NZ739056B2 - Enhancing microbial metabolism of c5 organic carbon - Google Patents

Abstract

Provided herein are recombinant microorganisms having two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is an exogenous nucleic acid. Optionally, the recombinant microorganisms include at least one nucleic acid sequence encoding a xylulose kinase 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. coding a xylulose kinase 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.

Description

ENHANCING MICROBIAL METABOLISM OF C5 ORGANIC CARBON CROSS-REFERENCE TO D ATIONS This application claims the benefit of 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 by reference herein in their entireties.

BACKGROUND OF THE INVENTION Heterotrophic tation of rganisms is an efficient way of generating high value oil and biomass products. Under certain cultivation conditions, microorganisms synthesize intracellular oil, which can be extracted and used to produce fuel (e.g., biodiesel, tfuel, and the like) and ional lipids (e.g., polyunsaturated fatty acids such as DHA, EPA, and DPA). The biomass of some microorganisms is of great ional value due to high polyunsaturated fatty acid (PUFA) and protein content, and can be used as a nutritional supplement for animal feed. Thraustochytrids are eukaryotic, single-cell, microorganisms which can be used in such fermentation processes to produce lipids. Heterotrophic fermentations with Thraustochytrids convert organic carbon provided in the growth medium to lipids, which are harvested from the biomass at the end of the fermentation process.

However, existing microorganism fermentations use mainly ive carbohydrates, such as glucose, as the carbon source.

BRIEF Y OF THE INVENTION sed herein are recombinant microorganisms having two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding the xylose isomerase is an exogenous nucleic acid. Optionally, the recombinant microorganisms include at least one nucleic acid sequence encoding a xylulose kinase and/or at least one nucleic acid sequence encoding a xylose transporter. The disclosed recombinant microorganisms are capable of growing on xylose as a carbon source.

A first aspect provides a recombinant Thraustochytrium microorganism comprising: a) two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the c acid encoding xylose isomerase is an ous nucleic acid; 17746603_1 (GHMatters) P107943.NZ 1 04/06/2021 b) two or more copies of a nucleic acid sequence ng a xylulose kinase; and c) at least one nucleic acid ce encoding a xylose transporter, wherein the isomerase, kinase and transporter are integrated into the genome of the Thraustochytrium rganism.

A second aspect provides a method of making a recombinant xylose-metabolizing Thraustochytrium microorganism comprising: i. transforming the tochytrium microorganism with one or more nucleic acid constructs comprising a c acid sequence encoding a xylose isomerase, a nucleic acid sequence encoding a xylulose kinase and a nucleic acid sequence encoding a xylose transporter; and ii. isolating the Thraustochytrium microorganism comprising at least two or more copies of the nucleic acid sequences encoding the xylose isomerase, at least two copies of the nucleic acid ce encoding the xylulose kinase and at least one copy of the nucleic acid sequence encoding the xylose transporter, wherein the isomerase, kinase and transporter are integrated into the genome of the Thraustochytrium microorganism.

A third aspect provides a method of producing oil comprising: culturing the recombinant Thraustochytrium rganism of the first aspect in a culture medium to produce the oil, wherein the microorganism grows on xylose as the sole carbon source. 17746603_1 (GHMatters) P107943.NZ 1a 04/06/2021 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a tic of the xylose metabolism pathway.

Figure 2 is a graph showing expression of xylose isomerase in WT ONC-T18 during cycles of glucose starvation.

Figure 3 is a graph showing expression of the putative xylulose kinase in WT ONCT18 during cycles of glucose starvation.

Figure 4 is a schematic showing an alpha-tubulin ble-isomerase d construct.

Figure 5 is a schematic showing an alpha-tubulin xylB plasmid construct.

Figure 6 is a schematic showing a nucleic acid construct having an alpha-tubulin promoter a ble sequence a 2A sequence an xylose isomerase sequence and an alpha-tubulin ator.

Figure 7 is an image of a Southern blot to probe the xylose isomerase His-tagged gene within recombinant ONC-T18 strains “6” and “16”.

Figure 8 is a graph showing the qPCR determination of the number of xylose isomerase His-tagged gene insertions in recombinant ONC-T18 strains.

Figure 9 is an image of a Southern blot to probe the xylB gene within recombinant 8 strains containing both xylose isomerase and xylulose kinase referred to in the graph as “7-3” and “7-7”.

Figure 10 is a graph of qPCR determination of the number of xylB gene insertions in recombinant 7-3 and 7-7 ONC-T18 strains.

Figure 11 is a graph showing the expression of the xylose ase gene transcript in recombinant ONC-T18 strains “6” and “16.” Figure 12 is a graph showing the in vitro xylose isomerase activity in Wt ONC-T18 and inant ONC-T18 strains “6” and “16.” Figure 13 is a graph showing the combined xylose isomerase and se kinase activity in vitro of recombinant ONC-T18 strain “16” encoding only xylose isomerase and recombinant 8 strains “7-3” and “7-7” encoding xylose isomerase and xylulose kinase.

Figures 14A and 14B are graphs showing xylose uptake improvement and decreased xylitol production in recombinant 8 strain “16” (squares). The Wild Type (WT) strain is represented by ds.

Figures 15A and 15B are graphs showing xylose usage improvement and decreased xylitol production in recombinant 8 strain “16” (squares) and recombinant ONC-T18 17746603_1 (GHMatters) P107943.NZ 2 04/06/2021 strains “7-3” (triangles) and “7-7” (asterisks). The Wild Type (WT) strain is represented by Figure 16 is a graph g accumulation of xylitol during a glucose:xylose fermentation with recombinant ONC-T18 strain “16” and recombinant ONC-T18 strain “7- Figure 17 is a schematic of different versions of the ucts used for transformation of ONC-T18.

Figure 18 is a graph showing the ent of the xylB sequence from E. coli (SEQ ID NO:20) with the codon optimized version of E. coli xylB (SEQ ID NO:5).

Figures 19A, 19B, and 19C are graphs g xylose usage (Fig. 19A), glucose usage (Fig. 19B) and percent xylitol made (Fig. 19C) in strains comprising xylose isomerase, xylulose kinase and the sugar transporter Gxs1. WT is wild-type; IsoHis XylB “7-7” contains the xylose isomerase and xylB sequences, 36-2, 36-9 and 36-16 are ormants containing Gxs1, xylose isomerase and the xylB sequences (xylulose ).

Figures 20A and 20B are graphs showing the impact of temperature incubation on the activity of isomerase from T18 (Figure 20A) and E. coli (Figures 20B) with xylose (diamond) and xylulose (square).

Figures 21A and 21B are graphs g dose dependency of isomerase from T18 (Figure 21A) and E. coli (Figure 21B) with xylose (diamond) and xylulose (square).

Figures 22A and 22B are graphs showing xylose use (Figure 22A) and decreased l production (Figure 22B) in a T18B strain engineered with xylose isomerases (“16” (squares), “B” (x), and “6” (crosses)). s 22C (xylose) and 22D (xylitol production) show the same data expressed relative to wild type (diamonds) at 4 (gray) and 7 (black) days.

Figures 23A and 23B are graphs showing xylose use and decreased xylitol tion in a T18B strain ered with a xylose isomerase “16” (squares) and strains engineered to express a xylose isomerase and xylulose kinase “7-7” (x) and “7-3” (triangles).

Figures 23C (xylose) and 23D (xylitol production) show the same data relative to wild type (diamonds) at 9 (gray) and 11 (black) days.

Figure 24 is a graph showing improved xylose usage and decreased xylitol production in a T18B strain engineered to express a xylose isomerase and xylulose kinase “7- 7” in fermentation. The wild type strain is represented by diamonds and the dotted line and the strain “7-7” is represented by circles.

Figure 25 is a schematic showing α-tubulin aspTx-neo and α-tubulin gxs1-neo constructs. 03_1 (GHMatters) P107943.NZ 3 04/06/2021 Figure 26A is an image of a Southern blot to probe the Gxs1 gene within “7-7” T18B strains engineered with the xylose transporter Gxs1. Figure 26B is an image of a Southern blot to probe the AspTx gene within “7-7” T18B strains engineered with the xylose transporter AspTx.

Figures 27A is a graph showing the use of xylose in T18 strains engineered with a xylose isomerase, a se kinase and either the Gxs1 transporter (triangles) or AspTx transporter (circles). Strain “7-7” is represented by diamonds. Figure 27B is a bar graph of the ratio of xylitol production versus xylose use for each of the 3 modified strains. Figure 27C is a bar graph showing xylose use relative to strain “7-7.” Figures 27D is a bar graph showing xylitol production made relative to strain “7-7.” Figures 28 is a graph showing growth of wild type (WT) (diamonds), isohis strain “16” (squares), strain “7-7” (x), and transporter strains Gxs1 (asterisks) and AspTx (triangles) in media containing xylose as sole carbon source.

Figure 29A is a graph g remaining e in alternative feedstock containing e and xylose during growth of WT (squares), strain “7-7” (triangles), and transporter strains Gxs1 (asterisks) and AspTx (crosses). Figure 29B is a graph showing xylose remaining and xylitol produced over time when WT (squares) strain “7-7” (triangles) and transporter s Gxs1 (asterisks) and AspTx (crosses) are grown on alternative feedstock containing glucose and xylose.

DETAILED DESCRIPTION OF THE INVENTION Microorganisms such as Thraustochytrids encode genes required for the metabolism of xylose. However, the microorganism’s innate metabolic pathways produce a large amount of the sugar alcohol, xylitol, which is secreted and potentially hinders growth of the microorganisms (see Figure 14, WT). Furthermore, carbon atoms sequestered into xylitol are atoms that are diverted away from the target product in this process, namely, lipid tion.

In nature, two xylose metabolism pathways exist, the xylose reductase/xylitol dehydrogenase pathway and the xylose isomerase/xylulose kinase y e 1). Thraustochytrids have genes that encode proteins active in both pathways; however, the former pathway appears to be dominant as evidenced by a up of xylitol when grown in a xylose medium. In other organisms, the build-up of xylitol has been shown to be due to a redox cofactor imbalance ed for xylose reductase/xylitol dehydrogenase y. Since the isomerase/kinase pathway does not depend on redox co-factors, xpression of the isomerase gene removes co-factor dependence in the conversion of xylose to xylulose. As shown herein, transcriptomic s with ONC-T18 showed that its xylose isomerase and 17746603_1 (GHMatters) P107943.NZ 4 04/06/2021 putative xylulose kinase genes are mostly expressed during glucose tion (Figure 2 and Figure 3); whereas, the putatively identified genes encoding for the xylose reductase and xylitol dehydrogenase are constitutively expressed. To se the expression of the ase and kinase hout all growth stages, microorganisms were engineered to include ONC-T18 isomerase gene and an E. coli xylulose kinase gene (xylB) such that they are under the control of the constitutively expressed promoter and terminator, e.g., an αtubulin promoter and terminator. Optionally, the genes can be under the control of a inducible promoter and/or terminator.

The provided recombinant microorganisms demonstrate a level of control of the amount of expression of a gene of st via the number of integrated transgene copies. As shown in the examples below, a recombinant ONC-T18 strain (Iso-His #16) harbouring eight (8) transgene copies demonstrates higher levels of xylose ase transcript sion, enzyme activity and xylose metabolism than a strain harbouring a single copy of the transgene (Iso-His #6). When Iso-His #16 was further ed to incorporate the xylB gene, a similar phenomenon is observed. Multiple copies of the xylB gene conferred greater enzyme ty and xylose metabolism tivity compared to single insertions. Thus, unexpectedly, it was not only necessary to recreate a xylose metabolism pathway, but to do so with multiple copies of the necessary transgenes. It was not pated that the Thraustrochytrid genome could accommodate multiple transgene copies and remain viable; therefore, it was not expected to observe such variability in expression levels amongst ormant strains. However, as provided , recombinant microorganisms can be produced that allow for controlled expression levels of transgenes indirectly by ing among transformant strains that possess a transgene copy number “tailored” to a particular expression level optimized for the metabolic engineering of a ular pathway, e.g., the xylose pathway.

Provided herein are nucleic acids encoding one or more genes involved in xylose metabolism. The present application provides recombinant microorganisms, methods for making the microorganisms, and methods for producing oil using the microorganisms that are capable of metabolizing xylose. Specifically, provided herein are nucleic acids and polypeptides encoding xylose isomerase, xylulose kinase and xylose transporters for modifying microorganisms to be capable of lizing xylose and/or growing on xylose as the sole carbon . Thus, provided are nucleic acids encoding a xylose isomerase. The nucleic acid sequences can be endogenous or heterologous to the microorganism. Exemplary nucleic acids sequences of xylose isomerases include, but are not limited to, those from 17746603_1 (GHMatters) P107943.NZ 5 04/06/2021 Piromyces sp., Streptococcus sp., and Thraustochytrids. For example, exemplary nucleic acid sequences encoding xylose isomerases include, but are not d 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 acids sequences of se kinases include, but are not limited to, those from E. coli, Piromyces sp., Saccharomyces sp., and Pichia sp. For example, exemplary c acid sequences encoding xylulose 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 ces encoding sugar transporters, e.g., xylose transporters, include, but are not limited to, those from Aspergillus sp., Gfx1, Gxs1 and Sut1. For example, ary 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.

Nucleic acid, as used herein, refers to deoxyribonucleotides or cleotides and polymers and complements thereof. The term includes deoxyribonucleotides or ribonucleotides in either single- or double-stranded form. The term encompasses c acids containing known nucleotide analogs or modified backbone residues or es, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, conservatively modified variants of nucleic acid ces (e.g., degenerate codon substitutions) and complementary ces can be used in place of a particular nucleic acid sequence recited herein. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or nosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); a 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.

A nucleic acid is operably linked when it is placed into a functional relationship with another c acid sequence. For example, DNA that encodes a presequence or secretory leader is operably linked to DNA that encodes a polypeptide if it is expressed as a preprotein that ipates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a 03_1 ters) 3.NZ 6 04/06/2021 me binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase.

However, enhancers do not have to be contiguous. For example, a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such second sequence, gh any effective three-dimensional association is acceptable. A single c acid sequence can be operably linked to multiple other sequences. For example, a single promoter can direct transcription of multiple RNA species.

Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic ucleotide adaptors or linkers are used in accordance with conventional ce.

The terms identical or t identity, in the t of two or more nucleic acids or polypeptide ces, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for m correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be substantially identical. This tion also refers to, or may be applied to, the compliment of a test sequence. The definition also includes ces that have deletions and/or additions, as well as those that have tutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, lly one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default m parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. 17746603_1 (GHMatters) P107943.NZ 7 04/06/2021 A comparison window, as used herein, includes nce to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988); by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., n, WI); or by manual ent and visual inspection (see, e.g., Current ols in lar Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of an algorithm that is le for determining t sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are 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 ters described herein, to determine percent sequence identity for nucleic acids or proteins. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm involves first identifying high scoring sequence pairs (HSPs) by fying short words of a selected length (W) in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., . These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching es; always > 0) and N ty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its m achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue ents; or the end of either sequence is reached. The BLAST algorithm ters W, T, and X determine the 17746603_1 ters) P107943.NZ 8 04/06/2021 sensitivity and speed of the alignment. The Expectation value (E) represents the number of different alignments with scores equivalent to or better than what is expected to occur in a database search by chance. The BLASTN program (for tide sequences) uses as defaults a ngth (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid ces, the BLASTP program uses as defaults a ngth of 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), ents (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The term polypeptide, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids and is intended to include peptides and proteins. r, the term is also used to refer to specific functional classes of polypeptides, such as, for example, desaturases, elongases, etc. For each such class, the present disclosure provides several examples of known sequences of such polypeptides. Those of ordinary skill in the art will appreciate, however, that the term polypeptide is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a nce or database specifically mentioned herein), but also to ass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that s activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually ing at least one region of much higher ty, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term polypeptide as used herein. Those in the art can determine other regions of similarity and/or identity by analysis of the sequences of various polypeptides described herein. As is known by those in the art, a variety of strategies are known, and tools are available, for performing comparisons of amino acid or nucleotide sequences in order to assess degrees of identity and/or similarity. These strategies include, for example, manual alignment, computer assisted ce ent and combinations thereof. A number of algorithms (which are generally computer implemented) for performing sequence alignment are widely ble, or can be produced by one of skill in the art. Representative algorithms e, e.g., the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482); the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443); the search for 17746603_1 (GHMatters) P107943.NZ 9 04/06/2021 similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. (USA), 1988, 85: 2444); and/or by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package e 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). Readily available computer ms incorporating such algorithms include, for e, BLASTN, BLASTP, Gapped BLAST, PILEUP, CLUSTALW, etc. When utilizing BLAST and Gapped BLAST programs, default parameters of the tive programs may be used. Alternatively, the practitioner may use non-default ters depending on his or her experimental and/or other requirements (see for example, the Web site having URL www.ncbi.nlm.nih.gov).

As discussed above, the nucleic acids encoding the xylose transporter, xylulose kinase and xylose isomerase, can be linked to a er and/or terminator. Examples of ers and terminators include, but are not limited to, tubulin promoters and terminators. By way of example, the promoter is a tubulin promoter, e.g., an tubulin promoter. ally, the promoter is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. ally, 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.

As used herein, the terms er, promoter t, and regulatory sequence refer to a polynucleotide that regulates expression of a selected polynucleotide sequence operably linked to the promoter, and that effects expression of the selected polynucleotide sequence in cells. The term Thraustochytrium promoter, as used herein, refers to a promoter that functions in a Thraustochytrium cell. In some embodiments, a promoter element is or comprises untranslated s (UTR) in a position 5’ of coding sequences. 5’ UTRs form part of the mRNA transcript and so are an integral part of protein expression in eukaryotic organisms. Following transcription 5’UTRs can regulate protein expression at both the transcription and translation levels.

As used herein, the term terminator refers to a polynucleotide that abrogates expression of, targets for maturation (e.g., adding a polyA tail), or imparts mRNA stability to a selected polynucleotide sequence operably linked to the terminator in cells. A terminator sequence may be downstream of a stop codon in a gene. The term Thraustochytrium terminator, as used herein, refers to a terminator that functions in a Thraustochytrium cell.

Provided herein are also nucleic acid constructs that include c acid sequences encoding xylose isomerase, xylulose kinase and xylose transporter as well as promoters, terminators, selectable markers, 2A peptides or any combination f. By way of example, provided is a first c acid construct including a promoter, a able marker, a nucleic acid 17746603_1 (GHMatters) P107943.NZ 10 04/06/2021 sequence encoding a 2A peptide, a nucleic acid sequence encoding a xylose ase, and a terminator. Also provided is a second nucleic acid construct including a promoter, selectable , a nucleic acid ce encoding a 2A peptide, a c acid sequence encoding a xylulose kinase, and a terminator. Further provided is a third nucleic acid uct including a promoter, a nucleic acid sequence encoding a xylose transporter, a nucleic acid sequence encoding a 2A peptide, a able marker, and a ator. These constructs are ary and the nucleic acid sequences encoding the xylose isomerase, xylulose kinase and xylose transporter can be included on the same construct under control of the same or different promoters. Optionally, each of the nucleic acid ces encoding the xylose isomerase, xylulose kinase and xylose transporter are on the same construct and are separated by 2A polypeptide sequences, e.g., as shown in SEQ ID NO:6. Thus, by way of example, a nucleic acid construct can include a tubulin promoter, a nucleic acid sequences encoding a xylose isomerase, xylulose kinase, and xylose transporter ted by a c acid sequence encoding SEQ ID NO:6, a tubulin terminator and a selectable marker. Optionally, the selectable marker is the ble gene. Optionally, the selectable marker comprises SEQ ID NO:29.

The phrase selectable marker, as used herein, refers either to a nucleotide sequence, e.g., a gene, that encodes a product (polypeptide) that allows for selection, or to the gene product (e.g., polypeptide) itself. The term selectable marker is used herein as it is lly understood in the art and refers to a marker whose presence within a cell or organism confers a significant growth or survival advantage or antage on the cell or sm under certain defined culture conditions tive conditions). For example, the conditions may be the presence or absence of a particular compound or a ular environmental condition such as increased temperature, increased radiation, presence of a compound that is toxic in the absence of the marker, etc. The presence or absence of such nd(s) or environmental condition(s) is referred to as a ive condition or selective conditions. By growth advantage is meant either enhanced viability (e.g., cells or organisms with the growth advantage have an increased life span, on average, relative to otherwise identical cells), increased rate of proliferation (also referred to herein as growth rate) relative to otherwise identical cells or organisms, or both. In general, a population of cells having a growth advantage will exhibit fewer dead or nonviable cells and/or a greater rate of cell proliferation than a population of otherwise identical cells lacking the growth advantage. Although typically a selectable marker will confer a growth age on a cell, certain selectable markers confer a growth disadvantage on a cell, e.g., they make the cell more susceptible to 17746603_1 (GHMatters) P107943.NZ 11 04/06/2021 the deleterious effects of certain compounds or environmental conditions than otherwise cal cells not sing the marker. Antibiotic resistance markers are a non-limiting example of a class of selectable marker that can be used to select cells that express the marker. In the ce of an appropriate concentration of antibiotic (selective conditions), such a marker confers a growth advantage on a cell that expresses the marker. Thus, cells that express the antibiotic resistance marker are able to survive and/or erate in the presence of the antibiotic while cells that do not express the antibiotic resistance marker are not able to survive and/or are unable to proliferate in the presence of the antibiotic.

Examples of able markers include common bacterial antibiotics, such as but not limited to ampicillin, kanamycin and chloramphenicol, as well as ive compounds known to function in microalgae; examples include rrnS and AadA (Aminoglycoside 3’- adenylytranferase), which may be isolated from E. coli plasmid R538-1, conferring resistance to spectinomycin and streptomycin, respectively in E. coli and some microalgae (Hollingshead and Vapnek, d 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, a GC rich gene isolated from Streptoalloteichus hindustanus that s resistance to zeocin (Stevens, Purton et al., Mol. Gen. Genet., 251:23-30 1996). Aph7 is yet another example, which is a Streptomyces hygroscopicus-derived aminoglycoside phosphotransferase gene that confers resistance to hygromycin B (Berthold, Schmitt et al., Protist 153(4):401-412 2002).

Additional examples include: AphVIII, a Streptomyces rimosus d lycoside 3′- otransferase type VIII that confers ance to Paromycin in E. 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 & Sat-1, which encode nourseothricin acetyl transferase from Streptomyces noursei and streptothricin acetyl transferase from E. coli, which confer ance to nourseothricin (Zaslavskaia, Lippmeier et al., Journal of Phycology 379- 386, 2000); Neo, an aminoglycoside sphotransferase, conferring resistance to the aminoglycosides; cin, neomycin, and the analog G418 (Hasnain, Manavathu et al., Molecular and Cellular Biology 5(12):3647-3650, 1985); and Cry1, a mal protein S14 that confers resistance to emetine (Nelson, Savereide et al., Molecular and Cellular Biology 14(6):4011-4019, 1994).

Other selectable s include nutritional markers, also referred to as auto- or auxotrophic markers. These include photoautotrophy markers that impose selection based on the 17746603_1 (GHMatters) P107943.NZ 12 04/06/2021 restoration of photosynthetic activity within a photosynthetic organism. Photoautotrophic markers include, but are not d 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 ch Communications :966-975, 2005). Alternative or additional nutritional markers include ARG7, which encodes argininosuccinate lyase, a critical step in arginine biosynthesis hy, Purton et al., EMBO J 8(10):2803-2809, 1989); NIT1, which encodes a nitrate reductase essential to nitrogen metabolism (Fernández, Schnell et al., PNAS, 86(17):6449-6453, 1989); THI10, which is essential to thiamine biosynthesis (Ferris, Genetics 141(2):543-549, 1995); and NIC1, which catalyzes an essential step in nicotinamide biosynthesis (Ferris, Genetics 141(2):543-549, 1995). Such markers are generally s that function in a biosynthetic pathway to produce a compound that is needed for cell growth or survival. In general, under nonselective conditions, the required nd is present in the environment or is ed by an alternative pathway in the cell. Under ive conditions, functioning of the biosynthetic pathway, in which the marker is involved, is needed to produce the nd.

The phrase selection agent, as used herein refers to an agent that introduces a selective pressure on a cell or populations of cells either in favor of or against the cell or population of cells that bear a selectable . For example, , the selection agent is an antibiotic and the selectable marker is an antibiotic resistance gene. Optionally, zeocin is used as the selection agent.

Suitable microorganisms that can be transformed with the provided nucleic acids encoding the genes involved in xylose metabolism and nucleic acid constructs containing the same include, but are not limited to, algae (e.g., lgae), fungi (including yeast), bacteria, or protists. Optionally, the microorganism includes Thraustochytrids of the order Thraustochytriales, more specifically Thraustochytriales of the genus Thraustochytrium.

Optionally, the population of microorganisms includes tochytriales as described in U.S. Patent Nos. 5,340,594 and 5,340,742, which are incorporated herein by reference in their ties. The microorganism can be a Thraustochytrium species, such as the Thraustochytrium s ted as ATCC Accession No. PTA-6245 (i.e., ONC-T18) as described in U.S. Patent No. 8,163,515, which is incorporated by reference herein in its entirety. Thus, the microorganism can have an 18s rRNA sequence that is at least 95%, 96%, 17746603_1 (GHMatters) P107943.NZ 13 04/06/2021 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more (e.g., including 100%) cal to SEQ ID NO:1.

Microalgae are acknowledged in the field to represent a diverse group of sms.

For the purpose of this document, the term microalgae will be used to describe unicellular microorganisms derived from aquatic and/or terrestrial environments (some cyanobacteria are terrestrial/soil dwelling). Aquatic nments extend from oceanic environments to freshwater lakes and rivers, and also include brackish nments such as estuaries and river mouths. Microalgae can be photosynthetic; optionally, microalgae are heterotrophic.

Microalgae can be of eukaryotic nature or of prokaryotic nature. Microalgae can be nonmotile or motile.

The term thraustochytrid, as used herein, refers to any member of the order tochytriales, which includes the family Thraustochytriaceae. Strains described as thraustochytrids include the following organisms: Order: Thraustochytriales; Family: Thraustochytriaceae; Genera: tochytrium (Species: sp., arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, ermum, proliferum, , striatum), Ulkenia (Species: sp., amoeboidea, lensis, minuta, profunda, radiata, sailens, sarkariana, schizochytrops, visurgensis, yorkensis), Schizochytrium (Species: sp., aggregatum, limnaceum, mangrovei, minutum, oruni), chytrium (Species: sp., marinum), Aplanochytrium (Species: sp., haliotidis, kerguelensis, profunda, stocchinoϊ), Althornia (Species: sp., crouchii), or Elina (Species: sp., marisalba, sinorifica). Species described within Ulkenia will be considered to be members of the genus Thraustochytrium.

Strains described as being within the genus Thrautochytrium may share traits in common with and also be described as falling within the genus Schizochytrium. For example, in some taxonomic classifications ONC-T18 may be considered within the genus Thrautochytrium, while in other classifications it may be bed as within the genus Schizochytrium because it comprises traits indicative of both genera.

The term transformation, as used herein refers to a s by which an exogenous or heterologous c acid molecule (e.g., a vector or inant nucleic acid molecule) is introduced into a recipient cell or microorganism. The exogenous or heterologous nucleic acid molecule may or may not be integrated into (i.e., covalently linked to) chromosomal DNA making up the genome of the host cell or microorganism. For example, the exogenous or heterologous polynucleotide may be maintained on an episomal element, such as a plasmid. Alternatively or onally, the exogenous or heterologous polynucleotide may become integrated into a chromosome so that it is inherited by daughter cells through 17746603_1 (GHMatters) P107943.NZ 14 04/06/2021 chromosomal replication. Methods for ormation e, but are not limited to, calcium ate precipitation; Ca2+ treatment; fusion of recipient cells with bacterial protoplasts ning the recombinant nucleic acid; treatment of the recipient cells with liposomes containing the recombinant nucleic acid; DEAE dextran; fusion using polyethylene glycol (PEG); electroporation; magnetoporation; biolistic delivery; retroviral infection; lipofection; and micro-injection of DNA directly into cells.

The term transformed, as used in reference to cells, refers to cells that have one transformation as described herein such that the cells carry ous or heterologous c material (e.g., a recombinant nucleic acid). The term transformed can also or alternatively be used to refer to microorganisms, strains of microorganisms, tissues, organisms, etc. that contain exogenous or heterologous genetic material.

The term introduce, as used herein with reference to uction of a c acid into a cell or organism, is intended to have its broadest meaning and to encompass introduction, for example by transformation methods (e.g., calcium-chloride-mediated transformation, electroporation, particle bombardment), and also introduction by other methods including transduction, conjugation, and mating. ally, a construct is utilized to introduce a nucleic acid into a cell or organism.

The microorganisms for use 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, xanthophyls (e.g., oxycarotenoids), hydrocarbons, and other lipids known to one of ordinary skill in the art.

Optionally, the lipid compounds include unsaturated lipids. The unsaturated lipids can include polyunsaturated lipids (i.e., lipids containing at least 2 unsaturated carbon-carbon bonds, e.g., double bonds) or highly unsaturated lipids (i.e., lipids containing 4 or more unsaturated carbon-carbon bonds). Examples of unsaturated lipids include omega-3 and/or omega-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 nds.

Provided herein are recombinant microorganisms engineered to express polypeptides for lizing C5 carbon sugars such as xylose. Specifically, provided is a inant microorganism having one or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid encoding xylose isomerase is a exogenous c acid. Optionally, the recombinant microorganism comprises two or more copies of the nucleic acid sequence encoding xylose isomerase. Optionally, the recombinant 17746603_1 (GHMatters) P107943.NZ 15 04/06/2021 microorganisms also contains one or two copies of an endogenous nucleic acid ce encoding xylose isomerase. By way of example, the recombinant microorganisms can contain one or two copies of an endogenous c acid sequence encoding xylose ase and one copy of an exogenous nucleic acid sequence encoding xylose isomerase. Optionally, the recombinant rganism includes three copies of a nucleic acid sequence encoding xylose isomerase, one being exogenously introduced and the other two being nous.

The term recombinant when used with reference to a cell, nucleic acid, polypeptide, vector, or the like indicates that the cell, nucleic acid, polypeptide, vector or the like has been modified by or is the result of laboratory methods and is non-naturally occurring. Thus, for example, recombinant microorganisms include microorganisms produced by or modified by laboratory methods, e.g., transformation methods for introducing nucleic acids into the microroganism. Recombinant microorganisms can include nucleic acid sequences not found within the native (non-recombinant) form of the microroganisms or can include nucleic acid sequences that have been modified, e.g., linked to a non-native promoter.

As used herein, the term exogenous refers to a substance, such as a nucleic acid (e.g., nucleic acids including regulatory sequences and/or genes) or polypeptide, that is cially uced into a cell or organism and/or does not naturally occur in the cell in which it is present. In other words, the substance, such as nucleic acid or polypeptide, ates from outside a cell or organism into which it is introduced. An exogenous c acid can have a tide ce that is identical to that of a nucleic acid naturally present in the cell. For example, a Thraustochytrid cell can be engineered to include a nucleic acid having a Thraustochytrid or tochytrium regulatory sequence. In a particular example, an endogenous Thraustochytrid or Thraustochytrium regulatory sequence is operably linked to a gene with which the regulatory sequence is not involved under natural conditions. Although the tochytrid or Thraustochytrium regulatory sequence may naturally occur in the host cell, the uced nucleic acid is exogenous according to the present disclosure. An exogenous nucleic acid can have a nucleotide sequence that is different from that of any nucleic acid that is naturally present in the cell. For example, the exogenous nucleic acid can be a heterologous nucleic acid, i.e., a nucleic acid from a different species or organism. Thus, an exogenous nucleic acid can have a nucleic acid sequence that is identical to that of a nucleic acid that is naturally found in a source organism but that is different from the cell into which the exogenous nucleic acid is uced. As used herein, the term endogenous, refers to a nucleic acid sequence that is native to a cell. As used herein, the term heterologous refers to a c acid sequence that is not native to a cell, i.e., is from a different organism 17746603_1 (GHMatters) P107943.NZ 16 04/06/2021 than the cell. The terms exogenous and endogenous or logous are not mutually exclusive. Thus, a nucleic acid sequence can be exogenous and endogenous, meaning the nucleic acid sequence can be introduced into a cell but have a sequence that is the same as or similar to the sequence of a nucleic acid naturally present in the cell. Similarly, a nucleic acid sequence can be exogenous and heterologous meaning the nucleic acid sequence can be introduced into a cell but have a sequence that is not native to the cell, e.g., a sequence from a different organism.

As discussed above, the provided recombinant microorganisms n at least two copies of a nucleic acid sequence encoding a xylose isomerase. The provided microorganisms optionally also contain at least one c acid sequence encoding a xylulose kinase. Optionally, the recombinant microorganisms comprise at least one nucleic acid sequence encoding a xylose orter. The nucleic acid sequences encoding the xylose isomerase, xylulose , and/or xylose transporter are, optionally, exogenous nucleic acid sequences. Optionally, the nucleic acid sequence encoding the xylose ase is an endogenous nucleic acid sequence. Optionally, the nucleic acid sequence encoding the xylulose kinase and/or xylose transporter is a heterologous nucleic acid. Optionally, the microorganism contains at least two copies of a nucleic acid sequence encoding a xylose isomerase, at least two copies of a nucleic acid sequence encoding a xylulose kinase, and at least one nucleic acid sequence encoding a xylose transporter. Optionally, the heterologous nucleic acid ce encoding the xylose isomerase is at least 90% identical to SEQ ID NO:2. Optionally, the heterologous nucleic acid sequence ng the xylulose kinase is at least 90% identical to SEQ ID NO:5. As noted above, optionally, the nucleic acid encoding the xylose transporter is a heterologous nucleic acid. ally, the xylose transporter encoded by the heterologous nucleic acid is GXS1 from Candida intermedia. ally, the heterologous nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23.

The provided recombinant microorganisms not only contain nucleic acid ces encoding genes ed in xylose metabolism, they can include multiple copies of such sequences. Thus, the microorganism ses 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 ce 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, , 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 xylulose kinase. Optionally, the microorganism 17746603_1 (GHMatters) 3.NZ 17 04/06/2021 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, , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the c acid sequence encoding the xylose transporter.

In the provided microorganisms, the nucleic acids, e.g., xylose isomerase, xylulose kinase or xylose transporter can be operably linked to a promoter and/or terminator.

Optionally, the exogenous nucleic acid sequence encoding the xylose isomerase is operably linked to a promoter. Optionally, the nucleic acid ce ng the xylulose kinase and/or the nucleic acid sequence encoding the xylose transporter are also operably linked to a er. 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 exogenous c acid sequence encoding the xylose isomerase comprises a terminator. Optionally, the nucleic acid sequence encoding the xylulose kinase comprises a terminator. Optionally, the nucleic acid sequence encoding the xylose transporter comprises a terminator. Optionally, the terminator is a n terminator. Optionally, the terminator is at least 80% identical to SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30.

The provided microorganisms can include a selectable marker to confirm transformation of genes of interest. Thus, the microorganism can further include a selectable marker. Optionally, the selectable marker is an antibiotic resistance gene. Optionally, the otic is zeocin, ycin B, kanamycin or neomycin. Optionally, the microorganism is either a Thraustochytrium or a Schizochytrium microorganism. ally, the microorganism is ONC-T18.

The provided microorganisms have distinguishing features over wild type microorganisms. For e, the inant microorganisms can have increased xylose transport activity as compared to a non-recombinant control (or wild type) microorganism, increased xylose isomerase activity as ed to a non-recombinant control (or wild type) microorganism, sed xylulose kinase activity as compared to a non-recombinant control (or wild type) microorganism, or any combination of these activities. Optionally, the recombinant rganism grows with xylose as the sole carbon source.

Also provided are methods of making the recombinant microorganisms. Thus, provided is a method of making a recombinant xylose-metabolizing microorganism including providing one or more c acid constructs comprising a nucleic acid sequence encoding a xylose ase, a nucleic acid sequence encoding a xylulose kinase and a nucleic acid sequence encoding a xylose transporter; transforming the microorganism with the one or more nucleic acid constructs; and isolating microorganisms comprising at least two copies of 17746603_1 ters) P107943.NZ 18 04/06/2021 the nucleic acid sequences encoding the xylose isomerase. Optionally, the methods further include isolating microorganisms comprising at least two copies of the nucleic acid ce encoding the xylulose kinase. Optionally, the method es isolating microorganisms comprising at least one copy of the xylose transporter. Optionally, the one or more nucleic acid constructs further comprise a selectable marker.

In the provided methods, the nucleic acid sequences encoding the xylose isomerase, xylulose kinase and xylose transporter can be d on the same or different constructs.

Optionally, the method includes ing a first nucleic acid construct comprising a nucleic acid sequence encoding a xylose isomerase, a second nucleic acid construct sing a nucleic acid sequence encoding a xylulose kinase and a third nucleic acid construct comprising a nucleic acid sequence encoding a xylose transporter. Optionally, the first, second and third nucleic acid constructs comprise the same selectable marker. Optionally, the first nucleic acid construct comprises a er, a selectable marker, a nucleic acid sequence encoding a 2A peptide, the nucleic acid ce ng the xylose isomerase, and a terminator. Optionally, the second nucleic acid construct comprises a promoter, selectable marker, a nucleic acid sequence encoding a 2A peptide, the nucleic acid sequence encoding the xylulose kinase, and a terminator. Optionally, the third nucleic acid construct comprises a promoter, the nucleic acid sequence encoding the xylose transporter, a nucleic acid sequence encoding a 2A peptide, a selectable marker, and a terminator. As noted above, selectable markers include, but are not limited to, antibiotic resistance genes. Optionally, the antibiotic is zeocin, hygromycin B, kanamycin or neomycin. Promoters used for the constructs include, but are not limited to, a tubulin promoter. Optionally, the er is at least 80% identical to SEQ ID NO:25 or SEQ ID NO:26. Terminators used for the constructs e, but are not limited to, 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.

In the provided methods, the isolated recombinant microorganisms can include one or more copies of the xylose isomerase, xylulose kinase and xylose transporter. Optionally, the isolated recombinant microorganism comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, , 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 c acid sequence encoding xylose ase. Optionally, the xylose isomerase is an endogenous xylose isomerase or a heterologous xylose isomerase.

Optionally, the c acid sequence encoding the xylose isomerase is at least 90% identical to SEQ ID NO:2. Optionally, the isolated recombinant microorganism ses 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, 17746603_1 ters) P107943.NZ 19 04/06/2021 , 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 copies of the nucleic acid sequence encoding the xylulose kinase. Optionally, the se kinase is a heterologous xylulose kinase.

Optionally, the c acid sequence encoding the 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 the xylose transporter. Optionally, the xylose transporter is a logous xylose transporter. ally, the xylose transporter is GXS1 from a intermedia. Optionally, the nucleic acid sequence encoding the xylose transporter is at least 90% identical to SEQ ID NO:23.

Optionally, the microorganism is either a Thraustochytrium or a Schizochytrium microorganism. Optionally, the rganism is ONC-T18.

As noted above, the isolated recombinant microorgansims can have increased xylose transport activity as compared to a control non-recombinant microorganism, increased xylose isomerase activity as ed to a control combinant microorganism, increased xylulose kinase activity as compared to a control non-recombinant microorganism, or a combination thereof. Optionally, the isolated recombinant microorganism grows with xylose as the sole carbon .

As described herein, a control or standard control refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test rganism, e.g., a microorganism transformed with c acid sequences encoding genes for metabolizing xylose can be compared to a known normal (wild-type) microorganism (e.g., a standard control rganism). A standard control can also represent an average measurement or value gathered from a population of microorganisms (e.g., standard control microorganisms) that do not grow or grow poorly on xylose as the sole carbon source or that do not have or have minimal levels of xylose isomerase activity, xylulose kinase activity and/or xylose transport activity. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g., RNA levels, polypeptide levels, specific cell types, and the like). ed herein are also s of producing oil using the recombinant microorganisms. The method includes providing the recombinant microorganism, wherein the microorganism grows on xylose as the sole carbon source, and culturing the microorganism in a culture medium under suitable conditions to produce the oil. Optionally, the oil comprises triglycerides. ally, the oil comprises alpha linolenic acid, 17746603_1 (GHMatters) P107943.NZ 20 04/06/2021 arachidonic acid, docosahexanenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, nic acid, or a ation thereof. ally, the method further includes isolating the oil.

The provided s include or can be used in conjunction with additional steps for culturing microorganisms according to methods known in the art. For example, a Thraustochytrid, e.g., a Thraustochytrium sp., can be cultivated ing to methods described in U.S. Patent Publications 2009/0117194 or 2012/0244584, which are herein incorporated by reference in their entireties for each step of the methods or composition used therein.

Microorganisms are grown in a growth medium (also known as culture medium).

Any of a variety of medium can be suitable for use in culturing the microorganisms described herein. Optionally, the medium supplies s nutritional components, including a carbon source and a nitrogen source, for the microorganism. Medium for Thraustochytrid culture can include any of a variety of carbon s. Examples of carbon sources include fatty acids, lipids, glycerols, triglycerols, carbohydrates, polyols, amino , and any kind of 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, n, starch (corn or wheat), acetate, m-inositol (e.g., derived from corn steep liquor), galacturonic acid (e.g., derived from pectin), L-fucose (e.g., derived from galactose), biose, glucosamine, alpha-D-glucose- 1-phosphate (e.g., derived from glucose), cellobiose, n, cyclodextrin (e.g., derived from starch), and e (e.g., from molasses). Polyols include, but are not d to, maltitol, erythritol, and adonitol. Amino sugars include, but are not limited to, N-acetyl- D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl-beta-D-mannosamine.

Optionally, the microorganisms provided herein are cultivated under conditions that increase biomass and/or production of a compound of interest (e.g., oil or total fatty acid (TFA) content). Thraustochytrids, for example, are typically cultured in saline medium. ally, Thraustochytrids can be cultured in medium having a salt tration from about 0.5 g/L to about 50.0 g/L. Optionally, Thraustochytrids are cultured in medium having a salt concentration from about 0.5 g/L to about 35 g/L (e.g., from about 18 g/L to about 35 g/L). Optionally, the Thraustochytrids described herein can be grown in low salt conditions.

For example, the Thraustochytrids can be cultured in a medium having a salt concentration from about 0.5 g/L to about 20 g/L (e.g., from about 0.5 g/L to about 15 g/L). The culture 17746603_1 (GHMatters) P107943.NZ 21 04/06/2021 medium optionally includes NaCl. Optionally, the medium includes natural or artificial sea salt and/or cial seawater.

The culture medium can include non-chloride-containing sodium salts as a source of sodium. Examples of non-chloride sodium salts suitable for use in accordance with the present methods include, but are not limited to, soda ash (a mixture of sodium carbonate and sodium oxide), sodium ate, sodium bicarbonate, sodium sulfate, and mixtures thereof.

See, e.g., U.S. Pat. Nos. 5,340,742 and 6,607,900, the entire contents of each of which are incorporated by reference herein. A significant portion of the total sodium, for example, can be supplied by loride salts such that less than about 100%, 75%, 50%, or 25% of the total sodium in culture medium is supplied by sodium chloride.

Medium for Thraustochytrids culture can include any of a variety of nitrogen sources.

Exemplary en sources include ammonium solutions (e.g., NH4 in H2O), um or amine salts (e.g., (NH4)2SO4, (NH4)3PO4, NH4NO3, H2CH3 (NH4Ac)), peptone, tryptone, yeast extract, malt extract, fish meal, sodium glutamate, soy extract, casamino acids and distiller grains. Concentrations of nitrogen sources in suitable medium typically range between and including about 1 g/L and about 25 g/L.

The medium optionally includes a phosphate, such as potassium phosphate or sodiumphosphate.

Inorganic salts and trace nutrients in medium can include ammonium e, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenous acid, nickel sulfate, copper sulfate, zinc sulfate, cobalt chloride, iron chloride, manganese chloride calcium chloride, and EDTA. Vitamins such as pyridoxine hydrochloride, ne hydrochloride, calcium pantothenate, p-aminobenzoic acid, riboflavin, nic acid, biotin, folic acid and vitamin B12 can be included.

The pH of the medium can be adjusted to between and including 3.0 and 10.0 using acid or base, where appropriate, and/or using the nitrogen source. Optionally, the medium can be ized.

Generally a medium used for culture of a microorganism is a liquid medium.

However, the medium used for culture of a microorganism can be a solid medium. In addition to carbon and nitrogen sources as discussed herein, a solid medium can contain one or more components (e.g., agar or agarose) that provide structural support and/or allow the medium to be in solid form.

Optionally, the resulting biomass is rized to inactivate undesirable substances present in the biomass. For example, the biomass can be pasteurized to inactivate compound degrading substances. The biomass can be t in the tation medium or isolated 17746603_1 (GHMatters) P107943.NZ 22 04/06/2021 from the fermentation medium for the pasteurization step. The pasteurization step can be performed by heating the biomass and/or fermentation medium to an elevated ature.

For example, the biomass and/or fermentation medium can be heated to a temperature from about 50°C to about 95°C (e.g., from about 55°C to about 90°C or from about 65°C to about 80°C). Optionally, the s and/or fermentation medium can be heated from about 30 minutes to about 120 minutes (e.g., from about 45 minutes to about 90 minutes, or from about 55 minutes to about 75 minutes). The pasteurization can be performed using a suitable heating means, such as, for e, by direct steam injection.

Optionally, no pasteurization step is performed. Stated differently, the method taught herein optionally lacks a pasteurization step.

Optionally, the biomass can be harvested according to a variety of methods, including those currently known to one skilled in the art. For example, the biomass can be collected from the fermentation medium using, for example, centrifugation (e.g., with a solid-ejecting centrifuge) or filtration (e.g., cross-flow filtration). Optionally, the harvesting step es use of a precipitation agent for the accelerated collection of cellular biomass (e.g., sodium phosphate or calcium chloride).

Optionally, the biomass is washed with water. Optionally, the biomass can be concentrated up to about 20% solids. For example, the biomass can be concentrated to about % to about 20% solids, from about 7.5% to about 15% solids, or from about solids to about % solids, or any percentage within the recited ranges. Optionally, the biomass can 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 % 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.

The provided methods, optionally, include isolating the polyunsaturated fatty acids from the s or microorganisms. Isolation of the saturated fatty acids can be performed using one or more of a variety of methods, ing those currently known to one of skill in the art. For example, methods of isolating polyunsaturated fatty acids are bed in U.S. Patent No. 8,163,515, which is incorporated by reference herein in its entirety. Optionally, the medium is not sterilized prior to isolation of the polyunsaturated fatty acids. ally, sterilization comprises an increase in temperature. Optionally, the saturated fatty acids produced by the microorganisms and isolated from the provided 17746603_1 ters) P107943.NZ 23 04/06/2021 methods are medium chain fatty acids. ally, the one or more saturated fatty acids are selected from the group consisting of alpha linolenic acid, arachidonic acid, docosahexanenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic acid, and combinations thereof.

Oil including polyunsaturated fatty acids (PUFAs) and other lipids produced according to the method described herein can be utilized in any of a variety of applications exploiting their biological, nutritional, or al properties. Thus, the provided methods optionally include isolating oil from the harvested portion of the threshold volume.

Optionally, the oil is used to e fuel, e.g., biofuel. Optionally, the oil can be used in pharmaceuticals, food supplements, animal feed additives, cosmetics, and the like. Lipids ed according to the methods described herein can also be used as intermediates in the production of other compounds.

By way of e, the oil produced by the microorganisms cultured using the provided methods can comprise fatty acids. Optionally, the fatty acids are selected from the group ting of alpha nic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic acid, and ations thereof. Optionally, the oil comprises triglycerides. Optionally, the oil comprises fatty acids selected from the group consisting of palmitic acid (C16:0), myristic acid (C14:0), palmitoleic acid (C16:1(n-7)), cis-vaccenic acid (C18:1(n-7)), docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and combinations Optionally, the lipids produced according to the methods described herein can be incorporated into a final product (e.g., a food or feed supplement, an infant formula, a pharmaceutical, a fuel, etc.). Suitable food or feed supplements into which the lipids can be incorporated include beverages such as milk, water, sports drinks, energy drinks, teas, and juices; confections such as candies, jellies, and biscuits; fat-containing foods and beverages such as dairy products; processed food products such as soft rice (or porridge); infant formulae; ast cereals; or the like. Optionally, one or more ed lipids can be incorporated into a dietary supplement, such as, for example, a vitamin or multivitamin.

Optionally, a lipid produced according to the method described herein can be included in a dietary supplement and optionally can be directly incorporated into a component of food or feed (e.g., a food supplement).

Examples of feedstuffs into which lipids produced by the methods described herein can be incorporated include pet foods such as cat foods; dog foods; feeds for aquarium fish, 17746603_1 ters) 3.NZ 24 04/06/2021 cultured fish or crustaceans, etc.; feed for aised animals (including livestock and fish or crustaceans raised in aquaculture). Food or feed material into which the lipids produced according to the methods described herein can be incorporated is ably ble to the organism which is the intended recipient. This food or feed al can have any physical properties currently known for a food material (e.g., solid, liquid, soft).

Optionally, one or more of the produced compounds (e.g., PUFAs) can be incorporated into a nutraceutical or pharmaceutical product. Examples of such a euticals or pharmaceuticals e various types of tablets, capsules, drinkable agents, etc. Optionally, the nutraceutical or pharmaceutical is suitable for topical application.

Dosage forms can include, for example, capsules, oils, granula, a subtilae, pulveres, tabellae, pilulae, trochisci, or the like.

The oil or lipids produced according to the s described herein can be incorporated into products as described herein in combination with any of a variety of other agents. For instance, such compounds can be combined with one or more binders or fillers, chelating agents, pigments, salts, surfactants, moisturizers, viscosity modifiers, thickeners, emollients, fragrances, preservatives, etc., or any combination thereof.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other als are disclosed herein, and it is understood that when combinations, s, interactions, groups, etc. of these als are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of cations that can be made to a number of molecules including the method are discussed, each and every combination and ation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. se, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this sure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the sed methods, and that each such combination or subset of combinations is specifically contemplated and should be ered disclosed. 17746603_1 (GHMatters) P107943.NZ 25 04/06/2021 Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

The es below are intended to further illustrate certain aspects of the s and compositions described herein, and are not intended to limit the scope of the claims.

Example 1. C5 Carbon Metabolism by Recombinant tochytrids In nature, two xylose metabolism pathways exist, the xylose reductase/xylitol dehydrogenase pathway and the xylose isomerase/xylulose kinase pathway (Figure 1). ONCT18 encodes genes from both pathways, and, as described above, the xylose reductase/xylitol dehydrogenase y is dominant, as evidenced by a up of xylitol when grown in a xylose medium. Since the isomerase/kinase y does not depend on redox co-factors, over-expression of ONC-T18’s isomerase gene removes tor dependence in the conversion of xylose to xylulose. As shown herein in Figures 2 and 3, transcriptomic studies with ONC-T18 showed that its xylose isomerase and putative xylulose kinase genes were mostly expressed during glucose starvation; whereas, the putatively identified genes encoding for the xylose reductase and xylitol dehydrogenase were constitutively expressed.

T18 isomerase was purified by metal-affinity chromatography following gging and over-expression in yeast INVSc1. As a positive control, his-tagged XylA from E. coli strain W3110 was over-expressed and purified from E. coli strain BL21(DE3)plysS. The protein concentration of purified proteins was determined by a standard Bradford assay. The impact of temperature on the activity of T18 isomerase and E. coli isomerase was determined using 5 µg of protein and 0.75 g/L of either xylose or xylulose in 5mM MgATP, 50mM Hepes (pH 7.4), 10 mM MgCl2. ons were incubated overnight at 10 °C, 25 °C, 30 °C, 37 °C, 50 °C, 60 °C, and 80 °C. Reactions were stopped by heat vation at 95 ˚C for 5 mins. Reactions were analyzed by HPLC and the concentration of the sugars present was determined from the area under the peak relative to a rd curve. T18 isomerase had higher activity on both xylose and xylulose at temperatures at and above 37 °C (Figure 20A).

This is in contrast to E. coli ase, which had higher activity at temperatures between 25 °C and 30°C (Figure 20B).

Dose-dependency was determined by incubating increasing protein concentrations of the isomerase with 0.75 g/L xylose or xylulose in 5mM MgATP, 50mM Hepes (pH 7.4), mM MgCl2. Reactions were incubated overnight at 30˚C (E. coli) or 50˚C (T18) then stopped by heat inactivation at 95 ˚C for 5 mins. Reactions were analyzed by HPLC and the concentration of the sugars present was ined from the area under the peak relative to a 17746603_1 (GHMatters) P107943.NZ 26 04/06/2021 standard curve. Observed was a dose ency of T18 isomerase on both xylose and se (Figures 21A and 21B).

This example describes the use of a Thraustochytrium ONC-T18-derived 8 ) alpha-tubulin promoter to express endogenous and/or heterologous xylose metabolism transgenes in Thraustochytrid species, including ONC-T18. However, as discussed throughout, other regulatory elements can be used. Figures 4 and 5 show constructs of the plasmids containing the xylose isomerase and xylulose kinase genes, respectively. As described herein, the xylose metabolism transgenes were present in multiple (≥8) copies within the genome of the host. In the case of 8, the modified organisms demonstrated an sed metabolism of xylose compared to wild-type (WT) cells. For example, a strain ed to express an endogenous xylose isomerase gene (SEQ ID NO:2) (strain s #16) and a strain ed to express an endogenous xylose isomerase gene (SEQ ID NO:2) and a se kinase gene (SEQ ID NO:5) (Iso-His+xylB, strain 7-7) both used 40% more xylose than the WT strain. Both Iso-His #16 and 7-7 converted less xylose to xylitol than the WT strain, 40% less and 420% less, respectively. The constructs used for transformation of ONC-T18 are shown in Figures 4 and 5. ONC-T18 tranformants were d using standard biolistics protocols as described by BioRad’s Biolistic PDS-1000/He Particle Delivery System (Hercules, CA). Briefly, 0.6 µm gold particles were coated with 2.5 µg of linerized plasmid DNA , 37˚C, overnight). The coated gold particles were used to bombard plates previously spread with 1 ml of ONC-T18 cells at an OD600 of 1.0. The bombardment parameters included using a helium pressure of 1350 or 1100 psi with a target distance of 3 or 6 cm. After an overnight recovery, the cells were washed off the plate and plated on media containing selection antibiotics (Zeo 250 µg/mL and hygro 400 µg/mL).

Plates were incubated for 1 week at 25˚C to identify resistant colonies. The resulting transformants were screened by PCR and Southern blot. rn blots were performed using standard protocols. Briefly, approximately 20 µg of genomic DNA were digested with 40 units of BamHI restriction enzyme in a total volume of 50 µL overnight at 37˚C. 7.2 µg of each digested sample was run on a 1.0% agarose gel at 50V for approximately 1.5. hours, with a digoxigenin (DIG) DNA molecularweight marker II (Roche, Basel, Switzerland). DNA was depurinated in the gel by submerging the gel 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) for two 15 minute washes. The on was then neutralized by incubation in 0.5 M Cl (pH 7.5) for two minute washes. y, the gel was equilibrated in 20X saline-sodium citrate (SSC) buffer 17746603_1 (GHMatters) P107943.NZ 27 04/06/2021 for 15 minutes. DNA was transferred to a positively charged nylon membrane using a standard transfer apparatus. DNA was fixed to the membrane using a UV Stratalinker at an exposure of 120,000 µJ. Southern blot probe was generated using a PCR DIG Probe Synthesis Kit (Roche, Basel, Switzerland) to generate a DIG-labelled probe according to the manufacturer’s instructions. The DNA affixed to the nylon ne was prehybridized with 20 mL of DIG EasyHyb solution (DIG EasyHyb Granules, Roche, Basel, Switzerland).

The belled probe was red by adding 40 µL of the ble-probe reaction mixture to 300 µL of ddH2O and incubated at 99˚C for 5 minutes. This solution was then added to 20 mL of DIG hybridization solution to create the probe solution. The probe solution was then added to the DNA-affixed nylon membrane and incubated at 53 ˚C overnight. The following day, the membrane was washed twice in 2X SSC, 0.1% SDS at room ature. The membrane was further washed twice in 0.1X SSC, 0.1% SDS at 68˚C for 15 minutes. For ion, the membrane was washed and blocked using DIG Wash and Block Buffer set (Roche, Basel, Switzerland) according to the manufacturer’s instructions. An anti-DIG-AP conjugated dy from a DIG Nucleic Acid Detection Kit (Roche, Basel, rland) was used for detection. 2 µL of the antibody solution was added to 20mL detection solution and incubated with the membrane at room temperature for 30 minutes. The blot was then immersed in a washing buffer provided with the kit. CDP-Star (Roche, Basel, Switzerland) was used for visualization. 10 µL of the CDP-star on was incubated on the membrane in 1 mL of detection solution, which was covered in a layer of ‘sheet-protector’ plastic to hold the on to the membrane. Signal was ately detected using a oc imaging system (BioRad Laboratories, Hercules, CA).

The codon optimized ble gene was cloned under the control of T18B α-tubulin promoter and terminator elements (Figure 6). The isomerase gene was cloned from T18B in such a way as to add a six-histidine tag on the N-terminus of the expressed protein (Iso-His).

Xylose isomerase tic activity was confirmed by over-expression and purification of the histidine-tagged protein in yeast. The isomerase gene (along with the introduced six- histidine tag) was cloned under the control of the α-tubulin promoter and terminators by g the gene downstream of the ble gene and a 2A sequence (Figure 4 and Figure 6).

Biolistic transformation of T18B with this plasmid (pALPHTB-B2G-hisIso) resulted in Zeocin (zeo) resistant transformants. Many transformant strains were obtained from this procedure. Two of these strains are shown as example #6 containing one copy of the transgene and e #16 containing eight copies of the transgene (Figure 7). 17746603_1 (GHMatters) P107943.NZ 28 04/06/2021 The insertion of the Iso-His transgene within the T18B genome was confirmed by PCR and Southern blot analysis (Figure 7). atively, these data showed the presence of a single copy of the transgene in strain #6 and multiple, concatameric, ene copies, at a single site, in strain #16. The precise number of Iso-His transgene insertions was determined by qPCR on c DNA (Figure 8). These data showed the presence of one copy of the transgene in strain #6 and eight copies of the transgene in strain #16 (Figure 8). To test whether an increase in copy number correlated with an increase in sion level, mRNA was isolated from WT, Iso-His #6 and Iso-His #16 T18B cells and qRT-PCR was performed.

Figure 11 shows significantly sed expression of the Iso-His transcript in strain #16 cells, ning eight copies of the transgene, compared to strain #6, containing a single copy of the transgene. No s transcript is detectable in WT cells (Figure 11). To assess whether increased mRNA expression correlated with increased isomerase enzymatic activity, cell extracts were harvested from WT, Iso-His #6 and s #16 cells. ed isomerase enzyme activity is observed in strain #16 cells compared with strain #6 and WT cells (Figure 12). Finally, the ability of strain #16 to metabolize xylose was examined in xylose depletion assays (Figure 14) and compared with WT cells. These flask fermentations demonstrated the ability to metabolise xylose and quantify the amount of xylose converted to xylitol. Thus, Figure 14 shows an increase in xylose metabolism in s strain #16 compared with WT cells and significantly less production of xylitol.

For flasks assays, cells were grown in media for 2 to 3 days. s were washed twice in Media 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) containing no sugar. Then, minimal media containing 20 g/L glucose & 50 g/L xylose was ated to an OD600 of 0.05 with the washed cells. s were taken at various time points and the amount of sugar remaining in the supernatant was analyzed by HPLC. As shown in Figures 22A, 22B, 22C and 22D, with increased xylose isomerase gene copy number, up to 40% more xylose usage and 20% decrease in xylitol production when compared to WT.

Iso-His strain #16 was then used as the parent strain for a second round of transformation to introduce the E. coli xylB gene. This gene was introduced under hygromycin (hygro) selection. The hygro gene from pChlamy_3, the 2A sequence, and the T18B codon optimized W3110 E. coli xylB gene were cloned under the control of the T18B α-tubulin promoter and terminator elements for expression in T18B iso-his #16 (Figure 5).

The in vitro ability of the E. coli xylulose kinase to work in concert with the T18B isomerase 17746603_1 (GHMatters) P107943.NZ 29 04/06/2021 was confirmed by over-expression and purification of the histidine-tagged proteins in yeast followed by enzymatic reactions with xylose and xylulose. Biolistic ormation of T18B iso-his strain #16 with the xylB plasmid (pJB47) resulted in hygro and zeo resistant transformants. The insertion of the hygro-2A-xylB genes within the T18B genome was confirmed by PCR and Southern blot analysis (Figure 9). Qualitatively, these data show the ce of a single copy of the transgene in strain #7-3 and multiple, concatameric, transgene copies, at a single site, in strain #7-7. The number of xylB gene insertions was determined by qPCR on genomic DNA isolations (Figure 10). Figure 10 shows sixteen ions of the transgene in strain 7-7 and one copy in strain 7-3. To determine whether le copies of the transgene confer enhanced xylose metabolism in vitro, cell extract assays were performed and the ability of the cells extracts to metabolise xylose was analysed e 13). The ability of the ormant cells to metabolize xylose was examined through flask-based xylose depletion assays (Figure 15). In this ment, WT cells consumed the least amount of xylose and made the most xylitol. Strain 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 make significant amounts of xylitol. Finally, strains Iso-His #16 and 7-7 were tested at in 5L fermentation vessels in media containing glucose and . During a seventy-seven (77) hour fermentation, strain Iso-His #16 ted approximately 8% of xylose to xylitol, whereas strain 7-7 converted approximately 2% of xylose to xylitol. Xylitol accumulation in this fermentation is shown in Figure 16.

For flasks assays, cells were grown in media for 2 to 3 days. Pellets were washed twice in media containing no sugar. Media containing 20 g/L: 50 g/L glucose: xylose was inoculated to an OD600 of 0.05 with the washed cells. s were taken at various time points and the amount of sugar ing in the supernatant was analyzed by HPLC. As shown in Figures 23A, 23B, 23C and 23D, up to 50% more xylose was used and an 80% reduction in xylitol was ed in strains over-expressing both a xylose isomerase and a xylulose kinase when compared to WT.

To further analyze these strains, the strains were grown in el 5L Sartorius fermenters. Initial media contained 20g/L Glucose and 50g/L xylose along with other basal media ents. Both cultures were maintained at 28°C and 5.5 pH, with constant mixing at 720 RPM and constant aeration at 1 Lpm of environmental air. The cultures were fed glucose for 16 hrs followed by 8hr starvation period. This cycle was completed 3 times.

During starvation periods, 10mL s were taken every 0.5 hr. Glucose, xylose and xylitol concentrations were quantified in these samples by HPLC. Larger 50mL samples were 17746603_1 (GHMatters) P107943.NZ 30 04/06/2021 taken ically for r biomass and oil content quantification. e feed rates d glucose ption rates, which was quantified by CO2 detected in the culture exhaust gas. As shown in Figure 24, the 7-7 strain used up to 52% more xylose than WT under these conditions.

By Southern blot analysis, it was observed that strain Iso-His #16 contains eight (8) insertions of the isomerase transgene (Figure 8). This unexpected multiple insertion resulted in an increase in isomerase gene expression relative to strains harbouring a single copy (Figure 11) as well as increased isomerase in vitro activity (Figure 12). Strain s #16 demonstrated increased xylose productivity than strains harbouring a single copy of the isomerase transgene (Figure 14).

Similarly, within the Iso-His + xylB transformants, one of the clones (Iso-His + xylB 7-7) also had multiple insertions of the xylB gene (Figure 10), which resulted in increased in vivo activity of both the xylose isomerase and xylulose kinase within the cell (Figure 13).

This clone was capable of using either as much or more xylose than the parental strain, Iso- His #16, while producing significantly less xylitol (Figure 15). rmore the Iso-His + xylB 7-7 produced more biomass than WT in the presence of xylose. These two strains showed that, not only is the presence of both the isomerase and the kinase genes important, but the number of insertions is as well.

To further optimize the iso-his & xylB containing “7-7” strain, this strain was transformed with a xylose transporter. Figure 17 shows exemplary constructs for transformation. Examples of xylose transporters to be used e, but are not limited to, At5g17010 and At5g59250 dopsis na), Gfx1 and GXS1 (Candida), AspTx (Aspergillus), and Sut1 (Pichia). Gxs1 (SEQ ID NO:23) was selected for transformation.

The results are shown in Figures 19A, 19B, and 19C. The transformants 36-2, 36-9, and 36- 16, containing GXS1 use more xylose than 7-7 and WT strains. They also use glucose slower than WT and 7-7 strains. The data demonstrate both xylose and glucose being used in the earlier stages by the GXS1 containing strains. Further, the percent of xylitol made by the GXS1 containing strains is lower than both WT and 7-7 strains.

To further analyze the effect of sugar transporters on the metabolism of xylose, codon optimized xylose transporters AspTX from Aspergillus (An11g01100) and Gxs1 from a were introduced in the 7-7 strain (isohis + xylB). Figure 25 shows the alpha-tubulin aspTx-neo and alpha-tubulin gxs1-neo constructs. T18 transformants were created using standard biolistics protocols as described by BioRad’s tic PDS-1000/He le Delivery System. Briefly, 0.6 µm gold particles were coated with 2.5 µg of linearized 17746603_1 (GHMatters) 3.NZ 31 04/06/2021 d DNA (EcoRI, 37˚C, o/n), The coated gold particles were used to bombard WD plates previously spread with 1 ml of T18 cells at an OD600 of 1.0. The bombardment parameters included using a Helium pressure of 1350 or 1100 psi with a target distance of 3 or 6 cm.

After an overnight ry, the cells were washed off the plate and plated on media containing selection antibiotics (G418 at 2 mg/mL). Plates were incubated for 1 week at 25˚C to identify ant colonies. The resulting transformants were screened by PCR and Southern blot (Figure 26).

Southern blots were med using standard protocols. Briefly, approximately 20 µg of genomic DNA were digested with 40 units of BamHI restriction enzyme in a total volume of 50 µL o/n/ at 37˚C. 7.2 µg of each digested sample was run on a 1.0% agarose gel at 50V for approximately 1.5. hours, with a digoxigenin (DIG) DNA molecular-weight marker II (Roche). DNA was depurinated in the gel by submerging the gel in 250 mM HCl for 15 minutes. The gel was r denatured by incubation in a solution containing 0.5 M NaOH and 1.5 M NaCL (pH 7.5) for two 15 minute washes. The on was then neutralized by incubation in 0.5 M Tris-HCl (pH 7.5) for two 15 minute washes. Finally, the gel was equilibrated in 20X saline-sodium citrate (SSC) buffer for 15 minutes. DNA was transferred to a positively charged nylon membrane (Roche) using a standard er apparatus. DNA was fixed to the membrane using a UV Stratalinker at an exposure of 120,000 µJ. Southern blot probe was generated using a PCR DIG Probe Synthesis Kit (Roche) to generate a DIG-labelled probe ing to the manufacturer’s ctions. The DNA affixed to the nylon membrane was prehybridised with 20 mL of DIG EasyHyb solution (DIG EasyHyb Granules, Roche). The DIG-labelled probe was denatured by adding 40 µL of the ble-probe reaction mixture to 300 µL of ddH2O and incubated at 99˚C for 5 minutes. This solution was then added to 20 mL of DIG hybridization solution to create the probe solution. The probe solution was then added to the DNA-affixed nylon membrane and incubated at 53 ˚C overnight. The following day, the membrane was washed, twice, in 2X SSC, 0.1% SDS at RT. The membrane was further washed, twice, in 0.1X SSC, 0.1% SDS at 68˚C for 15 minutes. For detection, the membrane was washed and blocked using DIG Wash and Block Buffer set (Roche) according to the manufacturer’s instructions. An anti-DIG-AP conjugated antibody from a DIG Nucleic Acid Detection Kit (Roche) was used for detection. 2 µL of the antibody solution was added to 20mL detection solution and incubated with the membrane at RT for 30 minutes. The blot was then immersed in a washing buffer provided with the kit. CDP-Star ) was used for ization. 10 µL of the CDP-star solution was incubated on the ne in 1 mL of detection solution, which was covered in a layer 17746603_1 (GHMatters) P107943.NZ 32 04/06/2021 of ‘sheet-protector’ plastic to hold the on to the membrane. Signal was immediately detected using a ChemiDoc imaging system.

For flasks assays, cells were grown in media for 2 to 3 days. Pellets were washed twice in media 2 (9 g/L NaCl, 4 g/L MgSO4, 100 mg/L CaCl2, 5 mg/L FeCl3, 20 g/L SO4, 0.86 g/L KH2PO4, 150 µg/L n B12, 30 µg/L biotin, 6 mg/L thiamine hydrochloride, 1.5 mg/L cobalt (II) chloride, 3 mg/L manganese chloride) containing no sugar. Then, Media 2 containing 20 g/L Glucose and 20 g/L Xylose was inoculated to an OD600 of 0.05 with the washed cells. As shown in Figures 27A, 27B, 27C and 27D, the expression of the xylose isomerase, xylulose kinase, and either xylose transporters ed in up to 71% more xylose used and 40% less xylitol produced than the al strain 7-7.

For flasks assays, cells were grown in media for 2 to 3 days. Pellets were washed twice in saline. Then, media containing 60 g/L xylose instead of glucose was inoculated to an OD600 of 0.05 with the washed cells. Samples were taken at various time points and the amount of sugar remaining in the supernatant was ed by HPLC. Figure 28 shows T18 growth in media ning xylose as the main carbon source requires over-expression of both an isomerase and a kinase. The expression of the transporters in this background did not significantly increase xylose usage in this media.

Enhanced xylose usage by T18 7-7 and transporter strains was observed in media containing carbon from ative feed stocks. For flasks assays, cells were grown in media for 2 to 3 days. Pellets were washed twice in 0.9% saline solution. Media 2 containing 20 g/L glucose : 50 g/L xylose as a combination of lab grade glucose and glucose and xylose from an alternative feedstock from forestry, was inoculated to an OD600nm of 0.05 with the washed cells. Samples were taken at various time points and the amount of sugar remaining in the supernatant was analyzed by HPLC. As shown in s 29A and 29B, in media containing sugars from an alternative feedstock, the T18 7-7 strains encoding for transporters used more xylose than wild-type, or T18 7-7. 17746603_1 (GHMatters) P107943.NZ 33 04/06/2021

Claims (9)

What is Claimed

1. A recombinant Thraustochytrium microorganism comprising: a) two or more copies of a nucleic acid sequence encoding xylose isomerase, wherein the nucleic acid ng xylose isomerase is an exogenous nucleic acid; b) two or more copies of a nucleic acid sequence encoding a xylulose kinase; and c) at least one c acid sequence encoding a xylose transporter, wherein the isomerase, kinase and transporter are integrated into the genome of the Thraustochytrium microorganism.

2. The recombinant Thraustochytrium microorganism of claim 1, wherein the nucleic acid sequence encoding the xylulose kinase is an exogenous nucleic acid sequence.

3. The recombinant Thraustochytrium microorganism of claim 1 or 2, wherein the nucleic acid ce encoding the xylose transporter is an exogenous nucleic acid sequence.

4. The recombinant Thraustochytrium microorganism of any one of claims 1-3, wherein the nucleic acid ce encoding the xylose isomerase is a heterologous nucleic acid sequence.

5. The recombinant Thraustochytrium microorganism of claim 4, wherein the heterologous c acid sequence encoding the xylose isomerase comprises SEQ ID NO:2.

6. The inant Thraustochytrium rganism of any one of claims 2-5, wherein the microorganism comprises at least 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 ce encoding the xylulose kinase.

7. The recombinant Thraustochytrium microorganism of any one of claims 3-6, wherein the nucleic acid sequence encoding the xylulose kinase is a heterologous nucleic acid sequence.

8. The recombinant Thraustochytrium microorganism of claim 7, wherein the heterologous c acid sequence encoding the xylulose kinase comprises SEQ ID NO:5.

9. The recombinant Thraustochytrium microorganism of any one of claims 3-8, 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 ng the xylose orter. 17746603_1 (GHMatters) P107943.NZ 34