NOVEL XYLOSE ISOMERASE GENE AND POLYPEPTIDE AND USES THEREOF

Abstract

The present invention provides for a novel D-xylose isomerase (XI) gene that is suitable for metabolic engineering of Saccharomyces cerevisiae for an improved consumption of D-xylose.

Claims

1. An isolated or purified D-xylose isomerase (XI) having a maximal velocity equal to or more than about three times that of Piromyces XI, or any one of the XI comprising SEQ ID NO:3-5.

2. The isolated or purified XI of claim 1, wherein the XI has an amino acid sequence having at least 80% sequence identity with SEQ ID NO:2.

3. The isolated or purified XI of claim 2, wherein the XI has an amino acid sequence having at least 85% sequence identity with SEQ ID NO:2.

4. The isolated or purified XI of claim 3, wherein the XI has an amino acid sequence having at least 90% sequence identity with SEQ ID NO:2.

5. The isolated or purified XI of claim 4, wherein the XI has an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2.

6. The isolated or purified XI of claim 5, wherein the XI has an amino acid sequence having at least 99% sequence identity with SEQ ID NO:2.

7. The isolated or purified XI of claim 6, wherein the XI has an amino acid sequence comprising SEQ ID NO:2.

8. The isolated or purified XI of claim 2, wherein the XI comprises the indicated conserved amino acid residues shown in FIG. 3 with an asterisk or a bar.

9. A nucleic acid comprising an open reading frame (ORF) encoding the XI of claim 1.

10. The nucleic acid of claim 9, wherein the ORF is codon optimized for a microbe

11. The nucleic acid of claim 10, wherein the ORF is codon optimized for expression in a Sacchromyces species.

12. The nucleic acid of claim 11, wherein the ORF is codon optimized for expression in Sacchromyces cerevisae.

13. The nucleic acid of claim 12, wherein the ORF comprises a nucleotide sequence of SEQ ID NO:1.

14. A vector comprising the nucleic acid of claim 9.

15. The vector of claim 14, wherein the vector is a plasmid or an expression vector.

16. A host cell comprising the vector of claim 14.

17. The host cell of claim 16, wherein the host cell is a Sacchromyces species.

18. The host cell of claim 17, wherein the host cell is a Sacchromyces cerevisae.

19. A method for producing a D-xylose isomerase (XI), the method comprising: (a) optionally providing a vector of claim 14, (b) introducing the vector into a host cell, (c) optionally culturing or growing the host cell in a culture medium such that the host cell expresses the XI, and (d) optionally separating the XI from the rest of the host cell.

20. A method for treating a biomass, the method comprising: providing a composition comprising a biomass and an isolated or purified XI of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0026] FIG. 1. Growth rates of Saccharomyces cerevisiae cultures in defined media containing xylose (20 g/L) as the sole carbon source. Data points represent an average of at least 3 biological replicates with standard error of the mean indicated.

[0027] FIG. 2. Specific enzymatic activity of Xylose Isomerase enzymes 8454_2 and opt.PiXI (codon optimized Piromyces sp. XI gene). Data points represent an average of at least 3 biological replicates with standard error of the mean indicated.

[0028] FIG. 3. The amino acid sequence of SEQ IDNO:2 is compared to the amino acid sequences of Piromyces species xylose isomerase encoded by xy1A (Accession No. Q9P8C9; SEQ ID NO:5). Residues underlined are believed to form a coiled coil structure. Residues indicated by a bar are conserved. Residues indicated by an asterisk are conserved and are believed to contain eight manganese (Mn.sup.2+) ligands possibly involved in regulating the catalytic activity of XI.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0030] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0031] The terms optional or optionally as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0032] The term about when applied to a value, describes a value that includes up to 10% more than the value described, and up to 10% less than the value described.

[0033] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0034] Carbohydrate rich substrates such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses such as glucose and galactose. For low value/high volume products, yield is of paramount importance for process economy. In one particular industrial organism Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and so far only thirteen genes have been reported to be active. A novel D-xylose isomerase is synthesized and cloned from microorganisms in the gut of Odontotaenius disjunctus, a wood-feeding beetle, that is identified through analysis of genes present in metagenome assemblies with XI functional predictions. Although sharing 79% homology with the XI from Piromyces sp., metagenome scaffold gene neighborhoods and metagenome binning identified the gene as bacterial in origin and the host as a Clostridium species. Expression of the new XI enzyme results in faster aerobic growth of S. cerevisiae with D-xylose as the sole carbon source. Maximal velocity of the new enzyme is three times higher than the one measured with the Piromyces sp. enzyme. In some embodiments, the new XI is a useful addition to the molecular toolbox for genetic modification of S. cerevisiae for the metabolism of second-generation substrates. The new XI exhibits a Km for D-xylose of 19 mM and three times higher isomerization maximal velocity (Vmax) than the XI from Piromyces sp. under identical biological backgrounds and experimental conditions.

[0035] The present invention provides for:

[0036] An isolated or synthesized polypeptide comprising an amino acid sequence at least 95% identical to a sequence from a list consisting of: SEQ ID NO: 2, as a yeast growth enhancer.

[0037] An isolated or synthesized polynucleotide encoding a polypeptide according to the isolated or synthesized polypeptide of the present invention, wherein the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 1, as a yeast growth enhancer.

[0038] The polynucleotide of the present invention, wherein the polynucleotide is a deoxyribonucleic acid or a ribonucleic acid molecule, namely mRNA, tRNA or rRNA molecule.

[0039] The polypeptide or polynucleotide of the present invention wherein the Saccharomyces growth enhancer is a Saccharomyces cerevisiae growth enhancer.

[0040] The polypeptide or polynucleotide of the present invention wherein said amino acid or nucleotide sequence, respectively, is 96%, 97%, 98% or 99% identical to said SEQ ID NO:1, SEQ ID NO:2, or mixtures thereof.

[0041] The polypeptide or polynucleotide of the present invention wherein said amino acid or nucleotide sequence, respectively, is 100% identical to said SEQ ID NO:1, SEQ ID NO:2.

[0042] Protein of the present invention, comprising the amino acid sequence is SEQ ID NO:2.

[0043] A composition comprising at least one sequence 95% identical to the sequence from a list consisting of: SEQ ID NO:1, SEQ ID NO:2, or mixtures thereof.

[0044] Vector comprising the DNA sequence of the present invention.

[0045] Plasmid comprising the vector of the present invention.

[0046] Host cell comprising an expression vector or the plasmid of the present invention, wherein the host cell is a yeast.

[0047] Saccharomyces, such as Saccharomyces cerevisiae, comprising an expression vector or the plasmid of the present invention.

[0048] Use of the polypeptide of the present invention as a metabolism booster, particularly by accelerating the growth of Saccharomyces cerevisiae.

[0049] Use of the Saccharomyces of the present invention as a fermentation improver or a bakery improver, such as a D-xylose consumption improver.

[0050] Use of the Saccharomyces of the present invention in the production of biofuel.

[0051] A nucleotide sequence encoding SEQ ID NO:2 is as follows:

TABLE-US-00003 (SEQIDNO:1) ATGACATACTTTCCCACAGTGGAGAAGATAAAATTTGAGGGCAAGGAGTC CAAGAATCCGCTGGCGTTTAGGTATTACGACCCCGAGAAGATGGTCTACG GTAAAAAGATGAAGGACTGGTTCAAATTTTCCATGGCCTGGTGGCATACT TTGTGTGCGGAGGGCGGCGACCCTTTCGGTGGGGGTACAAAAACGTTCCC ATGGGCACAAGGTAGCTCTGCCTTAGAGGTGGCGAAACAGCGTCTGGATG CCGGCTTTGAGTTTATGCAGAAAATAGGCATCGAGTATTACTGCTTCCAC GATATTGATTTGATCTCAGAAGGTGATAGTATCGAGGAATACGAAAGCAA CCTGAAAGCGATTGTGGCATACGCTAAGCAAAAACAAGCGGAAACGGGAA TAAAGCTTCTATGGGGCACAGCGAACGTTTTCAGTCATAAAAGGTACATG AACGGGGCCGCGACAAACCCGGACTTCGAAGTGGTTAGCAGGGCAGCGCT ACAGATAAAGAATGCAATTGACGCGACCATCGAGTTGGGTGGTGAGAACT ACGTCTTCTGGGGAGGTAGAGAGGGGTACTCTTCCCTGCTTAACACAGAG ATGAAGAGAGAAAAAGATCATCTTGCGACCATCTTAACCAAGGCAAGAGA TTATGCTCGTAGTAAAGGCTTTAAGGGCAACTTTCTAATTGAACCAAAAC CTATGGAGCCCACCAAACATCAATACGATGTCGATACAGAAACGGTGATA GGATTTTTAAGGGCGCATGGTCTGGATAAGGATTTTAAAGTGAACATAGA AGTTAATCACGCAACGTTGGCTGGGCATACCTTTGAGCATGAATTGCAAT GCGCCGTAGACGCCGGCATGCTAGGCTCAATAGACGCGAACAGGGGGGAC TATCAAAATGGTTGGGACACAGATCAGTTCCCCGTTGATGTGAATGAACT TACTCAAGCGATGCTTGTAATTTTGAAGGGCGGCGGCTTGCAGGGTGGTG GTACTAATTTCGATGCGAAGACAAGGCGTAACTCCACAGACTTAGAAGAT ATTTTTATTGCGCATATAGCCGGAATGGATACTTTCGCCCGTGCACTTGA GTCTGCGGCAGCGCTATTGGAAGACTCTCCGTACGAGAAGATGTTAAAGG ACAGATATGCGTCATTCGATGCCGGTAAAGGCAAGGAGTTTGAAGATGGG AAGCTATCACTTGAAGACATTGTAGCATATGCGAAGTCCAAAGGGGGCGA ACCGGCCCAAATCAGTGGTAAACAGGAGCTGTATGAGGCGCTGGTAAACA TGTATATCTAA.

[0052] An amino acid sequence of the XI of the present invention is as follows:

TABLE-US-00004 (SEQIDNO:2) MTYFPTVEKIKFEGKESKNPLAFRYYDPEKMVYGKKMKDWFKFSMAWWHT LCAEGGDPFGGGTKTFPWAQGSSALEVAKQRLDAGFEFMQKIGIEYYCFH DIDLISEGDSIEEYESNLKAIVAYAKQKQAETGIKLLWGTANVFSHKRYM NGAATNPDFEVVSRAALQIKNAIDATIELGGENYVFWGGREGYSSLLNTE MKREKDHLATILTKARDYARSKGFKGNFLIEPKPMEPTKHQYDVDTETVI GFLRAHGLDKDFKVNIEVNHATLAGHTFEHELQCAVDAGMLGSIDANRGD YQNGWDTDQFPVDVNELTQAMLVILKGGGLQGGGTNFDAKTRRNSTDLED IFIAHIAGMDTFARALESAAALLEDSPYEKMLKDRYASFDAGKGKEFEDG KLSLEDIVAYAKSKGGEPAQISGKQELYEALVNMYI.

[0053] The amino acid sequence of Bacteroides stercoris xylose isomerase is as follows:

TABLE-US-00005 (SEQIDNO:3) MATKEYFPGIGKIKFEGKESKNPMAFRYYDAEKVIMGKKMKDWLKFSMAW WHTLCAEGGDQFGGGTKHFPWNGDADKLQAAKNKMDAGFEFMQKMGIEYY CFHDVDLCDEADTIEEYEANLKAIVAYAKQKQEETGIKLLWGTANVFGHA RYMNGAATNPDFDVVARAAVQIKNAIDATIELGGSNYVFWGGREGYMSLL NTDQKREKEHLAQMLTIARDYARARGFKGTFLIEPKPMEPTKHQYDVDTE TVVGFLKAHGLDKDFKVNIEVNHATLAGHTFEHELAVAVDNGMLGSIDAN RGDYQNGWDTDQFPIDNYELTQAMMQIIRNGGFGDGGTNFDAKTRRNSTD LEDIFIAHIAGMDVMARALESAAKLLEESPYKKMLADRYASFDSGKGKEF EEGKLTLEDVVAYAKANGEPKQTSGKQELYEAIVNMYC.

[0054] The amino acid sequence of Porphyromonadaceae bacterium xylose isomerase is as follows:

TABLE-US-00006 (SEQIDNO:4) MATKTYFPTVEKIKFEGKESKNPLAFRYYDPEKVVYGKKMKEWFKFSMAW WHTLCAEGGDPFGGGTKTFPWTDGNSALEIAKQRMDAGFEFMQKIGIEYY CFHDIDLIDEGGSIEEYEANLKAIVAYAKQKQEETGIKLLWGTANVFGHK RYMNGAATNPDFDVVARAAVQIKNAIDATIELGGENYVFWGGREGYSSLL NTDMKREKEHLAAMLKAARDYARSKGFNGTFLIEPKPMEPTKHQYDVDAE TVIGFLRAHGLDKDFKLNIEVNHATLAGHTFEHELQCAADAGLLGSIDAN RGDYQNGWDTDQFPIDVNELTQAMLVILKSGGLQGGGTNFDAKTRRNSTD PEDIFIAHVAGMDAFARALEVAAAILENSPYQGMIQNRYASFDAGKGKEF EQGQLSLEDLVAYAKQKGEPAQISGKQELYEAIVNMYI.

Microbe

[0055] In some embodiments, the microbe is any prokaryotic or eukaryotic cell, with any genetic modifications, taught in U.S. Pat. Nos. 7,985,567; 8,420,833; 8,852,902; 9,109,175; 9,200,298; 9,334,514; 9,376,691; 9,382,553; 9,631,210; 9,951,345; and 10,167,488; and PCT International Patent Application Nos. PCT/US14/48293, PCT/US2018/049609, PCT/US2017/036168, PCT/US2018/029668, PCT/US2008/068833, PCT/US2008/068756, PCT/US2008/068831, PCT/US2009/042132, PCT/US2010/033299, PCT/US2011/053787, PCT/US2011/058660, PCT/US2011/059784, PCT/US2011/061900, PCT/US2012/031025, and PCT/US2013/074214 (all of which are incorporated in their entireties by reference).

[0056] Generally, although not necessarily, the microbe is a yeast or a bacterium. In some embodiments, the microbe is Rhodosporidium toruloides or Pseudomonas putida. In some embodiments, the microbe is a Gram negative bacterium. In some embodiments, the microbe is of the phylum Proteobactera. In some embodiments, the microbe is of the class Gammaproteobacteria. In some embodiments, the microbe is of the order Enterobacteriales. In some embodiments, the microbe is of the family Enterobacteriaceae. Examples of suitable bacteria include, without limitation, those species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus taxonomical classes. Suitable eukaryotic microbes include, but are not limited to, fungal cells. Suitable fungal cells are yeast cells, such as yeast cells of the Saccharomyces genus.

[0057] Yeasts suitable for the invention include, but are not limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces and Pichia cells. In some embodiments, the yeast is Saccharomyces cerevisae. In some embodiments, the yeast is a species of Candida, including but not limited to C. tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. panapsilosis and C. zeylenoides. In some embodiments, the yeast is Candida tropicalis. In some embodiments, the yeast is a non-oleaginous yeast. In some embodiments, the non-oleaginous yeast is a Saccharomyces species. In some embodiments, the Saccharomyces species is Saccharomyces cerevisiae. In some embodiments, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is a Rhodosporidium species. In some embodiments, the Rhodosporidium species is Rhodosporidium toruloides.

[0058] In some embodiments the microbe is a bacterium. Bacterial host cells suitable for the invention include, but are not limited to, Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, the Escherichia cell is an E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the Corynebacterium cell is Corynebacterium glutamicum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Corynebacterium amycolatum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale. In some embodiments, the Pseudomonas cell is a P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Streptomyces cell is a S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus, or S. scabies. In some embodiments, the Bacillus cell is a B. subtilis, B. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, or B. pumilus.

[0059] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0060] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0061] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.