpXI (Figure 5B). This demonstrates the superior performance of Es7XI over CpXI, at least in the background of the tested strain. Table 2. Clustal alignment of xylose isomerase amino acid sequences by MUSCLE / 3.8) PiXI MAKEYFPQIQKIKFEGKDSKNPLAFHYYDAEKEVMGKKMKDWLRFAMAWWHTLCAEGADQ Cc10XI -MKEYFSNIPKVRYEGPDSKNPFAFKFYNPEEKIAGKTMREQLKFSLAYWHTLDAEGTDM Am6XI -MREHFLEINKIKFEGGDSTNPLAFKYYDANRIVAGKKMKDHLRFALSYWHTLTGNGTDP Fm9XI —MEFFKGIDKVKYEGVKTNNLLAFAHYNPEEVILGKKMKDHLKFAMSYWHTLTGEGTDP Cs8XI -MKEYFGNVSKINYEGPGSKNPYSFKYYNPDEVIGGKTMKEHLRFSLSYWHTLTANGADP Cl11XI -MAEFFKGIGVIPFEGADSVNPLAFKHYNKDEKVGDKTMAEHLRFAMSYWHTLCAEGGDP Pcd12XI -MSEIFKGIGQIKFEGVKSDNELAFRYYNPEQVVGNKTMKEHLRFAMSYWHTLCGEGNDP Es7XI ---MYFNNIEKIKFEGVNSKNPLAFKYYDADRIIAGKKMSEHLKFAMSYWHTMCADGTDM Es5XI -MVNGLTNIPPVKFEGRDSKKALSFKYYNPDEMIQGKKMKDYLKFAMSYWHTLCGDGTDP Mi3XI —MKFFENVPKVKYEGSKSTNPFAFKYYNPEAVIAGKKMKDHLKFAMSWWHTMTATGQDQ Ca2XI -MKEYFKGIPEVKYEGKDSINPFAFKFYDAKRVIDGKSMEEHLKFAMSWWHTMTATGTDP Lp1XI -MKNYFPNVPEVKYEGPNSTNPFAFKYYDAERIVAGKTMKEHCRFALSWWHTLCAGGADP CpXI -MKNYFPNVPEVKYEGPNSTNPFAFKYYDANKVVAGKTMKEHCRFALSWWHTLCAGGADP • · · ·** · · · * * · · * * *....***. * * : : : : : : : .: . : ♦ ♦ : .::::: . PiXI FGGGTKSFPWNEGTDAIEIAKQKVDAGFEIMQKLGIPYYCFHDVDLVSEGNSIEEYESNL Cc10XI FGRATMDKSFGETD-PMAIYKNKAYAAFELMDKLDIDYFCFHDRDIAPEGPTLSETNKNL Am6XI FGQPTMERDYNSLD-GIELSKARVDAAFELMTKLGIEFFCFHDLDIAPEGNSLQEKLDNL Fm9XI FGNATMDREWNEYT-PMEKAKARVKAGFEFMEKLGLEYFCFHDKDIAPEAETLEEYHRNL Petition 870190079230, dated 08 / 15 / 2019, pages 87 / 101 80 / 86 Cs8XI C111XI Pcd12XI Es7XI Es5XI Mi3XI Ca2XI LplXI CpXI PiXI CclOXI Am6XI Fm9XI Cs8XI C111XI Pcd12XI Es7XI Es5XI Mi3XI Ca2XI Lp1XI CpXI PiXI Cc10XI Am6XI Fm9XI Cs8XI C111XI FGAGTMLRPWDDITNEMDLAKARMEAAFELMDKLNIEYFCFHDRDIAPEGKTLQETNENL FGSTTAARPWNQIANPIEMAKAKVDAGFEFMQKLGIEYFCFHDRDIAPEGKDLAETNQIL FGVGTVERPWNNITDPIEIAKIKVDAGFEFMSKMGIEYFCFHDRDIAPEGRDLEETNKIL FGRGTINKSFGGKT-AIEIYEHKVYAAFELMEKLGMQYFCFHDRDIAPEGATLKETNENL FGSTSTIDRDYSGQT-PMEKAKTKADVAFALMQILGIEYFCFHDLDIAPTGNSLKELKNNL FGSGTMSRIYDGQTEPLALAKARVDAAFDFMEKLNIEYFCFHDADLAPEGNSLQERNENL FGAGTIDRNYGQTE-SMEIARAKVDAAFELMKKLGIKYFCFHDVDIVPEGKDLKETKENL FGVTTMDRTYGNITDPMEFAKAKVDAGFELMTKLGIEYFCFHDADIAPEGDTFEESKKNL FGVTTMDRTYGNITDPMELAKAKVDAGFELMTKLGIEFFCFHDADIAPEGDTFEESKKNL ** * :. : . ..* :* :.: ::**** *:.. .: * * KAVVAYLKEKQKETGIKLLWSTANVFGHKRYMNGASTNPDFDVVARAIVQIKNAIDAGIE DEIVSLLKKLMAEHNKKLLWGTANTFSHPRYVHGAGTSCNASVFAFAAAQIKKALEITKE DTILERIEDKMKETGIKCLWGTTNAFSHPRFMHGAATSPNADVFAFAAAQVKKALEITHR DEIVDLIEEEMKRTGIKLLWGTSNMFSHPRFMHGAATSCNADVFAYAAAQTKKALEITKR DEIVAYCKELMKKYNKKLLWGTANCFTNPRYVHGAGTSCNADVFAYAAAQIKKALEVTKE DEVVAYIKVKMQETGIKLLWGTANCFNNKRFMHGAGTTCNAEVFAYAAAQIKKALEVTKE DEIVEYIKVNMEKTGIKLLWGTANMFGNPRFVHGASTTCNADVYAYAAAQVKKAMEITKY ERIVPIIKSEMKRTGIKLLWGTANCFNHPRYMCGAGTAPSADVFAYAAAQIKKALEITVE IEITDYIKGLMDKTGIKLLWGTANCFSHPRYMNGAGTSPQADIFACAAAQIKNAIDATIK QEMVSYLKQKMAGTSIKLLWGTSNCFSNPRFMHGAATSCEADVFAWTATQLKNAIDATIA SVIVDYIEEKMKGTDIKLLWGTANCFSSPRYMHGAGTSCNADSFSYAASQIKNAIDATIQ FVIVDYIKEKMDQTGIKLLWGTANNFGHPRFMHGASTSCNADVFAYAAAKIKNALDATIK FEIVDYIKEKMDQTGIKLLWGTANNFSHPRFMHGASTSCNADVFAYAAAKIKNALDATIK : : . * **.*:* * *:: **.* . . : : : *::*. LGAENYVFWGGREGYMSLLNTDQKREKEHMATMLTMARDYARSKGFKGTFLIEPKPMEPT LDGCGYVFWGGREGYETLLNTDMELELDNMARLLKMAVDYARSIGFKGEFFIEPKPKEPT LRGENYVFWGGREGYETLLNTDIALENDNLAKFLKMAKDYARNIGFEGQFLIEPKPKEPT LNGTGYVFWGGREGYETLLNTDIGLELDNLARFLQMAVDYAKKIGFEGQFFIEPKPKEPT LGGENYVFWGGREGYETLLNTDMGLELDNFARLLQMAVDYAKEIGFTGQFLIEPKPKEPT LGGENYVFWGGREGYETLLNTDTGLELDNFARLLQMAVDYAKEIGFTGQFLIEPKPKEPT Petition 870190079230, dated 08 / 15 / 2019, pages 88 / 101 81 / 86 Pcd12XI Es7XI Es5XI Mi3XI Ca2XI Lp1XI CpXI PiXI CclOXI Am6XI Fm9XI Cs8XI ClllXI Pcd12XI Es7XI Es5XI Mi3XI Ca2XI LplXI CpXI PiXI Cc10XI Am6XI Fm9XI Cs8XI ClllXI Pcd12XI Es7XI LGGENFVFWGGREGYETLLNTNTELEMDNFARFLQMAVDYAKEIGFTGQFLIEPKPKEPT LGGQGYVFWGGREGYDTILNTDMAKEQDNMAYLMRMAVDYGRSIGFTGDFYIEPKPKEPT LGGTGYVFWGGREGYETLLNTNMEIELDNMAKLMHMAVDYARSKGFTGDFYIEPKPKEPT LGGKGYVFWGGREGYETLLNTDVGLEMDNYARMLKMAVAYARSKGYTGDFYIEPKPKEPT LGGSGYVFWGGREGYETLLNTDMGFELDNMARLMKMAVKYARKKGFNGDFYIEPKPKEPT LGGKGYVFWGGREGYETLLNTDLGLELDNMARLMKMAVEYGRANGFDGDFYIEPKPKEPT LGGKGYVFWGGREGYETLLNTDLGLELDNMARLMKMAVEYGRANGFDGDFYIEPKPKEPT * . .:********* ::***: * :: * :: ** *..*: * * ***** *** KHQYDVDTETAIGFLKAHNLDKDFKVNIEVNHATLAGHTFQHELRVARINGVLGSVDANQ KHQYDFDTMTVLGFLRKYNLIDDFKLNIEANHATLAGHTFQHELAMARINGVLGSVDANQ KHQYDFDTTTVLEFLRKYNLDKYFKMNIEANHATLAGHTFQHELCTARINGVFGSIDANQ KHQYDFDTATVLGFLKYNLDKYFKVNIEANHATLAQHTFQHELNFARINNFLGSIDANQ KHQYDFDTATVLAFLRKYNLDTYFKMNIEANHATLAGHTFQHELNMSRINNVLGSIDANQ KHQYDFDTATVLGFLRKYNLDTYFKMNIEANHATLAGHTFQHELNMIANINNVLGSIDANQ KHQYDFDTATVLGFLRKYNLDTYFKMNIEANHATLAGHTFQHELNIANINNVLGSIDANQ KHQYDFDVSTVLAFLRKYNLDKDFKMNIEANHATLAGHTFQHELNMIANINNVLGSIDANQ KHQYDFDVATVVGFLRKYGLDKDFKMNIEANHATLAGHTFQHELNVARVNNVFGSIDANQ KHQYDFDVATCVAFLEKYDLMRDFKVNIEANHATLAGHTFQHELRMARTFGVFGSVDANQ KHQYDFDAATVIGFLRKYDLMDDFKLNIEANHATLAGHTFPHELAVARINGVFGSVDANQ KHQYDFDTATVLGFLRKYGLEKDFKMNIEANHATLAGHTFHEHELALARVNGVFGSVDANQ KHQYDFDTATVLAFLRKYGLEKDFKMNIEANHATLAGHTFHEHELAMARVNGAFGSVDANQ ***** *. * : ** :.* **:***.****** *** *** : . :**:***.GDYQNGWDTDQFPIDQYELVQAWMEIIRGGGFVTGGTNFDAKTRRNSTDLEDIIIAHVSG GDVMLGWDTDQFPTNVYDTALAMYEILKNGGLPSGGLNFDSKNRRGSFEPEDIFHGFIAG GDLLLGWDTDQFPTNIYDATLSMYEVLKNGGIAPGGLNFDAKVRRGSFKPDDLFIAYIVG GDMLLGWDTDQFPTNVYDAVLAMYETLLAGGFKEGGLNFDAKVRRGSFEPKDLFYAYISG GDPMLGWDTDQFPTNIYDATLAMYEILKNGGLAPGGVNFDAKVRRASFEKEDLFLAYIAG GDLMLGWDTDQFPTNIYDATMAMYEVLKAGGIAPGGFNFDSKVRRGSFEEADLFIAYIAG GDLLLGWDTDQFPTNIYDATLAMYEVLKQGGIAPGGFNFDSKVRRASFEVEDLFLAYIAG GDMLLGWDTDQFPTDLYSTTMCMYEVLKQGGFTNGGLNFDAKARRASNTYEDVFLSYIAG. Petition 870190079230, dated 08 / 15 / 2019, pages 89 / 101 82 / 86 Es5XI Mi3XI Ca2XI LplXI CpXI PiXI CclOXI Am6XI Fm9XI Cs8XI ClllXI Pcd12XI Es7XI Es5XI Mi3XI Ca2XI LplXI CpXI PiXI CclOXI Am6XI Fm9XI Cs8XI ClllXI Pcdl2XI Es7XI Es5XI Mi3XI GDLLLGWDTDQFPTNVYDTTLCMLEVIKAGGFTNGGLNFDAKVRRASYTMEDIILAYISG GDSNLGWDTDQFPGNIYDTTLAMYEILKAGGFTNGGLNFDAKVRRPSFTPEDIAYAYILG GDSLLGWDTDQFPTDVKEATLSMLEIIKAGGFTNGGLNFDAKVRRPSFTFEDIVYGYISG GDPNLGWDTDQFPTDVHSATLAMLEVLKAGGFTNGGLNFDAKVRRGSFEFDDIAYGYIAG GDPNLGWDTDQFPTDVHSATLAMLEVLKAGGFTNGGLNFDAKVRRGSFEFDDIAYGYIAG ** ******** : . . . * : **: ** ***.* ** * *: ..: * MDAMARALENAAKLLQESPYTKMKKERYASFDSGIGKDFEDGKLTLEQVYEYGKKNGEPMDAFALGLRIADRIIRDGRLEQFVKDRYKSYQSGIGADIVSGRAKIEDLEKYALKLGEVN **::* .* * :: : : .* *: *** : : :: * KQTSGKQELYEAIVA—MYQ------AIGSGRQEYLEDILNSIMFGK-----VLESGRQEMLEDIVNRYIYK------KNSSGRQEMLENILNRYIYE------TNKSGRQELLEAIVNQYIFED-----VNKSGRQEWLETVVNQYIYNNK----KNKSGRQEMLESILNRYIYEVDTISNK TAESGKQEYLEALVNQYIISAGREL — EPHPGKQEYLEAVFNNVMFTV-----KLYSGRQEYLESVVNNVIFNGNL---Petição 870190079230, de 15 / 08 / 2019, pág. 90 / 101 83 / 86 Ca2XI PMESGRQEYLETILNQILYK------Lp1XI VMQSGRQEVLESIVNNILFR------CpXI VMQSGRQEVLETIVNNILFR------.*.** * :. : Referências 1. Zaldivar J, Nielsen J, Olsson L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol. 2001 Jul;56(1-2):17-34. 2. Hahn-Hagerdal B, Karhumaa K, Fonseca C, SpencerMartins I, Gorwa-Grauslund MF. Towards industrial pentosefermenting yeast strains. Appl Microbiol Biotechnol. 2007 Apr;74(5):937-53. 3. Lau MW, Gunawan C, Balan V, Dale BE. Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production. Biotechnol Biofuels. 2010 May 27;3(1):11. 4. Bettiga M, Hahn-Hagerdal B, Gorwa-Grauslund MF. Comparing the xylose reductase / xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains. Biotechnol Biofuels. 2008 Oct 23;1(1):16. 5. Hector RE, Mertens JA, Bowman MJ, Nichols NN, Cotta MA, Hughes SR. Saccharomyces cerevisiae engineered for Petição 870190079230, de 15 / 08 / 2019, pág. 91 / 101 84 / 86 xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation. Yeast. 2011 Sep 1;28(9):645-60. 6. Hou J, Suo F, Wang C, Li X, Shen Y, Bao X. Finetuning of NADH oxidase decreases byproduct accumulation in respiration deficient xylose metabolic Saccharomyces cerevisiae. BMC Biotechnol. 2014 Feb 14;14(1):13. 7. Jeppsson M, Bengtsson O, Franke K, Lee H, HahnHãgerdal B, Gorwa-Grauslund MF. The expression of a Pichia stipitis xylose reductase mutant with higher KM for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng. 2006;93(4):66573. 8. Walfridsson M, Bao X, Anderlund M, Lilius G, Bulow L, Hahn-Hãgerdal B. Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an ative xylose (glicose) isomerase. Appl Environ Microbiol. 1996 Dec;62(12):4648-51. 9. Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MSM, de Laat WTAM, et al. High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae? FEMS Yeast Res. 2003;4(1):69-78. 10. Peng B, Huang S, Liu T, Geng A. Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster Petição 870190079230, de 15 / 08 / 2019, pág. 92 / 101 85 / 86 function in Saccharomyces cerevisiae for effective xylose fermentation. Microb Cell Factories. 2015 May 17;14(1):70. 11. Brat D, Boles E, Wiedemann B. Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae. Appl Environ Microbiol. 2009 Feb 13;75(8):230411. 12. Demeke MM, Dietz H, Li Y, Foulquié-Moreno MR, Mutturi S, Deprez S, et al. Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering. Biotechnol Biofuels. 2013 Jun 21;6(1):89. 13. Glanemann C, Loos A, Gorret N, Willis LB, O’Brien XM, Lessard PA, et al. Disparity between changes in mRNA abundance and enzyme ativity in Corynebacterium glutamicum: implications for DNA microarray analysis. Appl Microbiol Biotechnol. 2002 Dec 21;61(1):61-8. 14. Glanemann03.pdf [Internet]. [cited 2016 Nov 6]. Available from: http: / / web.mit.edu / biology / sinskey / www / Glanemann03.pdf 15. Demeke MM, Foulquié-Moreno MR, Dumortier F, Thevelein JM. Rapid Evolution of Recombinant Saccharomyces cerevisiae for Xylose Fermentation through Formation of Extra-chromosomal Circular DNA. PLoS Genet. 2015 Mar 4;11(3):e1005010. Petição 870190079230, de 15 / 08 / 2019, pág. 93 / 101 86 / 86 16. Demeke MM, Dumortier F, Li Y, Broeckx T, FoulquiéMoreno MR, Thevelein JM. Combining inhibitor tolerance and D-xylose fermentation in industrial Saccharomyces cerevisiae for efficient lignocellulose-based bioethanol production. Biotechnol Biofuels. 2013 Aug 26,6(1):120. 17. Austin MN, Rabe LK, Srinivasan S, Fredricks DN, Wiesenfeld HC, Hillier SL. Mageeibacillus indolicus gen. nov., sp. nov.: A novel bacterium isolated from the female genital tract. Anaerobe. 2015 Apr,-32:37—42. 18. Gietz RD, Schiestl RH, Willems AR, Woods RA. Studies on the transformation of intact yeast cells by the LiAc / SS-DNA / PEG procedure. Yeast. 1995 Apr 15,11(4):355-60. Petição 870190079230, de 15 / 08 / 2019, pág. 94 / 101

Claims

1 / 4 CLAIMS 1. A eukaryotic cell of filamentous fungus or yeast characterized by comprising a polypeptide with xylose isomerase activity, wherein the polypeptide consists of an amino acid sequence of SEQ ID NO: 7, and wherein said amino acid sequence is obtainable from a bacterium of the species Eubacterium sp. CAG_180, and wherein said cell is a cell of the genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Yarrowia, Kazachstania Naumovia, Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, and Penicillium.

2. Cell, according to claim 1, characterized in that the cell further comprises a polypeptide with xylose isomerase activity, wherein the polypeptide consists of an amino acid sequence with SEQ ID NO: 10, and wherein said amino acid sequence is obtainable from a bacterium of the species Clostridium cellulosii.

3. A cell, according to either claim 1 or 2, characterized in that the cell is a yeast capable of anaerobic alcoholic fermentation.

4. Cell, according to claim 3, characterized in that the cell is a yeast cell, wherein said yeast belongs to a Saccharomyces species selected from the group consisting of S. cerevisiae, S. bayanus, S. bulderi, S. cervazzii, S. cariocanus, S. castellii, S. dairenensis, S. exiguus, S. kluyveri, S. kudriazevii, S. mikatae, S. paradoxus, S. pastorianus, S. turicensis and S. unisporus.

5. A cell according to any one of claims 1 to 4, characterized in that the cell comprises at least one genetic modification selected from: a) a genetic modification that increases the specific activity of xylulose kinase; b) a genetic modification that increases the flux of the pentose phosphate pathway; and, c) a genetic modification that reduces the non-specific aldose reductase activity in the cell.

6. A cell, according to any one of claims 1 to 5, characterized in that the cell further comprises at least one genetic modification that results in a selected characteristic of the group consisting of: a) increased tolerance to ethanol; b) increased tolerance to acetic acid; c) reduced glycerol production; d) increased rate of xylose fermentation into ethanol; Petition 870260045160, dated 05 / 13 / 2026, page 15 / 23 3 / 4 and, e) increased thermo-tolerance.

7. A cell according to claim 6, characterized in that: a) the genetic modification is a modification that introduces an allele of one or more of the genes ADE1, KIN3, MKT1, VPS70, SWS2, and APJ1 that confer increased tolerance to ethanol; b) the genetic modification is a modification that introduces an allele of one or more of the genes GLO1, DOT5, CUP2, and HAA1 that confer increased tolerance to acetic acid; c) the genetic modification is a modification that introduces a mutant SSK1 gene that encodes a truncated ssk1 protein; d) the genetic modification is a modification that introduces an allele of the NNK1 gene that confers an increased rate of xylose fermentation into ethanol; and, e) the genetic modification is the overexpression of at least one of a gene encoding the Prp42 protein and a gene encoding the Smd2 protein.

8. Cell, according to any one of claims 1 to 7, characterized in that the cell is a cell of an industrial yeast strain or derived from an industrial yeast strain. Petition 870260045160, dated 05 / 13 / 2026, page 16 / 23 4 / 4 9. A cell, according to any one of claims 1 to 8, characterized in that the cell is a diploid, aneuploid, or polyploid cell.

10. A cell, according to any one of claims 1 to 9, characterized in that the cell is improved in at least one industrially relevant phenotype by evolutionary engineering, wherein the industrially relevant phenotype is the xylose utilization rate.

11. A cell, according to any one of claims 1 to 10, characterized in that the cell has 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, amino acids, 1,3-propanediol, ethylene, glycerol, butyric acid, caproate, butanol, glyoxylate, muconic acid, fatty alcohols, fatty acids, beta-lactam antibiotics, and cephalosporins. Petition 870260045160, dated 05 / 13 / 2026, pp. 17 / 23

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