Enhanced xylose metabolism in microalgae

Abstract

Provided herein are recombinant microorganisms having two or more copies of a nucleic acid sequence encoding a xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is an exogenous nucleic acid. Optionally, the recombinant microorganism comprises at least one nucleic acid sequence encoding a xylulokinase and / or at least one nucleic acid sequence encoding a xylose transporter. The provided recombinant microorganism is capable of growing on xylose as a carbon source.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 191,983, filed July 13, 2015, and U.S. Provisional Application No. 62 / 354,444, filed June 24, 2016, which are incorporated herein by reference in their entirety. Background of the Invention

[0003] Heterotrophic fermentation by microorganisms is an effective way to produce high-value oils and biomass products. Under certain culture conditions, microorganisms synthesize intracellular oils that can be extracted and used to produce fuels (e.g., biodiesel, biojet fuel, etc.) and nutritional lipids (e.g., polyunsaturated fatty acids such as DHA, EPA, and DPA). Due to their high polyunsaturated fatty acid (PUFA) and protein content, the biomass of some microorganisms has significant nutritional value and can be used as a nutritional supplement in animal feed. *Tetranychids* are eukaryotic single-celled microorganisms that can be used in such fermentation processes to produce lipids. Heterotrophic fermentation using *Tetranychids* converts organic carbon provided in the growth medium into lipids, which are harvested from the biomass at the end of the fermentation process. However, existing microbial fermentations primarily use expensive carbohydrates (such as glucose) as carbon sources. Invention Overview

[0004] This document provides recombinant microorganisms having two or more copies of a nucleic acid sequence encoding a xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is a foreign nucleic acid. Optionally, the recombinant microorganisms comprise at least one nucleic acid sequence encoding a xylulokinase and / or at least one nucleic acid sequence encoding a xylose transporter. The provided recombinant microorganisms are capable of growing on xylose as a carbon source. Brief description of the attached diagram

[0005] Figure 1 This is a schematic diagram of the xylose metabolic pathway.

[0006] Figure 2 This is a diagram showing the expression of xylose isomerase in wild-type ONC-T18 during glucose starvation cycles.

[0007] Figure 3 This is a diagram showing the putative expression of xylulokine in wild-type ONC-T18 during glucose starvation cycles.

[0008] Figure 4 This shows α-tubulin ble - A schematic diagram of the isomerase plasmid construct.

[0009] Figure 5 This shows α-tubulin hygro- xylBA schematic diagram of the plasmid construct.

[0010] Figure 6 This shows the presence of the α-tubulin promoter, ble A schematic diagram of the nucleic acid construct containing the sequence, 2A sequence, xylose isomerase sequence, and α-tubulin terminator.

[0011] Figure 7 These are images of DNA imprints used to detect the His-tagged xylose isomerase genes in recombinant ONC-T18 strains “6” and “16”.

[0012] Figure 8 This is a graph showing the number of His-tagged xylose isomerase genes inserted in the recombinant ONC-T18 strain by qPCR.

[0013] Figure 9 It is used to detect the presence of both xylose isomerase and xylulokine in the recombinant ONC-T18 strain (referred to as "7-3" and "7-7" in the figure). xylB An image of the DNA imprint of a gene.

[0014] Figure 10 It is in the recombinant 7-3 and 7-7 ONC-T18 strains xylB A graph showing the number of gene insertions determined by qPCR.

[0015] Figure 11 This is a diagram showing the expression of xylose isomerase gene transcripts in recombinant ONC-T18 strains “6” and “16”.

[0016] Figure 12 This is a diagram showing the in vitro xylose isomerase activity in wild-type ONC-T18 and recombinant ONC-T18 strains “6” and “16”.

[0017] Figure 13 This is a diagram showing the in vitro combined xylose isomerase and xylose kinase activities of the recombinant ONC-T18 strain "16" encoding only xylose isomerase, and the recombinant ONC-T18 strains "7-3" and "7-7" encoding xylose isomerase and xylulokine, respectively.

[0018] Figure 14A and 14B This is a graph showing improved xylose uptake and reduced xylitol production in the recombinant ONC-T18 strain "16" (square). Wild-type (WT) strains are represented by diamonds.

[0019] Figure 15A and 15BThis is a graph showing the improved xylose use and reduced xylitol production in recombinant ONC-T18 strain "16" (square), as well as recombinant ONC-T18 strains "7-3" (triangle) and "7-7" (asterisk). Wild-type (WT) strains are represented by diamonds.

[0020] Figure 16 This is a diagram showing the accumulation of xylitol during glucose:xylose fermentation using recombinant ONC-T18 strain "16" and recombinant ONC-T18 strain "7-7".

[0021] Figure 17 This is a schematic diagram of different types of constructs used for converting ONC-T18.

[0022] Figure 18 It shows the bacteria from Escherichia coli (SEQ ID NO:20). xylB Sequence and Escherichia coli xylB A diagram showing the alignment of the codon optimization form (SEQ ID NO:5).

[0023] Figure 19A , 19B 19C is an example of xylose use in strains containing xylose isomerase, xylulokinase, and the sugar transporter Gxs1. Figure 19A ), glucose use ( Figure 19B ) and the percentage of xylitol prepared ( Figure 19C The image shows that WT is wild-type; IsoHis XylB "7-7" contains xylose isomerase and xylB Sequences 36-2, 36-9, and 36-16 contain Gxs1, xylose isomerase, and xylB Transformation of the sequence (xylulose kinase).

[0024] Figure 20A and 20B This illustrates the effect of temperature incubation on xylose (rhombus) and xylulose (square) under the conditions of xylose (rhombus) and xylulose (square). Figure 20A ) and Escherichia coli ( Figure 20B The figure shows the effect of isomerase activity on ).

[0025] Figure 21A and 21B This shows the results from T18 in the cases of xylose (rhombus) and xylulose (square). Figure 21A ) and Escherichia coli ( Figure 21B A dose-dependent plot of the isomerase of ).

[0026] Figure 22A and 22BThis illustrates the use of xylose in the T18B strain engineered with xylose isomerases (“16” (square), “B” (x), and “6” (cross)). Figure 22A ) and reduced xylitol production ( Figure 22B (The image is shown.) Figure 22C (Xylose) and 22D (xylitol production) are shown as the same data relative to wild type (diamond) at 4 days (grey) and 7 days (black).

[0027] Figure 23A and 23B The graph shows the reduced xylose usage and xylitol production in T18B strain (square) engineered with xylose isomerase "16" and strains engineered to express xylose isomerase and xylulokine "7-7" (x) and "7-3" (triangle). Figure 23C (Xylose) and 23D (xylitol production) show the same data relative to wild type (diamond) at 9 days (grey) and 11 days (black).

[0028] Figure 24 This is a diagram showing increased xylose use and decreased xylitol production in a T18B strain engineered to express xylose isomerase and xylulokine "7-7" in fermentation.

[0029] Wild-type strains are represented by diamonds and dashed lines, and strain "7-7" is represented by a circle.

[0030] Figure 25 This is a schematic diagram showing the constructs of α-tubulin aspTx-neo and α-tubulin gxs1-neo.

[0031] Figure 26A This is an image used to detect the DNA imprint of the Gxs1 gene in the “7-7” T18B strain engineered with the xylose transporter Gxs1. Figure 26B This is an image used to detect the DNA imprint of the AspTx gene in the "7-7" T18B strain engineered with the xylose transporter AspTx.

[0032] Figure 27A This diagram illustrates the use of xylose in T18 strains engineered with xylose isomerase, xylulokine, and either the Gxs1 transporter (triangle) or the AspTx transporter (circle). Strain "7-7" is indicated by a diamond. Figure 27B This is a bar graph showing the ratio of xylitol production to xylose usage for each of the three modified strains. Figure 27C This is a bar graph showing the xylose usage relative to strain "7-7". Figure 27D This is a bar graph showing the production of xylitol relative to strain "7-7".

[0033] Figure 28 This diagram shows the growth of wild-type (WT) (diamond), isohis strain "16" (square), strain "7-7" (x), transporter strain Gxs1 (asterisk), and AspTx (triangle) in a medium containing xylose as the sole carbon source.

[0034] Figure 29A This is a diagram showing the residual glucose in the alternative feedstock containing glucose and xylose during the growth of WT (square), strain "7-7" (triangle), and transporter strains Gxs1 (asterisk) and AspTx (cross). Figure 29B This is a diagram showing the residual xylose and produced xylitol over time when WT (square), strain "7-7" (triangle), and transporter strains Gxs1 (asterisk) and AspTx (cross) are grown on alternative feedstocks containing glucose and xylose. Invention Details

[0035] Microorganisms such as *Cypripedium* encode genes required for xylose metabolism. However, the innate metabolic pathway in microorganisms produces large amounts of the sugar alcohol xylitol, which is secreted and potentially inhibits microbial growth (see Figure 14, WT). Furthermore, the carbon atom chelated into xylitol is the atom that is transferred away from the target product during this process (i.e., lipid production). In nature, two xylose metabolic pathways exist: the xylose reductase / xylitol dehydrogenase pathway and the xylose isomerase / xylulose kinase pathway. Figure 1 *Cyclochytrium* possesses genes encoding proteins active in both pathways; however, when grown in xylose-rich media, the former pathway appears to be dominant, as evidenced by xylitol accumulation. In other organisms, xylitol accumulation has been shown to be due to an imbalance of redox cofactors required for the xylose reductase / xylitol dehydrogenase pathway. Since the isomerase / kinase pathway is independent of redox cofactors, overexpression of isomerase genes removes the cofactor dependence in the xylose-to-xylulose conversion. As shown in this paper, transcriptomic studies of ONC-T18 revealed that its xylose isomerase and putative xylulose kinase genes are mostly expressed during glucose starvation. Figure 2 and Figure 3 The genes encoding xylose reductase and xylitol dehydrogenase, which were presumed to be expressed, were constitutively expressed. To increase the expression of isomerases and kinases at all growth stages, the microorganisms were engineered to include the ONC-T18 isomerase gene and the *E. coli* xylulose kinase gene. xylB This allows them to be under the control of constitutively expressed promoters and terminators (e.g., α-tubulin promoters and terminators). Optionally, the genes may be under the control of inducible promoters and / or terminators.

[0036] The recombinant microorganisms provided demonstrate the level of control over the amount of target gene expression by the number of integrated transgenic copies. As shown in the examples below, the recombinant ONC-T18 strain (Iso-His #16) carrying eight (8) transgenic copies exhibited higher levels of xylose isomerase transcript expression, enzyme activity, and xylose metabolism than the strain carrying a single copy of the transgene (Iso-His #6). When Iso-His #16 was further modified to incorporate... xylB Similar phenomena were observed when working with genes. Compared to a single insertion, xylB Multiple copies of a gene confer higher enzyme activity and xylose metabolic productivity. Therefore, it is surprising that not only is it necessary to recreate the xylose metabolic pathway, but it is also necessary to do so with multiple copies of the essential transgene. The ability of the *Cyclochytrium* genome to accommodate multiple transgene copies and remain viable was unexpected; hence, this variation in expression levels observed in transformant strains is surprising. However, as presented herein, recombinant microorganisms allowing controlled levels of transgene expression can be indirectly generated by selecting transformant strains with transgene copy numbers “tailored” to specific expression levels optimized for a particular pathway (e.g., the xylose pathway) through metabolic engineering.

[0037] This document provides nucleic acids encoding one or more genes involved in xylose metabolism. This application provides recombinant microorganisms, methods for preparing said microorganisms, and methods for producing oil using microorganisms capable of metabolizing xylose. Specifically, this document provides nucleic acids and polypeptides encoding xylose isomerase, xylulokinase, and xylose transporter proteins for modifying microorganisms to be capable of metabolizing xylose and / or growing on xylose as the sole carbon source. Therefore, nucleic acids encoding xylose isomerase are provided. The nucleic acid sequences may be endogenous or heterologous for the microorganisms. Exemplary nucleic acid sequences of xylose isomerase include, but are not limited to, those from the genera *Pyroxytrichum*, *Streptococcus*, and *Cyclochytrium*. For example, exemplary nucleic acid sequences encoding xylose isomerase include, but are not limited to, SEQ ID NO:2 and SEQ ID NO:15; and exemplary polypeptide sequences of xylose isomerase include, but are not limited to, SEQ ID NO:16. Exemplary nucleic acid sequences of xylulokinase include, but are not limited to, those from *Escherichia coli*, *Pyroxytrichum*, *Saccharomyces*, and *Pichia pastoris*. For example, exemplary nucleic acid sequences encoding xylulokine kinases include, but are not limited to, SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. Exemplary nucleic acid sequences encoding sugar transport proteins (e.g., xylose transporters) include, but are not limited to, those from the genus Aspergillus, Gfx1, Gxs1, and Sut1. For example, exemplary nucleic acid sequences encoding xylose transporters include, but are not limited to, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

[0038] As used herein, nucleic acid refers to deoxyribonucleotides or ribonucleotides and their polymers and complements. The term includes deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, said nucleic acids being synthetic, naturally occurring, or non-natural, having similar binding properties to reference nucleic acids, and being metabolized in a similar manner to reference nucleotides. Examples of such analogs include, but are not limited to, thiophosphates, aminophosphates, methyl phosphates, chiral methyl phosphates, 2-O-methylribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, conserved variants of nucleic acid sequences (e.g., degenerate codon substitutions) and complementary sequences may be used instead of the specific nucleic acid sequences described herein. Specifically, degenerate codon substitution can be achieved by producing a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0039] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if the DNA encoding a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, the DNA is operably linked to the DNA encoding the polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is located to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally, operably linked means that the linked DNA sequences are adjacent to each other, and in the case of a secretory leader sequence, are contiguous and within the reading frame. However, enhancers do not have to be contiguous. For example, a nucleic acid sequence operably linked to a second nucleic acid sequence may be directly or indirectly covalently linked to the second sequence, but any effective three-dimensional association is acceptable. A single nucleic acid sequence can be operably linked to multiple other sequences. For example, a single promoter can guide the transcription of multiple RNA species. Linkage can be achieved by linking to a suitable restriction site. If the site is not present, a synthetic oligonucleotide adaptor or linker is used according to conventional practice.

[0040] In the context of two or more nucleic acid or polypeptide sequences, the term "identity" or "percentage of identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., approximately 60% identity relative to a specified region when comparing and aligning with the maximum correspondence relative to a comparison window or specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) when compared and aligned with the maximum correspondence relative to a comparison window or specified region. Such sequences are then considered "substantially identical." This definition also refers to, or can be applied to, the complement of the test sequence. The definition also includes sequences with deletions and / or additions, as well as those with substitutions. As described below, preferred algorithms can interpret vacancies, etc. Preferably, the identity exists in a region of at least about 25 amino acids or nucleotides in length, or more preferably in a region of 50-100 amino acids or nucleotides in length.

[0041] For sequence comparisons, typically one sequence serves as a reference sequence, and the test sequence is compared to the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences and the sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0042] As used herein, the comparison window includes any segment involving a plurality of consecutive positions, selected from groups of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150, wherein after optimal comparison of two sequences, one sequence can be compared with a reference sequence of the same number of consecutive positions. Methods for aligning sequences used for comparison are well known in the art. The best alignment of sequences for comparison can be achieved, for example, through the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); through the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); through the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); through computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or through manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., ed., Supplement 1995)).

[0043] Preferred examples of algorithms suitable for determining sequence identity percentages and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used with the parameters described herein to determine the sequence identity percentage of nucleic acids or proteins. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of a selected length (W) in the query sequence that match or satisfy a threshold score T with a positive value when compared to words of the same length in the database sequence. T is referred to as the neighbor string score threshold (Altschul et al., ibid.). These initial neighbor string hits act as seeds to initiate a search for longer HSPs containing them. As long as the cumulative alignment score can increase, the string hit extends along each sequence in both directions. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The string hit stops extending in each direction when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score tends to 0 or below due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The expected value (E) represents the number of distinct alignments whose scores are equal to or higher than the score expected to occur in a random database search. The default values ​​used by the BLASTN program (for nucleotide sequences) are a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and two-strand comparisons. For the amino acid sequence, the BLASTP program uses the following default values: word length 3, expected value (E) 10, and BLOSUM62 score matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), alignment (B) 50, expected value (E) 10, M=5, N=4, and comparison of the two strands.

[0044] As used herein, the term polypeptide generally has the meaning of a polymer having at least three amino acids, as is recognized in the art, and is intended to include both peptides and proteins. However, the term is also used to refer to polypeptides of specific functional classes, such as desaturases, elongases, etc. For each of these classes, this disclosure provides several examples of known sequences of said polypeptides. However, those skilled in the art will understand that the term polypeptide is intended to be sufficiently general to encompass not only polypeptides having the complete sequence listed herein (or in the references or databases mentioned herein), but also polypeptides representing functional fragments of said complete polypeptide (i.e., fragments retaining at least one activity). Furthermore, those skilled in the art will understand that protein sequences generally allow for some substitutions without destroying activity. Therefore, any polypeptide that retains activity and shares at least about 30%-40% total sequence identity with another polypeptide of the same class, typically greater than about 50%, 60%, 70%, or 80%, and further typically includes at least one region with higher identity, typically greater than 90% or even 95%, 96%, 97%, 98%, or 99%, typically covering at least 3-4 and typically up to 20 or more amino acids, is covered under the relevant term polypeptide as used herein. Those skilled in the art can determine other regions of similarity and / or identity by analyzing the sequences of the various polypeptides described herein. As is known to those skilled in the art, various strategies are known, and tools for performing comparisons of amino acid or nucleotide sequences to assess the degree of identity and / or similarity are available. These strategies include, for example, manual alignment, computer-aided sequence alignment, and combinations thereof. Many algorithms for performing sequence alignment (which are typically implemented by computers) are widely available or can be generated by those skilled in the art. Representative algorithms include, for example, Smith and Waterman's local homology algorithm (Adv. Appl. Math., 1981, 2: 482); Needleman and Wunsch's homology alignment algorithm (J. Mol. Biol., 1970, 48: 443); Pearson and Lipman's method for searching similarity (Proc. Natl. Acad. Sci. (USA), 1988, 85: 2444); and / or computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). Easily available computer programs incorporating such algorithms include, for example, BLASTN, BLASTP, vacancy BLAST, PILEUP, CLUSTALW, etc.When using the BLAST and vacancy BLAST procedures, the default parameters for the respective procedures can be used. Alternatively, the practitioner may use non-default parameters depending on his or her experimental requirements and / or other requirements (see, for example, URL: www.ncbi.nlm.nih.gov).

[0045] As described above, nucleic acids encoding xylose transporters, xylulokines, and xylose isomerases can be linked to promoters and / or terminators. Examples of promoters and terminators include, but are not limited to, tubulin promoters and terminators. For example, the promoter is a tubulin promoter, such as the α-tubulin promoter. Optionally, the promoter is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. Optionally, the terminator is a tubulin terminator. Optionally, the terminator is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30.

[0046] As used herein, the terms promoter, promoter element, and regulatory sequence refer to a polynucleotide that is regulated and operablely linked to a promoter and affects the expression of the selected polynucleotide sequence in the cell. The term *Cyclochytrium* (as used herein) Thraustochytrium A promoter is a promoter that functions in *Cyclochytrium* cells. In some implementations, the promoter element is the untranslated region (UTR) at position 5' of the coding sequence or contains the untranslated region (UTR) at position 5' of the coding sequence. The 5' UTR forms part of the mRNA transcript and is therefore a major component of protein expression in eukaryotes. Following transcription, the 5' UTR can regulate protein expression at both the transcriptional and translational levels.

[0047] As used herein, the term term terminator refers to a polynucleotide that prevents the expression of a target for maturation (e.g., by adding a polyA tail) or confers stability to a selected polynucleotide sequence of mRNA that is operatively linked to a terminator in a cell. The terminator sequence may be downstream of a stop codon in a gene. The term *Cyclochytrium* terminator, as used herein, refers to a terminator that functions in *Cyclochytrium* cells. This document also provides nucleic acid constructs comprising nucleic acid sequences encoding xylose isomerase, xylulose kinase, and xylose transporter, as well as a promoter, terminator, selectivity marker, 2A peptide, or any combination thereof. As an example, a first nucleic acid construct is provided, comprising a promoter, selectivity marker, nucleic acid sequence encoding a 2A peptide, nucleic acid sequence encoding xylose isomerase, and a terminator. A second nucleic acid construct is also provided, comprising a promoter, selectivity marker, nucleic acid sequence encoding a 2A peptide, nucleic acid sequence encoding xylulose kinase, and a terminator. A third nucleic acid construct is further provided, comprising a promoter, nucleic acid sequence encoding a xylose transporter, nucleic acid sequence encoding a 2A peptide, selectivity marker, and terminator. These constructs are exemplary, and the nucleic acid sequences encoding xylose isomerase, xylulokine, and xylose transporter may be contained within the same construct under the control of the same or different promoters. Optionally, each of the nucleic acid sequences encoding xylose isomerase, xylulokine, and xylose transporter is on the same construct and separated by 2A polypeptide sequences, such as those shown in SEQ ID NO:6. Thus, by way of example, the nucleic acid construct may comprise a tubulin promoter, nucleic acid sequences encoding xylose isomerase, xylulokine, and xylose transporter separated by the nucleic acid sequence encoding SEQ ID NO:6, a tubulin terminator, and a selectability marker. Optionally, the selectability marker is a ble gene. Optionally, the selectability marker comprises SEQ ID NO:29.

[0048] As used herein, the phrase "selective label" refers to a nucleotide sequence (e.g., a gene) encoding a product (peptide) that allows selection, or the gene product (e.g., the peptide) itself. The term "selective label" is used herein as it is generally understood in the art, and refers to a label present in a cell or organism that, under certain defined culture conditions (selective conditions), confers a significant growth or survival advantage or disadvantage to said cell or organism. For example, said conditions may be the presence or absence of a specific compound or specific environmental conditions, such as increased temperature, increased radiation, the presence of a toxic compound in the absence of a label, etc. The presence or absence of one or more such compounds or one or more environmental conditions is referred to as selective conditions or multiple selective conditions. "Growth advantage" refers to increased viability relative to other identical cells or organisms (e.g., cells or organisms with growth advantage have an increased average lifespan relative to other identical cells), increased proliferation rate (also referred to herein as growth rate), or both. Generally, a cell population with growth advantage will exhibit fewer dead or non-surviving cells and / or a larger cell proliferation rate compared to another identical cell population lacking growth advantage. While selective markers generally confer a growth advantage to cells, some selective markers confer a growth disadvantage, for example, by making cells more sensitive to the harmful effects of certain compounds or environmental conditions than other identical cells that do not express the marker. Antibiotic resistance markers are a non-limiting example of a class of selective markers that can be used to select cells that express the marker. In the presence of appropriate concentrations of antibiotics (selective conditions), such markers confer a growth advantage to cells that express the marker. Therefore, cells expressing antibiotic resistance markers can survive and / or proliferate in the presence of antibiotics, while cells that do not express antibiotic resistance markers cannot survive and / or proliferate in the presence of antibiotics.

[0049] Examples of selective labeling include common bacterial antibiotics, such as, but not limited to, ampicillin, kanamycin, and chloramphenicol, as well as selective compounds known to function in microalgae; examples include rrnS and AadA (aminoglycoside 3'-adenosyltransferases), which can be isolated from E. coli plasmid R538-1 and confer resistance to spectinomycin and streptomycin, respectively, in E. coli and some microalgae (Hollingshead and Vapunek, Plasmid 13:17-30, 1985; Meslet-Cladière and Vallon, Eukaryot Cell. 10(12):1670-8 2011). Another example is the 23S RNA protein rrnL, which confers resistance to erythromycin (Newman, Boynton et al., Genetics, 126:875–888 1990; Roffey, Golbeck et al., Proc. Natl Acad. Sci. USA, 88:9122–9126 1991). Another example is Ble, which confers resistance to bleomycin from *Streptococcus hirta* (Hindustan streptococcus). Streptoalloteichus hindustanusGC-rich genes isolated from Streptomyces hygromycin B (Stevens, Purton et al., Mol. Gen. Genet., 251:23-30 1996). Aph7 is another example, an aminoglycoside phosphotransferase gene from Streptomyces hygromycin B that confers resistance to hygromycin B (Berthold, Schmitt et al., Protist 153(4):401-412 2002). Other examples include: AphVIII, a type VIII aminoglycoside 3'-phosphotransferase from *Streptomyces schreberi* that confers resistance to paromomycin in *Escherichia coli* and some microalgae (Sizova, Lapina et al., Gene 181(1-2):13-18 1996; Sizova, Fuhrmann et al., Gene 277(1-2):221-229 2001); Nat and Sat-1, which encode norsin acetyltransferase from *Streptomyces northerly* and streptomycin acetyltransferase from *Escherichia coli*, conferring resistance to norsin (Zaslavskaia, Lippmeier et al., Journal of Phycology 36(2):379-386, 2000); Neo, an aminoglycoside 3'-phosphotransferase that confers resistance to aminoglycosides; kanamycin, neomycin, and the analogue G418 (Hasnain, Manavathu et al., Molecular and Cellular Biology 5(12):3647-3650, 1985); and Cry1, a ribosomal protein S14 that confers resistance to emetine (Nelson, Savereide et al., Molecular and Cellular Biology 14(6):4011-4019, 1994).

[0050] Other selective markers include trophic markers, also known as autotrophic markers or auxotrophic markers. These include selective photoautotrophic markers applied based on the restoration of photosynthetic activity in photosynthetic organisms. Photoautotrophic markers include, but are not limited to, AtpB, TscA, PetB, NifH, psaA, and psaB (Boynton, Gillham et al., Science 240(4858):1534-1538 1988; Goldschmidt-Clermont, Nucleic Acids Research 19(15):4083-4089,1991; Kindle, Richards et al., PNAS, 88(5):1721-1725, 1991; Redding, MacMillan et al., EMBO J 17(1):50-60, 1998; Cheng, Day et al., Biochemical and Biophysical Research Communications 329(3):966-975, 2005). Alternative or additional nutrient markers include ARG7, which encodes argininosuccinate lyase, a key step in arginine biosynthesis (Debuchy, Purton et al., EMBO J 8(10):2803-2809, 1989); NIT1, which encodes nitrate reductase, essential for nitrogen metabolism (Fernández, Schnell et al., PNAS, 86(17):6449-6453, 1989); THI10, essential for thiamine biosynthesis (Ferris, Genetics 141(2):543-549, 1995); and NIC1, which catalyzes an important step in nicotinamide biosynthesis (Ferris, Genetics 141(2):543-549, 1995). These markers are typically enzymes that function in biosynthetic pathways to produce compounds required for cell growth or survival. Generally, under nonselective conditions, the desired compounds are present in the environment or produced in the cell via alternative pathways. Under selective conditions, biosynthetic pathways involving labeling are required to function in order to produce compounds.

[0051] As used herein, the phrase "selective agent" refers to an agent that can introduce selective pressure on cells or cell populations to favor or oppose cells or cell populations carrying a selective marker. For example, a selective agent is an antibiotic and a selective marker is an antibiotic resistance gene. Optionally, bleomycin is used as a selective agent.

[0052] Suitable microorganisms, including but not limited to algae (e.g., microalgae), fungi (including yeast), bacteria, or protozoa, can be transformed using nucleic acids encoding genes involved in xylose metabolism and nucleic acid constructs containing said nucleic acids. Optionally, said microorganisms include Thraustochytrids of the order Thraustochytriales, and more specifically, Thraustochytrids of the genus Thraustochytrids. Thraustochytrium The microbial community includes, optionally, the order Thraustochytriales as described in U.S. Patent Nos. 5,340,594 and 5,340,742, which are incorporated herein by reference in their entirety. The microorganisms may be species of Thraustochytrium, such as the Thraustochytrium species deposited with ATCC Registry No. PTA-6245 (i.e., ONC-T18) as described in U.S. Patent No. 8,163,515, which is incorporated herein by reference in its entirety. Therefore, the microorganism may have the same 18S rRNA sequence as SEQ ID NO:1 by at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher (e.g., including 100%).

[0053] In this field, microalgae are generally recognized as representing a diverse group of organisms. For the purposes of this document, the term microalgae will be used to describe single-celled microorganisms originating from aquatic and / or terrestrial environments (some cyanobacteria are terrestrial / soil-dwelling). Aquatic environments extend from marine environments to freshwater lakes and rivers, and also include saline environments such as estuaries and river mouths. Microalgae can be photosynthetic; optionally, microalgae are heterotrophic. Microalgae can be eukaryotic or prokaryotic. Microalgae can be non-motile or motile.

[0054] As used herein, the term "hygrochycephalomycetes" refers to any member of the order Hygrochycephalomycetes, including the family Hygrochycephalomycetes. Strains described as Hygrochycephalomycetes include the following organisms: Order: Hygrochycephalomycetes; Family: Hygrochycephalomycetes; Genus: Hygrochycephalomycetes (Species: genus and species, Adimendo). arudimentale ), golden yellow ( aureum ), Bishi Koala ( benthicola ), Grobo ( globosum ), Kinney kinnei ), zoospores ( motivum ), multibasal proliferation ( multirudimentale ), thick skin ( pachydermum ), Prover ( proliferum ), Ross roseum ), Stott ( striatum )), genus *Wuken's Chrystridium* ( Ulkenia (Species: Genus and species, Emporio davidii) amoeboidea ), Cognis ( kerguelensis ), Minota ( minuta ), Profoda ( profunda ), Redian ( radiate ), Seren ( sailens ), Seraphim ( sarkariana ), Szokai ( schizochytrops ), Vigs visurgensis ), Unix ( yorkensis )), genus Schizochytrium ( Schizochytrium (Species: Dendrobium nobile) aggregatum ), Limnaeser ( limnaceum ), Munger ( mangrovei ), mini algae ( minutum ), Otto ( octosporum )), genus *Chytridactylum* ( Japonochytrium (Species: Genus and species, Mani ( marinum Acinetochytrium ( )), genus Acinetochytrium ( Aplanochytrium (Species: Genus and species, Herodia) haliotidis ), Coglunds ( kerguelensisi ), Profoda ( profunda ), Stoke ( stocchino )), genus Artonii ( Althornia (Species: Claude) crouchii or Chytridactylum genus ( Elina (Species: Genus and species, Marissa) marisalba ), Xinnuo Riverka ( sinorifica Species described within the genus *Wukenchitriga* will be considered members of the genus *Schizochytriga*. Strains described as belonging to the genus *Schizochytriga* may share common traits with strains described as belonging to the genus *Schizochytriga*, and may also be described as belonging to the genus *Schizochytriga*. For example, in some taxonomic classifications, ONC-T18 may be considered within the genus *Schizochytriga*, while in others it may be described as belonging to the genus *Schizochytriga* because it contains traits indicating both genera.

[0055] As used herein, the term transformation refers to the method of introducing exogenous or heterologous nucleic acid molecules (e.g., vectors or recombinant nucleic acid molecules) into recipient cells or microorganisms. Exogenous or heterologous nucleic acid molecules may or may not be integrated into (i.e., covalently linked to) the chromosomal DNA constituting the genome of the host cell or microorganism. For example, exogenous or heterologous polynucleotides may be maintained on addendum elements (e.g., plasmids). Alternatively, exogenous or heterologous polynucleotides may become integrated into the chromosome so that they are inherited by daughter cells through chromosomal replication. Methods for transformation include, but are not limited to, calcium phosphate precipitation; Ca 2+ Treatment; fusion of recipient cells with bacterial protoplasts containing recombinant nucleic acids; treatment of recipient cells with liposomes containing recombinant nucleic acids; DEAE dextran; fusion using polyethylene glycol (PEG); electroporation; magnetoporation; biological projectile delivery; retroviral infection; lipid transfection; and direct microinjection of DNA into cells.

[0056] The term "transformation" as used in relation to cells refers to cells that have been transformed as described herein to carry foreign or heterologous genetic material (e.g., recombinant nucleic acids). The term "transformation" may also, or alternatively, refer to microorganisms, microbial strains, tissues, organisms, etc., that contain foreign or heterologous genetic material.

[0057] As used herein, the term "introduction intent" has its broadest meaning and encompasses introduction, for example, by transformation methods (e.g., calcium chloride-mediated transformation, electroporation, particle bombardment), and also by other methods including transduction, conjugation, and mating. Optionally, a vector is used to introduce nucleic acids into cells or organisms.

[0058] The microorganisms used in the methods described herein can produce a variety of lipid compounds. As used herein, the term lipid includes phospholipids, free fatty acids, esters of fatty acids, triacylglycerols, sterols and sterol esters, carotenoids, lutein (e.g., oxycarotenoids), hydrocarbons, and other lipids known to those skilled in the art. Optionally, the lipid compounds include unsaturated lipids. The unsaturated lipids may include polyunsaturated lipids (i.e., lipids containing at least two unsaturated carbon-carbon bonds (e.g., double bonds)) or highly unsaturated lipids (i.e., lipids containing four or more unsaturated carbon-carbon bonds). Examples of unsaturated lipids include ω-3 and / or ω-6 polyunsaturated fatty acids, such as docosahexaenoic acid (i.e., DHA), eicosapentaenoic acid (i.e., EPA), and other naturally occurring unsaturated, polyunsaturated, and highly unsaturated compounds.

[0059] This document provides recombinant microorganisms engineered to express polypeptides for metabolizing C5 sugars (such as xylose). Specifically, a recombinant microorganism is provided having two or more copies of a nucleic acid sequence encoding a xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is exogenous. Optionally, the recombinant microorganism comprises two or more copies of a nucleic acid sequence encoding a xylose isomerase. Optionally, the recombinant microorganism also contains one or two copies of an endogenous nucleic acid sequence encoding a xylose isomerase. For example, the recombinant microorganism may contain one or two copies of an endogenous nucleic acid sequence encoding a xylose isomerase and one copy of an exogenous nucleic acid sequence encoding a xylose isomerase. Optionally, the recombinant microorganism comprises three copies of a nucleic acid sequence encoding a xylose isomerase, one exogenously introduced and the other two endogenous. When used with respect to cells, nucleic acids, polypeptides, vectors, etc., the term recombinant means that the cells, nucleic acids, polypeptides, vectors, etc., have been modified by laboratory methods or are the result of laboratory methods and are not naturally occurring. Therefore, for example, recombinant microorganisms include microorganisms produced or modified by laboratory methods (e.g., transformation methods for introducing nucleic acids into microorganisms). Recombinant microorganisms may contain nucleic acid sequences not present in the natural (non-recombinant) form of said microorganisms, or may contain nucleic acid sequences that have been modified (e.g., linked to a non-natural promoter).

[0060] As used herein, the term exogenous refers to substances artificially introduced into a cell or organism and / or not naturally present in the cell in which it is present, such as nucleic acids (e.g., nucleic acids containing regulatory sequences and / or genes) or polypeptides. In other words, substances such as nucleic acids or polypeptides originate from outside the cell or organism in which they are introduced. Exogenous nucleic acids may have the same nucleotide sequence as nucleic acids naturally present in cells. For example, *Cyclochytrium* cells may be engineered to contain nucleic acids having *Cyclochytrium* or *Cyclochytrium* genus regulatory sequences. In a specific instance, endogenous *Cyclochytrium* or *Cyclochytrium* genus regulatory sequences may be operatively linked to genes that do not involve regulatory sequences under natural conditions. Although *Cyclochytrium* or *Cyclochytrium* genus regulatory sequences may be naturally present in host cells, the nucleic acids introduced according to this disclosure are exogenous. Exogenous nucleic acids may have nucleotide sequences different from any nucleic acids naturally present in cells. For example, exogenous nucleic acids may be heterologous nucleic acids, i.e., nucleic acids from different species or organisms. Therefore, exogenous nucleic acids can have the same nucleic acid sequence as those naturally present in a source organism, but the source organism is different from the cell into which the exogenous nucleic acid is introduced. As used herein, the term endogenous refers to a nucleic acid sequence naturally present in a cell. As used herein, the term heterologous refers to a nucleic acid sequence not naturally present in a cell, i.e., originating from an organism different from the cell. The terms exogenous and endogenous or heterologous are not mutually exclusive. Thus, a nucleic acid sequence can be both exogenous and endogenous, meaning that a nucleic acid sequence can be introduced into a cell but has the same or similar sequence as those naturally present in the cell. Similarly, a nucleic acid sequence can be both exogenous and heterologous, meaning that a nucleic acid sequence can be introduced into a cell but has a sequence not naturally present in the cell, such as a sequence originating from a different organism.

[0061] As described above, the provided recombinant microorganism contains at least two copies of a nucleic acid sequence encoding xylose isomerase. Optionally, the provided microorganism also contains at least one nucleic acid sequence encoding xylulose kinase. Optionally, the recombinant microorganism contains at least one nucleic acid sequence encoding a xylose transporter. The nucleic acid sequences encoding xylose isomerase, xylulose kinase, and / or xylose transporter are optionally exogenous nucleic acid sequences. Optionally, the nucleic acid sequence encoding xylose isomerase is an endogenous nucleic acid sequence. Optionally, the nucleic acid sequences encoding xylulose kinase and / or xylose transporter are heterologous nucleic acids. Optionally, the microorganism contains at least two copies of a nucleic acid sequence encoding xylose isomerase, at least two copies of a nucleic acid sequence encoding xylulose kinase, and at least one nucleic acid sequence encoding a xylose transporter. Optionally, the heterologous nucleic acid sequence encoding xylose isomerase is at least 90% identical to SEQ ID NO:2. Optionally, the heterologous nucleic acid sequence encoding xylulose kinase is at least 90% identical to SEQ ID NO:5. As described above, optionally, the nucleic acid encoding the xylose transporter is a heterologous nucleic acid. Optionally, the xylose transporter encoded by the heterologous nucleic acid is derived from *Candida intermedius* (…). Candida intermedia GXS1 of SEQ ID NO:23. Optionally, the heteronucleotide sequence encoding the xylose transporter is at least 90% identical to that of SEQ ID NO:23.

[0062] The provided recombinant microorganisms not only contain nucleic acid sequences encoding genes involved in xylose metabolism, but they may also contain multiple copies of such sequences. Therefore, the microorganisms contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding xylose isomerase. Optionally, the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of a nucleic acid sequence encoding xylulokine. Optionally, the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of a nucleic acid sequence encoding a xylose transporter.

[0063] In the provided microorganisms, nucleic acids (e.g., xylose isomerase, xylulokine, or xylose transporter) can be operatively linked to a promoter and / or a terminator. Optionally, a foreign nucleic acid sequence encoding xylose isomerase is operatively linked to a promoter. Optionally, nucleic acid sequences encoding xylulokine and / or xylose transporter sequences are also operatively linked to a promoter. Optionally, the promoter is a tubulin promoter. Optionally, the promoter is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. Optionally, the foreign nucleic acid sequence encoding xylose isomerase contains a terminator. Optionally, the nucleic acid sequence encoding xylulokine contains a terminator. Optionally, the nucleic acid sequence encoding xylose transporter contains a terminator. Optionally, the terminator is a tubulin terminator. Optionally, the terminator is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30.

[0064] The provided microorganism may contain a selective marker to confirm the transformation of the target gene. Therefore, the microorganism may also contain a selective marker. Optionally, the selective marker is an antibiotic resistance gene. Optionally, the antibiotic is bleomycin, hygromycin B, kanamycin, or neomycin. Optionally, the microorganism is a genus of *Schizochytrium* or *Schizochytrium*. Optionally, the microorganism is ONC-T18.

[0065] The provided microorganisms possess significant characteristics relative to wild-type microorganisms. For example, the recombinant microorganisms may have increased xylose transport activity, increased xylose isomerase activity, increased xylulose kinase activity, or any combination of these activities compared to non-recombinant control (or wild-type) microorganisms. Optionally, the recombinant microorganisms grow using xylose as the sole carbon source.

[0066] A method for preparing the recombinant microorganism is also provided. Therefore, a method for preparing recombinant xylose-metabolizing microorganisms is provided, the method comprising providing one or more nucleic acid constructs comprising a nucleic acid sequence encoding a xylose isomerase, a nucleic acid sequence encoding a xylulose kinase, and a nucleic acid sequence encoding a xylose transporter protein; transforming the microorganism with the one or more nucleic acid constructs; and isolating a microorganism comprising at least two or more copies of the nucleic acid sequence encoding the xylose isomerase. Optionally, the method further comprises isolating a microorganism comprising at least two copies of the nucleic acid sequence encoding the xylulose kinase. Optionally, the method comprises isolating a microorganism comprising at least one copy of the xylose transporter protein. Optionally, the one or more nucleic acid constructs further comprise a selective marker.

[0067] In the provided method, the nucleic acid sequences encoding xylose isomerase, xylulokine, and xylose transporter may be located on the same or different constructs. Optionally, the method includes providing a first nucleic acid construct containing a nucleic acid sequence encoding xylose isomerase, a second nucleic acid construct containing a nucleic acid sequence encoding xylulokine, and a third nucleic acid construct containing a nucleic acid sequence encoding xylose transporter. Optionally, the first, second, and third nucleic acid constructs contain the same selective marker. Optionally, the first nucleic acid construct contains a promoter, a selective marker, a nucleic acid sequence encoding a 2A peptide, a nucleic acid sequence encoding xylose isomerase, and a terminator. Optionally, the second nucleic acid construct contains a promoter, a selective marker, a nucleic acid sequence encoding a 2A peptide, a nucleic acid sequence encoding xylulokine, and a terminator. Optionally, the third nucleic acid construct contains a promoter, a nucleic acid sequence encoding a xylose transporter, a nucleic acid sequence encoding a 2A peptide, a selective marker, and a terminator. As mentioned above, the selective marker includes, but is not limited to, antibiotic resistance genes. Optionally, the antibiotic is bleomycin, hygromycin B, kanamycin, or neomycin. Promoters used for the construct include, but are not limited to, tubulin promoters. Optionally, the promoter is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. Terminators used for the construct include, but are not limited to, tubulin terminators. Optionally, the terminator is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30.

[0068] In the provided method, the isolated recombinant microorganism may contain one or more copies of xylose isomerase, xylulose kinase, and xylose transporter. Optionally, the isolated recombinant microorganism contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding xylose isomerase. Optionally, the xylose isomerase is an endogenous xylose isomerase or a heteroxylose isomerase. Optionally, the nucleic acid sequence encoding xylose isomerase is at least 90% identical to SEQ ID NO:2. Optionally, the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding xylulose kinase. Optionally, the xylulose kinase is a heterologous xylulose kinase. Optionally, the nucleic acid sequence encoding xylulose kinase is at least 90% identical to SEQ ID NO:5. Optionally, the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding a xylose transporter. Optionally, the xylose transporter is a heterologous xylose transporter. Optionally, the xylose transporter is GXS1 from *Candida intermedia*. Optionally, the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23. Optionally, the microorganism is a *Schizochytrium* or *Schizochytrium* microorganism. Optionally, the microorganism is ONC-T18.

[0069] As described above, the isolated recombinant microorganisms may possess increased xylose transport activity, increased xylose isomerase activity, increased xylulokine activity, or any combination thereof compared to the control non-recombinant microorganisms. Optionally, the isolated recombinant microorganisms grow using xylose as the sole carbon source.

[0070] As described herein, a control or standard control refers to a sample, measurement, or value that serves as a reference (typically a known reference) for comparison with a test sample, measurement, or value. For example, a test microorganism (e.g., a microorganism transformed with a nucleic acid sequence encoding a gene for metabolizing xylose) may be compared with a known normal (wild-type) microorganism (e.g., a standard control microorganism). A standard control may also represent an average measurement or value collected from a microbial population (e.g., a standard control microorganism) that does not grow or grows poorly on xylose as its sole carbon source, or lacks or has the lowest levels of xylose isomerase activity, xylulose kinase activity, and / or xylose transport activity. Those skilled in the art will recognize that standard controls can be designed to assess any number of parameters (e.g., RNA levels, peptide levels, specific cell types, etc.).

[0071] This article also provides a method for producing oil using recombinant microorganisms. The method includes providing recombinant microorganisms, wherein the microorganisms are grown on xylose, which is the sole carbon source, and...

[0072] The microorganisms are cultured in a culture medium under suitable conditions to produce the oil. Optionally, the oil comprises triglycerides. Optionally, the oil comprises α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linolenic acid, linolenic acid, or combinations thereof. Optionally, the method further includes isolating the oil.

[0073] The provided methods include additional steps of culturing microorganisms according to methods known in the art, or may be used in combination with said additional steps. For example, *Cyclochytrium* (e.g., *Cyclochytrium* spp.) may be cultured according to the methods described in U.S. Patent Publications 2009 / 0117194 or 2012 / 0244584, the patent publications being incorporated herein by reference in their entirety with respect to each step of said methods or the compositions used therein.

[0074] Microorganisms are grown in a growth medium (also called a culture medium). Any of a variety of culture media is suitable for culturing the microorganisms described herein. Optionally, the culture medium supplies the microorganisms with various nutrient components, including carbon and nitrogen sources. The culture medium used for *Cyclochytrium* cultures may contain any of a variety of carbon sources. Examples of carbon sources include fatty acids, lipids, glycerol, triglycerides, carbohydrates, polyols, amino sugars, and any biomass or waste stream. Fatty acids include, for example, oleic acid. Carbohydrates include, but are not limited to, glucose, cellulose, hemicellulose, fructose, dextrose, xylose, lactulose, galactose, maltotriose, maltose, lactose, glycogen, gelatin, starch (corn or wheat), acetate, γ-inositol (e.g., derived from corn steep liquor), galacturonic acid (e.g., derived from pectin), L-fucose (e.g., derived from galactose), gentiobiose, glucosamine, α-D-glucose-1-phosphate (e.g., derived from glucose), cellobiose, dextrin, α-cyclodextrin (e.g., derived from starch), and sucrose (e.g., derived from molasses). Polyols include, but are not limited to, maltitol, erythritol, and arbutinol. Amino sugars include, but are not limited to, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl-β-D-mannosamine.

[0075] Optionally, the microorganisms provided herein are cultured under conditions that increase the production of biomass and / or target compounds (e.g., oil or total fatty acid (TFA) content). For example, *Cyclotridium* is typically cultured in a saline medium. Optionally, *Cyclotridium* can be cultured in a medium having a salt concentration of about 0.5 g / L to about 50.0 g / L. Optionally, *Cyclotridium* is cultured in a medium having a salt concentration of about 0.5 g / L to about 35 g / L (e.g., about 18 g / L to about 35 g / L). Optionally, the *Cyclotridium* described herein can grow under low-salt conditions. For example, *Cyclotridium* can be cultured in a medium having a salt concentration of about 0.5 g / L to about 20 g / L (e.g., about 0.5 g / L to about 15 g / L). The medium optionally contains NaCl. Optionally, the medium contains natural or artificial sea salt and / or artificial seawater.

[0076] The culture medium may contain a non-chlorinated sodium salt as a sodium source. Examples of non-chlorinated sodium salts suitable for use in the methods according to the invention include, but are not limited to, soda ash (a mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate, sodium sulfate, and mixtures thereof. See, for example, U.S. Patent Nos. 5,340,742 and 6,607,900, the entire contents of each of which are incorporated herein by reference. For example, a significant portion of the total sodium may be supplied by a non-chlorinated salt such that less than about 100%, 75%, 50%, or 25% of the total sodium in the culture medium is supplied by sodium chloride.

[0077] The culture medium for *Cyclotris* cultures may contain any of a variety of nitrogen sources. Exemplary nitrogen sources include ammonium solutions (e.g., H₂O solution of NH₄), ammonium or amine salts (e.g., (NH₄)₂SO₄, (NH₄)₃PO₄, NH₄NO₃, NH₄OOCH₂CH₃(NH₄Ac)), peptone, tryptone, yeast extract, malt extract, fish meal, monosodium glutamate, soybean extract, casein amino acids, and distillers' grains. The concentration of the nitrogen source in a suitable culture medium is typically between about 1 g / L and about 25 g / L, and is inclusive.

[0078] The culture medium optionally contains phosphates, such as potassium phosphate or sodium phosphate. Inorganic salts and micronutrients in the culture medium may include ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenite, nickel sulfate, copper sulfate, zinc sulfate, cobalt chloride, ferric chloride, manganese chloride, calcium chloride, and EDTA. Vitamins may be included, such as pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, para-aminobenzoic acid, riboflavin, niacin, biotin, folic acid, and vitamin B12.

[0079] Where appropriate, the pH of the culture medium can be adjusted to between 3.0 and 10.0, and inclusive, using an acid or alkali and / or a nitrogen source. Optionally, the culture medium can be sterilized.

[0080] Culture media typically used for culturing microorganisms are liquid media. However, culture media used for culturing microorganisms can also be solid media. In addition to carbon and nitrogen sources as discussed herein, solid media may contain one or more components (e.g., agar or agarose) that provide structural support and / or allow the media to be in solid form.

[0081] Optionally, the resulting biomass is pasteurized to inactivate unwanted substances present in the biomass. For example, the biomass can be pasteurized to inactivate substances that degrade compounds. The biomass can be present in a fermentation medium or isolated from a fermentation medium for the pasteurization step. The pasteurization step can be carried out by heating the biomass and / or fermentation medium to a high temperature. For example, the biomass and / or fermentation medium can be heated to a temperature of about 50°C to about 95°C (e.g., about 55°C to about 90°C or about 65°C to about 80°C). Optionally, the biomass and / or fermentation medium can be heated for about 30 minutes to about 120 minutes (e.g., about 45 minutes to about 90 minutes, or about 55 minutes to about 75 minutes). Suitable heating means can be used for pasteurization, such as direct steam injection.

[0082] Optionally, pasteurization is omitted. In other words, the method taught in this paper optionally lacks a pasteurization step.

[0083] Optionally, biomass can be harvested using various methods, including those currently known to those skilled in the art. For example, biomass can be collected from the fermentation medium using methods such as centrifugation (e.g., with a solid jet centrifuge) or filtration (e.g., cross-flow filtration). Optionally, the harvesting step includes using a precipitant (e.g., sodium phosphate or calcium chloride) to accelerate the collection of cellular biomass.

[0084] Optionally, the biomass is washed with water. Optionally, the biomass may be concentrated to about 20% solids. For example, the biomass may be concentrated to about 5% to about 20% solids, about 7.5% to about 15% solids, or about solids to about 20% solids, or any percentage within the listed range. Optionally, the biomass may be concentrated to about 20% solids or less, about 19% solids or less, about 18% solids or less, about 17% solids or less, about 16% solids or less, about 15% solids or less, about 14% solids or less, about 13% solids or less, about 12% solids or less, about 11% solids or less, about 10% solids or less, about 9% solids or less, about 8% solids or less, about 7% solids or less, about 6% solids or less, about 5% solids or less, about 4% solids or less, about 3% solids or less, about 2% solids or less, or about 1% solids or less.

[0085] The provided method optionally includes the isolation of polyunsaturated fatty acids from biomass or microorganisms. One or more of a variety of methods can be used to isolate polyunsaturated fatty acids, including those currently known to those skilled in the art. For example, a method for isolating polyunsaturated fatty acids is described in U.S. Patent No. 8,163,515, which is incorporated herein by reference in its entirety. Optionally, the culture medium is not sterilized prior to the isolation of the polyunsaturated fatty acids. Optionally, sterilization includes increasing the temperature. Optionally, the polyunsaturated fatty acids produced by microorganisms and isolated by the provided method are medium-chain fatty acids. Optionally, the one or more polyunsaturated fatty acids are selected from the group consisting of α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, γ-linolenic acid, linoleic acid, linolenic acid, and combinations thereof.

[0086] Oils containing polyunsaturated fatty acids (PUFAs) and other lipids produced according to the methods described herein can be used in any of a variety of applications utilizing their biological, nutritional, or chemical properties. Therefore, the provided methods optionally include separating the oil from a harvested portion of a threshold volume. Optionally, the oil is used to produce fuels, such as biofuels. Optionally, the oil can be used in pharmaceuticals, food supplements, animal feed additives, cosmetics, etc. Lipids produced according to the methods described herein can also be used as intermediates in the production of other compounds.

[0087] For example, oil produced by microorganisms cultured using the provided method may contain fatty acids. Optionally, the fatty acids are selected from the group consisting of: α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, γ-linolenic acid, linoleic acid, linolenic acid, and combinations thereof. Optionally, the oil contains triglycerides. Optionally, the oil contains fatty acids selected from the group consisting of: palmitic acid (C16:0), myristic acid (C14:0), palmitoleic acid (C16:1(n-7)), cis-octadecenoic acid (C18:1(n-7)), docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and combinations thereof.

[0088] Optionally, lipids generated according to the methods described herein may be incorporated into a final product (e.g., food or feed supplements, infant formula, pharmaceuticals, fuels, etc.). Suitable food or feed supplements into which lipids may be incorporated include beverages such as milk, water, sports drinks, energy drinks, tea, and fruit juices; sweets such as candy, jelly, and biscuits; fatty foods and beverages such as dairy products; processed food products such as soft rice (or porridge); infant formula; breakfast cereals, etc. Optionally, one or more of the generated lipids may be incorporated into dietary supplements, such as vitamins or multivitamins. Optionally, lipids generated according to the methods described herein may be included in dietary supplements and optionally may be directly incorporated into the components of food or feed (e.g., food supplements).

[0089] Examples of feeds in which lipids produced by the methods described herein can be incorporated include pet food, such as cat food; dog food; feed for aquarium fish, farmed fish, or crustaceans; and feed for farm-raised animals, including livestock and farmed fish or crustaceans. Food or feed materials in which lipids produced according to the methods described herein can be incorporated are preferably palatable to the organism intended as the recipient. Such food or feed materials may have any physical properties currently known for food materials (e.g., solid, liquid, soft).

[0090] Optionally, one or more of the resulting compounds (e.g., PUFA) may be incorporated into a nutritional food or pharmaceutical product. Examples of such pharmaceutical products include various types of tablets, capsules, drinkable preparations, etc. Optionally, the nutritional food or pharmaceutical product is suitable for topical application. Dosage forms may include, for example, capsules, oils, granules, concentrated fine granules, powders, tablets, pills, lozenges, etc.

[0091] Oils or lipids produced according to the methods described herein may be incorporated into the products described herein in any combination with a variety of other agents. For example, such compounds may be combined with one or more binders or fillers, chelating agents, pigments, salts, surfactants, humectants, viscosity modifiers, thickeners, softeners, fragrances, preservatives, etc., or any combination thereof.

[0092] Materials, compositions, and components are disclosed that can be used in, in combination with, or in the preparation of the disclosed compositions, or as products of the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to each distinct individual and common combination and arrangement of these compounds may not be explicitly disclosed, but each is specifically covered and described herein. For example, if a method is disclosed and discussed, and various modifications that can be made to the plurality of molecules including the method are discussed, every combination and arrangement of the method and possible modifications are explicitly covered unless explicitly indicated to the contrary. Similarly, any subset or combination of these molecules is specifically covered and disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in methods using the disclosed compositions. Therefore, if there are multiple additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is specifically covered and should be considered disclosed.

[0093] This application also relates to the following implementation schemes:

[0094] Implementation Scheme 1. A recombinant microorganism comprising two or more copies of a nucleic acid sequence encoding a xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is a foreign nucleic acid.

[0095] Implementation Scheme 2. The recombinant microorganism as described in Implementation Scheme 1 further comprises at least one nucleic acid sequence encoding xylulokine.

[0096] Implementation Scheme 3. The recombinant microorganism as described in Implementation Scheme 2, wherein the nucleic acid sequence encoding the xylulokine is an exogenous nucleic acid sequence.

[0097] Implementation Scheme 4. The recombinant microorganism as described in any one of Implementation Schemes 1 to 3 further comprises at least one nucleic acid sequence encoding a xylose transporter protein.

[0098] Implementation Scheme 5. The recombinant microorganism as described in Implementation Scheme 4, wherein the nucleic acid sequence encoding the xylose transporter is an exogenous nucleic acid sequence.

[0099] Implementation Scheme 6. The recombinant microorganism as described in Implementation Scheme 1, wherein the microorganism further comprises at least two or more copies of a nucleic acid sequence encoding xylulokine and at least one nucleic acid sequence encoding a xylose transporter.

[0100] Implementation Scheme 7. The recombinant microorganism as described in any one of Implementation Schemes 1 to 6, wherein the nucleic acid sequence encoding the xylose isomerase is a heterologous nucleic acid.

[0101] Implementation Scheme 8. The recombinant microorganism as described in any one of Implementation Schemes 1 to 7, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase.

[0102] Implementation Scheme 9. The recombinant microorganism as described in any one of Implementation Schemes 1 to 8, wherein the heterologous nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:2.

[0103] Implementation Scheme 10. A recombinant microorganism as described in any one of Implementation Schemes 2 to 9, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylanokinase.

[0104] Implementation Scheme 11. The recombinant microorganism as described in any one of Implementation Schemes 4 to 10, wherein the nucleic acid sequence encoding xylulokine is a heterologous nucleic acid.

[0105] Implementation Scheme 12. The recombinant microorganism as described in Implementation Scheme 11, wherein the heterologous nucleic acid sequence encoding the xylulokine is at least 90% identical to SEQ ID NO:5.

[0106] Implementation Scheme 13. The recombinant microorganism as described in any one of Implementation Schemes 5 to 12, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylose transporter.

[0107] Implementation Scheme 14. The recombinant microorganism as described in any one of Implementation Schemes 5 to 13, wherein the nucleic acid encoding the xylose transporter is a heterologous nucleic acid.

[0108] Implementation Scheme 15. The recombinant microorganism as described in Implementation Scheme 14, wherein the xylose transporter protein encoded by the heterologous nucleic acid is derived from *Candida intermedia* (…). Candida intermedia GXS1.

[0109] Implementation Scheme 16. The recombinant microorganism as described in Implementation Scheme 14, wherein the heterologous nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23.

[0110] Implementation Scheme 17. The recombinant microorganism as described in any one of Implementation Schemes 1 to 16, wherein the recombinant microorganism has increased xylose transport activity, increased xylose isomerase activity, increased xylulokine activity, or any combination thereof compared with the non-recombinant control microorganism.

[0111] Implementation Scheme 18. The recombinant microorganism as described in any one of Implementation Schemes 1 to 17, wherein the recombinant microorganism grows using xylose as the sole carbon source.

[0112] Implementation Scheme 19. The recombinant microorganism as described in any one of Implementation Schemes 1 to 18, wherein the exogenous nucleic acid sequence encoding the xylose isomerase is operatively linked to a promoter.

[0113] Implementation Scheme 20. The recombinant microorganism as described in any one of Implementation Schemes 11 to 19, wherein the nucleic acid sequence encoding the xylulokine is operatively linked to a promoter.

[0114] Implementation Scheme 21. The recombinant microorganism as described in any one of Implementation Schemes 14 to 20, wherein the nucleic acid sequence encoding the xylose transporter is operatively linked to a promoter.

[0115] Implementation Scheme 22. The recombinant microorganism as described in any one of Implementation Schemes 19 to 21, wherein the promoter is the tubulin promoter.

[0116] Implementation Scheme 23. The recombinant microorganism as described in Implementation Scheme 22, wherein the promoter is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26.

[0117] Implementation Scheme 24. The recombinant microorganism as described in any one of Implementation Schemes 1 to 23, wherein the exogenous nucleic acid sequence encoding the xylose isomerase contains a terminator.

[0118] Implementation Scheme 25. The recombinant microorganism as described in any one of Implementation Schemes 11 to 24, wherein the nucleic acid sequence encoding the xylulokine contains a terminator.

[0119] Implementation Scheme 26. The recombinant microorganism as described in any one of Implementation Schemes 14 to 25, wherein the nucleic acid sequence encoding the xylose transporter contains a terminator.

[0120] Implementation Scheme 27. The recombinant microorganism as described in any one of Implementation Schemes 24 to 26, wherein the terminator is a tubulin terminator.

[0121] Implementation Scheme 28. The recombinant microorganism as described in Implementation Scheme 27, wherein the terminator is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:30.

[0122] Implementation Scheme 29. The recombinant microorganism as described in any one of Implementation Schemes 1 to 28, wherein the microorganism further comprises a selective marker.

[0123] Implementation Scheme 30. The recombinant microorganism as described in Implementation Scheme 29, wherein the selective marker is an antibiotic resistance gene.

[0124] Implementation Scheme 31. The recombinant microorganism as described in Implementation Scheme 30, wherein the antibiotic is bleomycin, hygromycin B, kanamycin, or neomycin.

[0125] Implementation Scheme 32. The recombinant microorganism as described in any one of Implementation Schemes 1 to 31, wherein the microorganism is a member of the genus *Cypripedium* (…). Thraustochytrium ) or Schizochytrium ( Schizochytrium )microorganism.

[0126] Implementation Scheme 33. The recombinant microorganism as described in Implementation Scheme 32, wherein the microorganism is ONC-T18.

[0127] Implementation Scheme 34. A method for preparing recombinant xylose-metabolizing microorganisms, the method comprising:

[0128] Provide one or more nucleic acid constructs, said nucleic acid constructs comprising a nucleic acid sequence encoding a xylose isomerase, a nucleic acid sequence encoding a xylulose kinase, and a nucleic acid sequence encoding a xylose transporter;

[0129] Transform the microorganism using one or more of the nucleic acid constructs; and

[0130] Microorganisms containing at least two or more copies of the nucleic acid sequence encoding the xylose isomerase were isolated.

[0131] Implementation Scheme 35. The method of Implementation Scheme 34, further comprising isolating a microorganism containing at least one copy of the nucleic acid sequence encoding the xylulokine.

[0132] Implementation Scheme 36. The method of Implementation Scheme 35, further comprising isolating a microorganism containing at least one copy of the xylose transporter.

[0133] Implementation Scheme 37. The method of any one of Implementation Schemes 34 to 36, wherein one or more nucleic acid constructs further comprise a selective marker.

[0134] Implementation Scheme 38. The method of any one of Implementation Schemes 34 to 37, wherein the provision includes providing a first nucleic acid construct comprising a nucleic acid sequence encoding a xylose isomerase, a second nucleic acid construct comprising a nucleic acid sequence encoding a xylulokinase, and a third nucleic acid construct comprising a nucleic acid sequence encoding a xylose transporter.

[0135] Implementation Scheme 39. The method of Implementation Scheme 38, wherein the first nucleic acid construct, the second nucleic acid construct, and the third nucleic acid construct contain the same selective marker.

[0136] Implementation Scheme 40. The method of Implementation Scheme 38 or 39, wherein the first nucleic acid construct comprises a promoter, a selectable label, a nucleic acid sequence encoding a 2A peptide, the nucleic acid sequence encoding the xylose isomerase, and a terminator.

[0137] Implementation Scheme 41. The method of any one of Implementation Schemes 38 to 40, wherein the second nucleic acid construct comprises a promoter, a selective label, a nucleic acid sequence encoding a 2A peptide, the nucleic acid sequence encoding the xylulokinase, and a terminator.

[0138] Implementation Scheme 42. The method of any one of Implementation Schemes 38 to 41, wherein the third nucleic acid construct comprises a promoter, the nucleic acid sequence encoding the xylose transporter, the nucleic acid sequence encoding the 2A peptide, a selective label, and a terminator.

[0139] Implementation Scheme 43. The method of any one of Implementation Schemes 37 to 42, wherein the selective marker is an antibiotic resistance gene.

[0140] Implementation Scheme 44. The method as described in Implementation Scheme 43, wherein the antibiotic is bleomycin, hygromycin B, kanamycin, or neomycin.

[0141] Implementation Scheme 45. The method of any one of Implementation Schemes 40 to 44, wherein the promoter is a tubulin promoter.

[0142] Implementation Scheme 46. The method of implementation scheme 45, wherein the promoter is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26.

[0143] Implementation Scheme 47. The method of any one of Implementation Schemes 40 to 46, wherein the terminator is a tubulin terminator.

[0144] Implementation Scheme 48. The method as described in Implementation Scheme 47, wherein the terminator is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:30.

[0145] Implementation Scheme 49. The method of any one of Implementation Schemes 34 to 48, wherein the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase.

[0146] Implementation Scheme 50. The method of any one of Implementation Schemes 34 to 49, wherein the xylose isomerase is a heteroxylose isomerase.

[0147] Implementation Scheme 51. The method of any one of Implementation Schemes 34 to 49, wherein the nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:2.

[0148] Implementation Scheme 52. The method of any one of Implementation Schemes 34 to 51, wherein the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylanokinase.

[0149] Implementation Scheme 53. The method of any one of Implementation Schemes 34 to 52, wherein the xylulokine is a heterologous xylulokine.

[0150] Implementation Scheme 54. The method of any one of Implementation Schemes 34 to 52, wherein the nucleic acid sequence encoding the xylulokine is at least 90% identical to SEQ ID NO:5.

[0151] Implementation Scheme 55. The method of any one of Implementation Schemes 34 to 54, wherein the isolated recombinant microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylose transporter.

[0152] Implementation Scheme 56. The method of any one of Implementation Schemes 34 to 55, wherein the xylose transporter is a heteroxylose transporter.

[0153] 57. The method of any one of embodiments 34 to 56, wherein the xylose transporter is GXS1 from Candida intermedia.

[0154] 58. The method of any one of embodiments 34 to 56, wherein the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23.

[0155] 59. The method of any one of embodiments 34 to 58, wherein the isolated recombinant microorganism has increased xylose transport activity, increased xylose isomerase activity, increased xylulose kinase activity, or any combination thereof compared with the control non-recombinant microorganism.

[0156] 60. The method of any one of embodiments 34 to 59, wherein the isolated recombinant microorganism is grown using xylose as the sole carbon source.

[0157] 61. The method of any one of embodiments 34 to 60, wherein the microorganism is a genus of Schizochytrium or Schizochytrium.

[0158] 62. The method of any one of embodiments 34 to 60, wherein the microorganism is ONC-T18.

[0159] 63. A method for producing oil, the method comprising:

[0160] Provide recombinant microorganisms as described in any one of embodiments 1 to 33, wherein the microorganisms are grown on xylose as the sole carbon source, and

[0161] The microorganisms are cultured in a culture medium under suitable conditions to produce the oil.

[0162] 64. The method of embodiment 63, wherein the oil comprises triglycerides.

[0163] 65. The method of embodiment 63, wherein the oil comprises α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, γ-linolenic acid, linolenic acid, linolenic acid, or combinations thereof.

[0164] 66. The method of embodiment 63, further comprising separating the oil.

[0165] The publications cited in this article, and the materials cited in those publications, are hereby explicitly incorporated in their entirety by reference.

[0166] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.

[0167] Example

[0168] Example 1. C5 carbon metabolism via recombinant *Cyclophorus*

[0169] In nature, there are two xylose metabolic pathways: the xylose reductase / xylitol dehydrogenase pathway and the xylose isomerase / xylulose kinase pathway. Figure 1 ONC-T18 encodes genes from two pathways, and as mentioned above, the xylose reductase / xylitol dehydrogenase pathway is dominant, as evidenced by xylitol accumulation during growth in xylose-rich media. Since the isomerase / kinase pathway is independent of redox cofactors, overexpression of the isomerase genes in ONC-T18 removes the cofactor dependence in the xylose-to-xylulose conversion. (As in...) Figure 2 and 3As shown, transcriptomic studies using ONC-T18 revealed that its xylose isomerase and putative xylulose kinase genes are mostly expressed during glucose starvation; while the putatively identified genes encoding xylose reductase and xylitol dehydrogenase are constitutively expressed.

[0170] Following his-labeling and overexpression in yeast INVSC1, the T18 isomerase was purified by metal affinity chromatography. As a positive control, his-labeled XylA from E. coli strain W3110 was overexpressed and purified from E. coli strain BL21(DE3)plysS. The protein concentration of the purified protein was determined by the standard Bradford assay. The effect of temperature on the activities of T18 isomerase and E. coli isomerase was determined using 5 µg protein and 0.75 g / L xylose or xylulose in 5 mM MgATP, 50 mM Hepes (pH 7.4), and 10 mM MgCl2. The reaction mixture was incubated overnight at 10 °C, 25 °C, 30 °C, 37 °C, 50 °C, 60 °C, and 80 °C. The reaction was terminated by heat inactivation at 95 °C for 5 min. The reaction was analyzed by HPLC, and the concentration of sugars present was determined from the area under the peak relative to a standard curve. At temperatures of 37°C and above, the T18 isomerase exhibits higher activity on both xylose and xylulose. Figure 20A This is in contrast to E. coli isomerases, which exhibit higher activity at temperatures between 25°C and 30°C. Figure 20B ).

[0171] The dose-dependency of the protein isomerase was determined by incubating it with 0.75 g / L xylose or xylulose in 5 mM MgATP, 50 mM MgPeps (pH 7.4), and 10 mM MgCl2. The reactants were incubated overnight at 30°C (E. coli) or 50°C (T18), and then the reaction was stopped by heat inactivation at 95°C for 5 min. The reaction was analyzed by HPLC, and the concentration of sugars present was determined from the area under the peak relative to a standard curve. A dose-dependent effect of the T18 isomerase on both xylose and xylulose was observed. Figure 21A and 21B ).

[0172] This example describes the use of *Cyclophorus*. ONC-T18 The (ONC-T18) α-tubulin promoter can be used to express endogenous and / or heterologous xylose metabolism transgenes in *Cyclochytrium* species (including ONC-T18). However, as discussed throughout the text, other regulatory elements may be used. Figure 4 and 5Constructs of plasmids containing the xylose isomerase gene and the xylulose kinase gene are shown, respectively. As described herein, xylose metabolism transgenes are present in multiple (≥8) copies within the host genome. In the case of ONC-T18, the modified organisms exhibited increased xylose metabolism compared to wild-type (WT) cells. For example, strains modified to express the endogenous xylose isomerase gene (SEQ ID NO:2) (strain Iso-His #16) and strains modified to express both the endogenous xylose isomerase gene (SEQ ID NO:2) and the xylulose kinase gene (SEQ ID NO:5) (Iso-His+xylB, strain 7-7) used 40% more xylose than WT strains. Compared to WT strains, Iso-His #16 and 7-7 converted less xylose to xylitol, by 40% and 420%, respectively. Figure 4 and 5 The construct used for transforming ONC-T18 is shown. ONC-T18 transformants were generated using a standard bio-projectile protocol as described by BioRad's PDS-1000 / He particle delivery system (Hercules, CA). Briefly, 0.6 µm gold particles were coated with 2.5 µg of linearized plasmid DNA (EcoRI, 37 °C, overnight). The coated gold particles were used to bombard plates previously plated with 1 mL of ONC-T18 cells with an OD600 of 1.0. Bombardment parameters included a helium pressure of 1350 or 1100 psi and a target distance of 3 cm or 6 cm. After overnight recovery, the cells were washed off the plates and inoculated onto media containing selective antibiotics (Zeo 250 µg / mL and hygro 400 µg / mL). The plates were incubated at 25 °C for 1 week to identify resistant colonies. Transformants were screened by PCR and Western blot.

[0173] DNA blotting was performed using a standard protocol. Briefly, approximately 20 µg of genomic DNA was digested overnight at 37°C with 50 μL of 40 units of BamHI restriction enzyme. Using digoxigenin (DIG) DNA molecular weight marker II (Roche, Basel, Switzerland), 7.2 µg of each digested sample was run on a 1.0% agarose gel at 50 V for approximately 1.5 hours. The DNA was depurinated in the gel by immersing it in 250 mM HCl for 15 minutes. The gel was further denatured by incubation in a solution containing 0.5 M NaOH and 1.5 M NaCl (pH 7.5) followed by two 15-minute washes. The reaction mixture was then neutralized by incubation in 0.5 M Tris-HCl (pH 7.5) followed by two 15-minute washes. Finally, the gel was equilibrated in 20X saline-sodium citrate (SSC) buffer for 15 minutes. The DNA was then transferred to a positively charged nylon membrane using a standard transfer apparatus. DNA was immobilized onto the membrane using a UV Stratalinker at 120,000 µJ. DNA blot probes were generated using a PCR DIG probe synthesis kit (Roche, Basel, Switzerland) to produce DIG-labeled probes according to the manufacturer's instructions. The DNA immobilized on the nylon membrane was pre-hybridized with 20 mL of DIG EasyHyb solution (DIG EasyHybGranules, Roche, Basel, Switzerland). The DIG-labeled probes were denatured by adding 40 μL of the ble-probe reaction mixture to 300 μL of ddH2O and incubating at 99 °C for 5 min. This solution was then added to 20 mL of DIG hybridization solution to generate the probe solution. The probe solution was then added to the DNA-immobilized nylon membrane and incubated overnight at 53 °C. The next day, the membrane was washed twice at room temperature in 2X SSC, 0.1% SDS. The membrane was further washed twice twice at 68 °C for 15 min each time in 0.1X SSC, 0.1% SDS. For detection, the membrane was washed and blocked using the DIG wash and blocking buffer kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Detection was performed using anti-DIG-AP conjugated antibody from the DIG Nucleic Acid Detection Kit (Roche, Basel, Switzerland). 2 µL of antibody solution was added to 20 mL of detection solution and incubated with the membrane at room temperature for 30 minutes. The blot was then immersed in the wash buffer provided with the kit. CDP-Star (Roche, Basel, Switzerland) was used for visualization.Incubate 10 µL of CDP-star solution on the membrane in 1 mL of detection solution, then cover the membrane with a sheet-protected plastic layer to hold the solution on the membrane. Detect the signal immediately using a ChemiDoc imaging system (BioRad Laboratories, Hercules, CA).

[0174] The codon-optimized ble gene was cloned under the control of the T18B α-tubulin promoter and terminator elements. Figure 6 The isomerase gene was cloned from T18B by adding a hexahistine tag (Iso-His) to the N-terminus of the expressed protein. The enzymatic activity of the xylose isomerase was confirmed by overexpression and purification of the histidine-tagged protein in yeast. The isomerase gene (and the introduced hexahistine tag) was cloned under the control of the α-tubulin promoter and terminator by cloning the gene downstream of the ble gene and the 2A sequence. Figure 4 and Figure 6 Bioprojectile transformation of T18B using this plasmid (pALPHTB-B2G-hisIso) produced bleomycin-resistant transformants. Numerous transformant strains were obtained from this procedure. Two of these strains are shown as Example #6 containing one copy of the transgene and Example #16 containing eight copies of the transgene. Figure 7 ).

[0175] The insertion of the Iso-His transgene into the T18B genome was confirmed by PCR and DNA blot analysis. Figure 7 Qualitatively, these data indicate the presence of a single copy of the transgene in strain #6 and multiple copies of the transgene at a single site in strain #16. The exact number of Iso-His transgene insertions was determined by qPCR on genomic DNA. Figure 8 These data indicate that one copy of the transgene is present in strain #6 and eight copies of the transgene are present in strain #16. Figure 8 To test whether the increase in copy number was associated with the increase in expression level, mRNA was isolated from WT, Iso-His #6, and Iso-His #16 T18B cells and qRT-PCR was performed. Figure 11 This study demonstrates a significant increase in Iso-His transcript expression in strain #16 cells containing eight copies of the transgene compared to strain #6 containing a single copy. No Iso-His transcript was detected in WT cells. Figure 11To assess whether increased mRNA expression was associated with increased isomerase activity, cell extracts were harvested from WT, Iso-His#6, and Iso-His#16 cells. Enhanced isomerase activity was observed in strain #16 cells compared to strain #6 and WT cells. Figure 12 Finally, the ability of strain #16 to metabolize xylose was examined in a xylose reduction assay (Figure 14) and compared with WT cells. These flask fermentations demonstrate the ability to metabolize xylose and quantify the amount of xylose converted to xylitol. Thus, Figure 14 shows the increased xylose metabolism and significantly less xylitol production in Iso-His strain #16 compared to WT cells.

[0176] For flask assays, cells were grown in culture medium for 2 to 3 days. The clumps were washed twice with sugar-free medium 2 (9 g / L NaCl, 4 g / L MgSO4, 100 mg / L CaCl2, 5 mg / L FeCl3, 20 g / L (NH4)2SO4, 0.86 g / L KH2PO4, 150 µg / L vitamin B12, 30 µg / L biotin, 6 mg / L thiamine hydrochloride, 1.5 mg / L cobalt(II) chloride, 3 mg / L manganese chloride). The washed cells were then seeded in basal medium containing 20 g / L glucose and 50 g / L xylose at an OD600 of 0.05. Samples were collected at different time points, and the amount of residual sugar in the supernatant was analyzed by HPLC. Figure 22A , 22B As shown in 22C and 22D, with an increased xylose isomerase gene copy number, xylose usage was increased by up to 40% and xylitol production was reduced by 20% compared to WT.

[0177] Then, Iso-His strain #16 was used as the parent strain for the second round of transformation to introduce E. coli. xylB Gene. This gene was introduced under hygromycin (hygro) selection. The hygro gene from pChlamy_3 (2A sequence) and the T18B codon-optimized W3110 E. coli were used. xylB The gene was cloned under the control of T18B α-tubulin promoter and terminator elements for expression in T18B iso-his #16. Figure 5The in vitro synergistic ability of *E. coli* xylulose kinase with T18B isomerase was confirmed by overexpression and purification of the histidine-tagged protein in yeast, followed by enzymatic reactions with xylose and xylulose. Bioprojectile transformation of T18B iso-his strain #16 using the xylB plasmid (pJB47) produced hygro and zeo resistant transformants. The insertion of the hygro-2A-xylB gene into the T18B genome was confirmed by PCR and Western blot analysis. Figure 9 Qualitatively, these data indicate the presence of a single copy of the transgene in strain #7-3 and multiple copies of the transgene at a single site in strain #7-7. The number of xylB gene insertions was determined by qPCR on genomic DNA isolates. Figure 10 ). Figure 10 Sixteen insertions of the transgene in strain 7-7 and one copy in strain 7-3 are shown. To determine whether multiple copies of the transgene confer enhanced xylose metabolism in vitro, cell extract assays were performed and the ability of cell extracts to metabolize xylose was analyzed. Figure 13 The ability of transformed somatic cells to metabolize xylose was examined by a flask-based xylose reduction assay (Fig. 15). In this experiment, WT cells consumed the least amount of xylose and produced the most xylitol. Strains Iso-His #16, 7-3, and 7-7 all consumed similar amounts of xylose; however, only 7-7 (containing multiple copies of the xylB transgene) did not produce a large amount of xylitol. Finally, strains Iso-His #16 and 7-7 were tested in a 5 L fermentation vessel in a medium containing glucose and xylose. During a fermentation of seventy-seven (77) hours, strain Iso-His #16 converted approximately 8% of the xylose to xylitol, while strain 7-7 converted approximately 2% of the xylose to xylitol. Figure 16 This shows the accumulation of xylitol during this fermentation.

[0178] For flask assays, cells were grown in culture medium for 2 to 3 days. The clumps were washed twice in sugar-free medium. The washed cells were seeded in medium containing 20 g / L:50 g / L glucose:xylose at an OD600 of 0.05. Samples were collected at different time points, and the amount of residual sugar in the supernatant was analyzed by HPLC. Figure 23A , 23B As shown in 23C and 23D, when compared with WT, up to 50% more xylose was used and an 80% reduction in xylitol was observed in strains that overexpress both xylose isomerase and xylulokine.

[0179] To further analyze these strains, they were grown in parallel 5L Sartorius fermenters. The initial medium contained 20 g / L glucose and 50 g / L xylose, along with other basal medium components. Both cultures were maintained at 28°C and 5.5 pH, constantly mixed at 720 RPM, and constantly aerated at 1 Lpm ambient air. Glucose was fed to the cultures for 16 hours, followed by an 8-hour starvation period. This cycle was repeated three times. During the starvation period, 10 mL samples were taken every 0.5 hours. The concentrations of glucose, xylose, and xylitol in these samples were quantified by HPLC. Larger 50 mL samples were periodically taken for further quantification of biomass and oil content. The glucose feed rate was matched to the glucose consumption rate, which was quantified by CO2 detected in the culture exhaust gas. Figure 24 As shown, under these conditions, strain 7-7 uses up to 52% more xylose than WT.

[0180] DNA blotting analysis revealed that strain Iso-His #16 contained eight (8) insertions of isomerase transgenes. Figure 8 This unexpected multiple insertion makes it possible to carry a single copy ( Figure 11 The strain showed increased expression of isomerase genes and increased in vitro activity of isomerases. Figure 12 Compared to strains carrying a single copy of the isomerase transgene, strain Iso-His #16 exhibited increased xylose production (Figure 14).

[0181] Similarly, in Iso-His + xylB In the transformant, one of the clones ( Iso-His + xylB 7-7) also has xylB Multiple insertion of genes ( Figure 10 This increases the in vitro activity of both xylose isomerase and xylulokine in cells. Figure 13 This clone was able to use the same or more xylose as its parent strain Iso-His #16 while producing significantly less xylitol (Figure 15). Furthermore, in the presence of xylose, Iso-His + xylB 7-7 produced more biomass than WT. These two strains demonstrate that not only the presence of isomerase and kinase genes is important, but the number of insertions is equally crucial.

[0182] To further optimize the strain containing iso-his and xylB "7-7", this strain was transformed with xylose transporter protein. Figure 17Exemplary constructs used for transformation are shown. Examples of xylose transporters to be used include, but are not limited to, At5g17010 and At5g59250 (Arabidopsis thaliana), Gfx1 and GXS1 (Candida), AspTx (Aspergillus), and Sut1 (Pichia pastoris). Gxs1 (SEQ ID NO:23) was selected for transformation. Results are shown in Figure 19A , 19B As shown in 19C, transformants 36-2, 36-9, and 36-16 containing GXS1 used more xylose than strains 7-7 and WT. They also used glucose more slowly than strains WT and 7-7. The data indicate that strains containing GXS1 use both xylose and glucose in the early stages. Furthermore, the percentage of xylitol produced from strains containing GXS1 was lower than that from both strains WT and 7-7.

[0183] To further analyze the effects of sugar transporters on xylose metabolism, codon-optimized xylose transporters AspTX from Aspergillus (An11g01100) and Gxs1 from Candida were introduced into strain 7-7 (isohis + xylB). Figure 25 The α-tubulin aspTx-neo and α-tubulin gxs1-neo constructs are shown. T18 transformants were generated using a standard bio-projectile protocol as described by the BioRad PDS-1000 / He particle delivery system. Briefly, 0.6 µm gold particles were coated with 2.5 µg of linearized plasmid DNA (EcoRI, 37 °C, overnight). The coated gold particles were used to bombard WD plates previously coated with 1 mL of T18 cells with an OD600 of 1.0. Bombardment parameters included a helium pressure of 1350 or 1100 psi and a target distance of 3 cm or 6 cm. After overnight recovery, the cells were washed off the plates and inoculated onto medium containing a selective antibiotic (G418, 2 mg / mL). The plates were incubated at 25 °C for 1 week to identify resistant colonies. Transformants were screened by PCR and DNA blotting (Figure 26).

[0184] DNA blotting was performed using a standard protocol. Briefly, approximately 20 µg of genomic DNA was digested overnight at 37°C with 50 μL of 40 units of BamHI restriction enzyme. Using digoxigenin (DIG) DNA molecular weight marker II (Roche), 7.2 µg of each digested sample was run on a 1.0% agarose gel at 50 V for approximately 1.5 hours. The DNA was depurinated in the gel by immersing it in 250 mM HCl for 15 minutes. The gel was further denatured by incubation in a solution containing 0.5 M NaOH and 1.5 M NaCl (pH 7.5) followed by two 15-minute washes. The reaction mixture was then neutralized by incubation in 0.5 M Tris-HCl (pH 7.5) followed by two 15-minute washes. Finally, the gel was equilibrated in 20X saline-sodium citrate (SSC) buffer for 15 minutes. The DNA was then transferred to a positively charged nylon membrane (Roche) using a standard transfer apparatus. DNA was immobilized onto the membrane using a UV Stratalinker at 120,000 µJ. DNA blot probes were generated using a PCR DIG probe synthesis kit (Roche) to produce DIG-labeled probes according to the manufacturer's instructions. The DNA immobilized on the nylon membrane was pre-hybridized with 20 mL of DIG EasyHyb solution (DIG EasyHyb Granules, Roche). The DIG-labeled probes were denatured by adding 40 μL of the ble-probe reaction mixture to 300 μL of ddH2O and incubating at 99 °C for 5 min. This solution was then added to 20 mL of DIG hybridization solution to generate the probe solution. The probe solution was then added to the DNA-immobilized nylon membrane and incubated overnight at 53 °C. The next day, the membrane was washed twice at room temperature in 2X SSC, 0.1% SDS. The membrane was further washed twice twice at 68 °C for 15 min each time in 0.1X SSC, 0.1% SDS. For detection, the membrane was washed and blocked using the DIG wash and block buffer kit (Roche) according to the manufacturer's instructions. Detection was performed using anti-DIG-AP conjugated antibody from the DIG Nucleic Acid Detection Kit (Roche). 2 µL of antibody solution was added to 20 mL of detection solution and incubated with the membrane at room temperature for 30 minutes. The blot was then immersed in the wash buffer provided with the kit. CDP-Star (Roche) was used for visualization. 10 µL of CDP-Star solution was incubated on the membrane in 1 mL of detection solution, and the membrane was covered with a sheet protection plastic layer to hold the solution on the membrane. The signal was detected immediately using the ChemiDoc imaging system.

[0185] For flask assays, cells were grown in culture medium for 2 to 3 days. The clumps were washed twice with sugar-free medium 2 (9 g / L NaCl, 4 g / L MgSO4, 100 mg / L CaCl2, 5 mg / L FeCl3, 20 g / L (NH4)2SO4, 0.86 g / L KH2PO4, 150 µg / L vitamin B12, 30 µg / L biotin, 6 mg / L thiamine hydrochloride, 1.5 mg / L cobalt(II) chloride, 3 mg / L manganese chloride). Then, the washed cells were seeded in medium 2 containing 20 g / L glucose and 20 g / L xylose at an OD600 of 0.05. Figure 27A , 27B As shown in 27C and 27D, compared with the parent strain 7-7, the expression of xylose isomerase, xylulose kinase, and any xylose transporter resulted in the use of up to 71% more xylose and the production of 40% less xylitol.

[0186] For flask assays, cells were grown in culture medium for 2 to 3 days. The clumps were washed twice with saline. The washed cells were then seeded with culture medium containing 60 g / L xylose instead of glucose at an OD600 of 0.05. Samples were collected at different time points, and the amount of remaining sugar in the supernatant was analyzed by HPLC. Figure 28 The results show that T18 growth in a medium containing xylose as the primary carbon source requires overexpression of both isomerase and kinase. In this context, the expression of transport proteins did not significantly increase xylose utilization in this medium.

[0187] Enhanced xylose use was observed in T18 7-7 and transporter strains in media containing carbon from alternative feedstocks. For flask assays, cells were grown in the medium for 2–3 days. Agglomerates were washed twice in 0.9% saline solution. Washed cells were seeded in medium 2 containing 20 g / L glucose: 50 g / L xylose as laboratory-grade glucose and a combination of glucose and xylose from forestry alternative feedstocks at 0.05 OD 600 nm. Samples were collected at different time points, and the amount of residual sugar in the supernatant was analyzed by HPLC. Figure 29A and 29B As shown, in a culture medium containing sugars from alternative sources, the T18 7-7 strain encoding the transporter protein used more xylose than the wild type or T18 7-7.

Claims

1. A recombinant microorganism comprising two or more copies of a nucleic acid sequence encoding a xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is a foreign nucleic acid.

2. The recombinant microorganism of claim 1, further comprising at least one nucleic acid sequence encoding xylulokine.

3. The recombinant microorganism of claim 2, wherein the nucleic acid sequence encoding the xylulokine is an exogenous nucleic acid sequence.

4. The recombinant microorganism according to any one of claims 1 to 3, further comprising at least one nucleic acid sequence encoding a xylose transporter protein.

5. The recombinant microorganism of claim 4, wherein the nucleic acid sequence encoding the xylose transporter is an exogenous nucleic acid sequence.

6. The recombinant microorganism of claim 1, wherein the microorganism further comprises at least two or more copies of a nucleic acid sequence encoding xylulokine and at least one nucleic acid sequence encoding a xylose transporter.

7. The recombinant microorganism according to any one of claims 1 to 6, wherein the nucleic acid sequence encoding the xylose isomerase is a heterologous nucleic acid.

8. The recombinant microorganism according to any one of claims 1 to 7, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 copies of the exogenous nucleic acid sequence encoding xylose isomerase.

9. The recombinant microorganism according to any one of claims 1 to 8, wherein the heterologous nucleic acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:

2.

10. The recombinant microorganism according to any one of claims 2 to 9, wherein the microorganism comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylulokine.

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