Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

Abstract

Modified Saccharomyces cerevisiae yeast that produce terminal alkenes are described. The modification of the Saccharomyces cerevisiae yeast includes insertion of at least one heterologous fatty acid decarboxylase gene, deletion of FAA1 and FAA4, overexpression of HEM3, and triple-deletion of CTT1, CTA1 and CCP1. Methods of producing terminal alkenes by culturing and fermenting the modified Saccharomyces cerevisiae yeast and optionally harvesting the terminal alkenes are also described. Mixtures of terminal alkenes produced by the modified Saccharomyces cerevisiae yeast, and methods of metabolically engineering a yeast for optimizing overexpression of one or more alkenes are also described.

Claims

1. A modified Saccharomyces cerevisiae yeast wherein the modification comprises: insertion of at least one heterologous fatty acid decarboxylase gene encoding a fatty acid decarboxylase that synthesizes terminal alkenes selected from 1-undecene, 1-tridecene, 1-pentadecene, 1-heptadecene or 1-nonadecene, deletion of fatty acyl-Coenzyme A synthetases, FAA1 and FAA4, overexpression of porphobilinogen deaminase, HEM3, and triple-deletion of catalase T, CTT1, catalase A CTA1 and cytochrome c peroxidase, CCP1.

2. The modified Saccharomyces cerevisiae yeast of claim 1, wherein the yeast produces at least one terminal alkene.

3. The modified Saccharomyces cerevisiae yeast of claim 1, wherein the fatty acid decarboxylase is selected for terminal alkene synthesis via a one-step fatty acid decarboxylation pathway.

4. The modified Saccharomyces cerevisiae yeast of claim 3, wherein the fatty acid decarboxylase is OleT.sub.SM (SEQ ID NO 1), OleT.sub.MC (SEQ ID NO 2), OleT.sub.SP (SEQ ID NO 3), OleT.sub.BS (SEQ ID NO 4), OleT.sub.CE (SEQ ID NO 6), OleT.sub.JE (SEQ ID NO 7) or OleT.sub.JE-CO (SEQ ID NO 8).

5. The modified Saccharomyces cerevisiae yeast of claim 1, characterized by BY22 (BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS41K-P.sub.TEF1-OleT.sub.JE-CO).

6. A method of producing at least one terminal alkene, the method comprising: culturing the modified Saccharomyces cerevisiae yeast of claim 1 in a rich growth medium; fermenting the culture of modified Saccharomyces cerevisiae yeast at a temperature of about 25 C. to about 35 C. under aerobic conditions to produce at least one terminal alkene, wherein the terminal alkene is 1-undecene, 1-tridecene, 1-pentadecene, 1-heptadecene or 1-nonadecene; and harvesting the at least one terminal alkene, wherein the harvesting comprises lysing the yeast cells and extracting the terminal alkene.

7. The method of claim 6, wherein the rich growth medium is selected from SC-U+GAL, YPG+G418, YPD+G418 or YPD.

8. The method of claim 6, wherein the fermenting is performed with a dissolved oxygen concentration of about 60%.

9. The method of claim 6, wherein the fermenting is performed at a temperature of about 30 C.

10. The method of claim 6, wherein the fermenting is performed without pH control.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

(2) FIGS. 1A-1B. (FIG. 1A) Schematic view of the metabolic pathway for the production of terminal alkenes in the genetically engineered strain. Solid-thin arrows represent the native pathway in S. cerevisiae; Solid-thick arrows represent the overexpression of genes in this study; Crosses represent the gene deletion performed. Dashed arrows represent cofactor transfer for OleT utilization. (AbbreviationsACC1: acetyl-CoA carboxylase; FAS1/2: fatty acid synthase; FAA1/4: fatty acyl-CoA synthetase; PDX1: fatty acyl-CoA oxidase; FOX2: 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase; POT1: 3-ketoacyl-CoA thiolase; CCP1: cytochrome c peroxidase; CTA1: catalase A; CTT1: catalase T; HEM1: 5-aminolevulinate synthase; HEM2: aminolevulinate dehydratase; HEM3: porphobilinogen deaminase; HEM4: uroporphyrinogen III synthase; HEM12: uroporphyrinogen decarboxylase; HEM13: coproporphyrinogen oxidase; HEM14: protoporphyrinogen oxidase; HEM15: ferrochelatase; OleT: fatty acid decarboxylase) (FIG. 1B) Synthesis of terminal alkene via fatty acid decarboxylase-OleT catalyzed reaction.

(3) FIG. 2. Production of alkenes by recombinant S. cerevisiae expressing oleT.sub.JE homologs. Distributions of different chain length alkenes produced by the overexpression of oleT.sub.SM, oleT.sub.MC, oleT.sub.SP, oleT.sub.BS, oleT.sub.CE, oleT.sub.JE and oleT.sub.JE-CO are shown. Alkenes with different chain lengths from C11 to C19 are represented. Results are the average of three biological replicates with error bars showing the standard deviation from the mean value.

(4) FIGS. 3A-3C. Effects of fatty acid pool engineering on alkene production. (FIG. 3A) Total alkene titers of the strains without (BY10) and with the engineered fatty acid synthesis pathway (BY11, BY12, BY13 and BY14) are shown in bars. White bar and grey horizontal dash line indicates the alkene titers of the control strain BY10. Alkene fold changes are shown in lines. For alkene fold changes, BY10 was set equal to 1.0 and all values were determined relative to BY10. + and indicate with and without engineering respectively. (FIG. 3B) Gas chromatography (GC) profile of the alkene products obtained by batch culture of BY14 (upper trace) and BY10 (lower trace). Filled peaks indicated by arrows were shown as specific alkenes. (FIG. 3C) The comparison of total alkenes produced by the expression of oleT.sub.JE homologs in wild-type BY4741 (white bar) and BY4741 faa1faa4 double-deletion strain (grey bar). Alkenes were detected and quantified by GC-MS after growing for 48 h. Results represent the mean of three biological replicates; standard deviations are presented.

(5) FIG. 4. Production of alkenes by cofactor engineering. Total alkene titers are shown in bars and alkene fold changes are shown in lines. White bars and grey horizontal dash lines indicate the alkene titers of the control strain BY10. Lattice bars represent samples with fatty acid overproduction; Grey color bars represent samples with fatty acid overproduction and cofactor supplementation; Black color bars represent samples with fatty acid overproduction and cofactor genetic engineering. For alkene fold changes, BY10 was set equal to 1.0 and all values were determined relative to BY10. + and indicate with and without engineering respectively. Error bars represent the standard deviation of three biological replicates.

(6) FIG. 5. Alkene production using strains with tuned gene expression in rich medium. Total alkene titers are shown in bars and alkene fold changes are shown in lines. White bar and grey horizontal dash line indicates the alkene titers of control strain BY10. For alkene fold change, BY10 was set equal to 1.0 and all values were determined relative to BY10. Promoter strengths, plasmid copy numbers and respective growth medium are listed for each sample. Data shown are the meanSD of three biological replicates.

(7) FIGS. 6A-6B. (FIG. 6A) Production of alkenes and cell optical density in 1-L fed-batch fermentation using the engineered strain BY22. Samples were withdrawn and analyzed at the indicated time intervals. Diamond-marked line indicates alkene titers and triangle-marked line indicates cell OD. All of the fermentation experiments were performed in triplicate. (FIG. 6B) Titer and fold change summary for alkene production in S. cerevisiae.

DETAILED DESCRIPTION OF THE INVENTION

(8) A description of example embodiments of the invention follows.

(9) The invention pertains, in one aspect, to modified Saccharomyces cerevisiae yeast wherein the modification comprises: insertion of at least one heterologous fatty acid decarboxylase gene, deletion of FAA1 and FAA4, overexpression of HEM3, and triple-deletion of CTT1, CTA1 and CCP1. The modified Saccharomyces cerevisiae yeast can produce at least one terminal alkene, for example, the terminal alkene is 1-undecene, 1-tridecene, 1-pentadecene, 1-heptadecene or 1-nonadecene.

(10) In one aspect, the at least one terminal alkene is produced via a one-step fatty acid decarboxylation pathway. For instance, the decarboxylation is catalyzed by at least one fatty acid decarboxylase. Example fatty acid decarboxylases include OleT.sub.SM (SEQ ID NO 1), OleT.sub.MC (SEQ ID NO 2), OleT.sub.SP (SEQ ID NO 3), OleT.sub.BS (SEQ ID NO 4), OleT.sub.MP (SEQ ID NO 5), OleT.sub.CE (SEQ ID NO 6), OleT.sub.JE (SEQ ID NO 7) or OleT.sub.JE-CO (SEQ ID NO 8).

(11) In one embodiment, a modified Saccharomyces cerevisiae yeast is characterized by BY22 (BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS41K-P.sub.TEF1-OleT.sub.JE-CO).

(12) In another aspect, the invention pertains to a mixture of terminal alkenes comprising at least two terminal alkenes produced by the modified Saccharomyces cerevisiae yeast described herein. The amount of terminal alkenes in the mixture produced by the modified Saccharomyces cerevisiae yeast represents at least a 7-fold increase, at least a 38-fold increase or at least a 67-fold increase, as compared to an amount of terminal alkenes produced by a non-modified Saccharomyces cerevisiae yeast. The mixture of at least two terminal alkenes can be are selected from 1-undecene, 1-tridecene, 1-pentadecene, 1-heptadecene or 1-nonadecene. In some versions the mixture of terminal alkenes comprises at least three terminal alkenes or at least five terminal alkenes, selected from 1-undecene, 1-tridecene, 1-pentadecene, 1-heptadecene or 1-nonadecene.

(13) Methods of producing at least one terminal alkene are also described. In one aspect, the method comprising: culturing the modified Saccharomyces cerevisiae yeast of claim 1 in a rich growth medium; fermenting the culture of modified Saccharomyces cerevisiae yeast at a temperature of about 25 C. to about 35 C. under aerobic conditions to produce at least one terminal alkene, wherein the terminal alkene is 1-undecene, 1-tridecene, 1-pentadecene, 1-heptadecene or 1-nonadecene; and optionally, harvesting the terminal alkene, wherein the harvesting comprises lysing the yeast cells and extracting the terminal alkene.

(14) The rich growth medium can be selected from SC-U+GAL, YPG+G418, YPD+G418 or YPD.

(15) The method of fermenting can be performed with a dissolved oxygen concentration of about 60%. The fermenting can be performed at a temperature of about 30 C. The fermenting can be performed without pH control.

(16) The invention also pertains to methods of metabolically engineering a yeast for optimizing overexpression of one or more alkenes. The method comprises selecting a yeast having inserted therein one or more heterologous decarboxylase genes for alkene biosynthesis in the yeast via free fatty acid decarboxylation; enhancing the metabolic flux towards free fatty acid production in the yeast by disrupting the fatty acid metabolic pathway by deleting at least one synthetase and optionally overexpressing at least one carboxylase; supplying at least one decarboxylase cofactor to the alkene biosynthesis pathway to enhance the metabolic flux towards alkene production in the yeast; tuning expression levels of the one or more heterologous decarboxylase genes by at least one of promoter strength tuning, plasmid copy number tuning and growth medium tuning; and optimizing yeast fermentation conditions by at least one of temperature control, dissolved oxygen concentration control and pH control.

(17) In one version, the supplying of the at least one decarboxylase cofactor is performed internally by the yeast and is performed by at least one of overexpression of one or more rate-limiting enzymes responsible for cofactor biosynthesis and deletion of one or more utilization enzymes that utilize cofactor.

(18) The overexpression of the one or more alkenes by the metabolically engineered yeast can be optimized as compared to a non-engineered yeast.

(19) In light of the foregoing, the inventors aimed to engineer the yeast S. cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and to improve the alkene production using combinatorial engineering strategies (see FIG. 1A). First, the inventors screened and characterized eight fatty acid decarboxylases (OleT) to enable and enhance alkene production in S. cerevisiae. Then they developed a fatty acid-overproducing strain to boost the precursor availability, which could enhance the metabolic flux (Scalcinati et al., 2012) and resulted in a higher production titer. The inventors then improved the enzyme cofactor accumulation through cofactor genetic engineering [24, 25]. Then they enhanced the cell growth in rich medium and tuned the enzyme expression by optimizing the combinations of the promoters and plasmids. Finally, they further increased the alkene production by optimizing the culturing conditions in bioreactors. This represents the first report of terminal alkene biosynthesis in the yeast S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer of the alkene production of S. cerevisiae 67.4-fold.

(20) Materials and Methods

(21) Strains and Media

(22) Escherichia coli TOP10 (Invitrogen) and Luria-Bertani (BD) were used for cloning experiments unless otherwise stated. 100 mg/L ampicillin was used for selection of positive colonies if applicable. Jeotgalicoccus sp. ATCC 8456 (NCIMB) was used for oleT.sub.JE cloning. The yeast strain S. cerevisiae BY4741 (ATCC) was used for functional characterization of OleT enzymes.

(23) S. cerevisiae BY4741 wild-type and mutant strains were cultured in rich medium (YPD/YPG), synthetic minimal medium lacking uracil (SC-U), lysine (SC-L), adenine (SC-A), or synthetic minimal induction medium (SC-U-G). YPD/YPG medium (1% yeast extract, 2% peptone and 2% D-glucose/galactose) was used to routinely maintain wild-type strain or cells with pRS41K or pRS42K plasmids. SC-U medium (0.67% yeast nitrogen base, 0.192% uracil dropout and 2% raffinose) was used for growing pESC-URA transformants. SC-L medium (0.67% yeast nitrogen base, 0.18% lysine dropout and 2% glucose) and SC-A medium (0.67% yeast nitrogen base, 0.078% adenine dropout and 2% glucose) was used for selecting positive integrants. SC-U-G medium (0.67% yeast nitrogen base, 0.192% uracil dropout, 1% raffinose and 2% galactose) was used for protein induction in pESC-URA transformants. 2% agar was supplemented for solid media. One mg/mL 5-Fluoroorotic acid (5-FOA, Fermentas) or 200 mg/L geneticin (G418, PAA Laboratories) was used for selection. Heme (20 ug/mL) [26, 27], hydrogen peroxide (0.4 mM every 12 h) [28], or both were supplemented into growth culture where necessary. Yeast growth media components were purchased from Sigma-Aldrich and MP Biomedicals. Yeast cells were cultivated at 30 C. in flasks and shaken at 250 rpm.

(24) Gene Deletion and Integration

(25) Genes were deleted by using the previously described gene disruption cassette containing loxP-kanMX-loxP module in S. cerevisiae [29]. Firstly, the gene disruption cassettes were constructed through fusing short homologous sequences with loxP-kanMX-loxP module from plasmid pUG6 (Euroscarf) via a PCR reaction. Following yeast transformation, colonies were selected on an YPD plate containing 200 mg/L G418. The kanMX marker was removed by inducing expression of Cre recombinase from plasmid pSH47 (Euroscarf), which enables subsequent rounds of gene deletion. Here, the correct gene deletion mutants were verified by PCR analysis and used for further gene deletion.

(26) Chromosomal integration was conducted based on the method previously reported by Sadowski et al. [30]. Briefly, genes were firstly cloned into plasmid pIS385 or pIS112 (Euroscarf) containing URA3 selectable marker. The recombinant plasmid was linearized and transformed into S. cerevisiae, followed by colony selection performed on SC-U medium. After non-selective growth on YPD plate, individual colonies were replica-plated onto 5-FOA and SC-L or SC-A plates to screen for positive colonies. Finally, the correct integrant was verified by PCR analysis. Oligonucleotide primers used for gene deletion and chromosomal integration are listed in Table 1.

(27) TABLE-US-00001 TABLE1 Primersusedinthisstudy.Restrictionsitesarebold. Primers SEQUENCE NO. Primersequences(5-3) ID.NO. OleT.sub.JE-F ACGCGGATCCTAAAAAATGTCTACACTTAAGAGGGAT SEQIDNO9 AAGGGCTTAG OleT.sub.JE-R ATAAGAATGCGGCCGCCTAATGGTGATGGTGATGATG SEQIDNO10 TGTTCTGTCTACAACTTCGCGAAC ACC1-SC-R AGAATTTTTGAAAATTCGAATTCAACCCTCACTAAAGG SEQIDNO11 GCGGCCGCACTAGTTAAAAAATGTCTGAAGAAAGCTT ATTCGAGTCTTCTCC ACC1-SC-R TAAGAGCTCAGATCTTATCGTCGTCATCCTTGTAATCCA SEQIDNO12 TCGATACTAGTCTAATGGTGATGGTGATGATGTTTCAA AGTCTTCAACAATTTTTC FAA1-deletion-F CAATAAAAACTAGAACAAACACAAAAGACAAAAAAAG SEQIDNO13 ACAACAATCAGCTGAAGCTTCGTACGC FAA1-deletion-R TGCTTTAGTATGATGAGGCTTTCCTATCATGGAAATGTT SEQIDNO14 GATCCAGCATAGGCCACTAGTGGATCTG FAA4-deletion-F TCTGTTCTTCACTATTTCTTGAAAAACTAAGAAGTACGC SEQIDNO15 ATCAAACAGCTGAAGCTTCGTACGC FAA4-deletion-R GTGTTTATGAAGGGCAGGGGGGAAAGTAAAAAACTAT SEQIDNO16 GTCTTCCTGCATAGGCCACTAGTGGATCTG pTEF1-F TTGAGAGCTCTTTCATAGCTTCAAAATGTTTCTACTCCT SEQIDNO17 TTT pTEF1-R TCAGGGCCCATTTTGTAATTAAAACTTAGATTAGATTGC SEQIDNO18 TATGCTTTC Hem3-F CTAATCTAAGTTTTAATTACAAAATGGGCCCTGAAACTC SEQIDNO19 TACATATTG HEM3-R CTTATTTAGTCAATGGTGATGGTGATGATGTTTGATTCT SEQIDNO20 GTCTAAATTAATTTCATCCAG TADH1-F CATCATCACCATCACCATTGACTAAATAAGCGAATTTCT SEQIDNO21 TATGATTTATGATTTTT TADH1-R ACGGGGTACCTTTCAGCTGAATTGGAGCGACC SEQIDNO22 CTT1-deletion-F TTCTCTTGTCTCATGCCAATAAGATCAATCAGCTCAGCT SEQIDNO23 TCACAACAGCTGAAGCTTCGTACGC CTT1-deletion-R TTATGGAGATATAATTACGAATAATTATGAATAAATAG SEQIDNO24 TGCTCTCCGCATAGGCCACTAGTGGATCTG CTA1-deletion-F AAATAAATATAATAGTACTTACAAATAAATTTGGAACC SEQIDNO25 CTAGAAGCAGCTGAAGCTTCGTACGC CTA1-deletion-R ATAATTGTCGTGGAAACAACGCCACTCATTTGTATATC SEQIDNO26 AGCGTTGCATAGGCCACTAGTGGATCTG CCP1-deletion-F ATTTCGCATTCATGCAGACGCAAACACACACGTATATC SEQIDNO27 TACAATTCAGCTGAAGCTTCGTACGC CCP1-deletion-R AATAATACGAAATATAACCAATAAATAATATCTTTCCT SEQIDNO28 CAGTGACGCATAGGCCACTACaGGATCTG pPGI1-F ATAAGAATGCGGCCGCTAACAAAAATCACGATCTGGG SEQIDNO29 TGG pPGI1-R TTATCTCTCTTCAAAGTAGCCATTTTAGGCTGGTATCTT SEQIDNO30 GATTCTAAA TCYC1-F AACTCATCATCACCATCACCATTAATAAGATCCGCTCTA SEQIDNO31 ACCGAAAAGG TCYC1-R AAACGAGCTCCTTCGAGCGTCCCAAAACCT SEQIDNO32
Fatty Acid Decarboxylase Selection

(28) Six more homologous enzymes from different organisms were selected for alkene biosynthesis in S. cerevisiae (Table 2). Among them, oleT.sub.BS, oleT.sub.MP and oleT.sub.CE were reported to produce 1-pentadecene when heterologously expressed in E. coli [1]; oleT.sub.SM, oleT.sub.MC and OleT.sub.SP were selected based on their protein sequence identity to oleT.sub.JE, and their histidine residue in position 85 (His85) which as mentioned, plays an important role in catalysis activity of OleT.sub.JE.

(29) TABLE-US-00002 TABLE 2 OleT used in this study Name Organism Accession no. Sequence ID No. OleT.sub.SM Staphylococcus WP_009381667 SEQ ID NO 1 massiliensis OleT.sub.MC Macrococcus YP_002560207 SEQ ID NO 2 caseolyticus JCSC5402 OleT.sub.SP Staphylococcus YP_006015679 SEQ ID NO 3 pseudintermedius ED99 OleT.sub.BS Bacillus subtilis 168 NP_388092 SEQ ID NO 4 OleT.sub.MP Methylobacterium populi ZP_02200540 SEQ ID NO 5 BJ001 OleT.sub.CE Corynebacterium NP_739069 SEQ ID NO 6 efficiens YS-314 OleT.sub.JE Jeotgalicoccus sp. HQ709266 SEQ ID NO 7 ATCC 8456
Plasmid Construction

(30) To clone oleT.sub.CE, genomic DNA of Jeotgalicoccus sp. ATCC 8456 was used as a PCR template performed with two primers OleT.sub.JE-F and OleT.sub.JE-R. One oleT.sub.JE codon optimized gene and six codon optimized oleT.sub.CE homologous genes, namely oleT.sub.JE-CO, oleT.sub.SM, oleT.sub.SP, oleT.sub.BS, oleT.sub.MP, and oleT.sub.CE, were synthesized from Life technologies. ACC1 and HEM3 were amplified from S. cerevisiae genome using two set of primers: ACC1-SC-F and ACC1-SC-R, Hem3-F and Hem3-R. A list of primers used was shown in Table 1. Plasmid pESC-URA (Agilent Technologies), pRS41K (Euroscarf) and pRS42K (Euroscarf) were used as expression vectors for oleT and/or ACC1 while plasmid pIS385 (Euroscarf) was used for HEM3 cloning. Either Gibson DNA assembly method [31] or digestion-ligation method was used for the construction of all the plasmids. The constructed recombinant plasmids are listed in Table 3.

(31) TABLE-US-00003 TABLE 3 Strains and plasmids used in this study Strains or plasmids Description Source Strains E. coli Top10 F mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 Invitrogen araD139 (ara-leu) 7697 galU galK rpsL(Str.sup.R) endA1 nupG S. cerevisiae BY4741 MATa his31 leu20 met150 ura30 ATCC BYSM BY4741 with pESC-OleT.sub.SM This study BYMC BY4741 with pESC-OleT.sub.MC This study BYSP BY4741 with pESC-OleT.sub.SP This study BYBS BY4741 with pESC-OleT.sub.BS This study BYCE BY4741 with pESC-OleT.sub.CE This study BYJE BY4741 with pESC-OleT.sub.JE This study BYFSM BY4741, faa1 faa4 with pESC-OleT.sub.SM This study BYFMC BY4741, faa1 faa4 with pESC-OleT.sub.MC This study BYFSP BY4741, faa1 faa4 with pESC-OleT.sub.SP This study BYFBS BY4741, faa1 faa4 with pESC-OleT.sub.BS This study BYFCE BY4741, faa1 faa4 with pESC-OleT.sub.CE This study BYFJE BY4741, faa1 faa4 with pESC-OleT.sub.JE This study BY10 BY4741 with pESC-OleT.sub.JE-CO This study BY11 BY4741 with pESC-OleT.sub.JE-CO-ACC1 This study BY12 BY4741, faa1 with pESC-OleT.sub.JE-CO This study BY13 BY4741, faa4 with pESC-OleT.sub.JE-CO This study BY14 BY4741, faa1 faa4 with pESC-OleT.sub.JE-CO This study BY15 BY4741, faa1 faa4 P.sub.TEF1-HEM3 with pESC-OleT.sub.JE-CO This study BY16 BY4741, faa1 faa4 ctt1 cta1 ccp1 with pESC-OleT.sub.JE-CO This study BY17 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pESC-OleT.sub.JE-CO This study BY18 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS41K-P.sub.GAL1-OleT.sub.JE-CO This study BY19 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS42K-P.sub.GAL1-OleT.sub.JE-CO This study BY20 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS41K-P.sub.PGH1-OleT.sub.JE-CO This study BY21 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS42K-P.sub.PGH1-OleT.sub.JE-CO This study BY22 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS41K-P.sub.TEF1-OleT.sub.JE-CO This study BY23 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3 with pRS42K-P.sub.TEF1-OleT.sub.JE-CO This study BY24 BY4741, faa1 faa4 ctt1 cta1 ccp1, P.sub.TEF1-HEM3, P.sub.TEF1-OleT.sub.JE-CO This study Plasmids pESC-URA P.sub.GAL1, P.sub.GAL10 promoter, 2 origin, AmpR, URA3 Agilent Technologies pIS385 AmpR, URA3 Euroscarf pIS112 AmpR, URA3 Euroscarf pUG6 AmpR, kanMX Euroscarf pSH47 CEN6/ARSH4 origin, CRE, AmpR, URA3 Euroscarf pRS41K ARS/CEN origin, kanMX Euroscarf pRS42K 2 origin, kanMX Euroscarf pESC-OleT.sub.JE pESC-URA carrying oleT.sub.JE under P.sub.GAL1 control This study pESC-OleT.sub.JE-CO pESC-URA carrying oleT.sub.JE-CO under P.sub.GAL1 control This study pESC-OleT.sub.SM pESC-URA carrying oleT.sub.SM under P.sub.GAL1 control This study pESC-OleT.sub.MC pESC-URA carrying oleT.sub.MC under P.sub.GAL1 control This study pESC-OleT.sub.SP pESC-URA carrying oleT.sub.SP under P.sub.GAL1 control This study pESC-OleT.sub.BS pESC-URA carrying oleT.sub.BS under P.sub.GAL1 control This study pESC-OleT.sub.MP pESC-URA carrying oleT.sub.MP under P.sub.GAL1 control This study pESC-OleT.sub.CE pESC-URA carrying oleT.sub.CE under P.sub.GAL1 control This study pESC-OleT.sub.JE-CO-ACC1 pESC-URA carrying oleT.sub.JE-CO under P.sub.GAL1 control and ACC1 under P.sub.GAL10 control This study pRS41K-P.sub.GAL1-OleT.sub.JE-CO pRS41K carrying oleT.sub.JE-CO under P.sub.GAL1 control This study pRS42K-P.sub.GAL1-OleT.sub.JE-CO pRS42K carrying oleT.sub.JE-CO under P.sub.GAL1 control This study pRS41K-P.sub.PGH1-OleT.sub.JE-CO pRS41K carrying oleT.sub.JE-CO under P.sub.PGH1 control This study pRS42K-P.sub.PGH1-OleT.sub.JE-CO pRS42K carrying oleT.sub.JE-CO under P.sub.PGH1 control This study pRS41K-P.sub.TEF1-OleT.sub.JE-CO pRS41K carrying oleT.sub.JE-CO under P.sub.TEF1 control This study pRS42K-P.sub.TEF1-OleT.sub.JE-CO pRS42K carrying oleT.sub.JE-CO under P.sub.TEF1 control This study
Alkene Extraction and Detection

(32) For alkene production, cells were pre-cultured in 10 ml medium overnight and then diluted in 50 ml induction medium using 250 ml flask to achieve an initial OD600 of 0.4. After growing for 48 h, yeast cells were harvested by centrifugation at 6000 g for 5 min. Cell pellets were re-suspended in HPLC grade methanol (Sigma), and 1-nonene was added into cell suspension as an internal standard. Acid-washed glass beads were added until the suspension was covered. Cells were then lysed by mechanical agitation using FastPrep-24 (MPBio) for 8 min at 6 m/s. HPLC grade hexane (Sigma) was then added and mixed thoroughly with crude extract for 5 min. The crude extract was separated into two phases by centrifugation, and the upper phase containing alkene was transferred into a clear GC vial.

(33) The alkenes dissolved in the upper layer were quantified using gas chromatography-mass spectrometry (GC-MS) under the following conditions. An HP-5 ms column (30 m by 0.25 mm; 0.25 m film; Agilent) was used with a helium flow rate set to 1.1 ml/min. Injections of 5 l were carried out under splitless injection condition with the inlet set to 250 C. The GC temperature profile was as follows: an initial temperature of 40 C. was maintained for 0.5 min, followed by ramping to 280 C. at a rate of 6 C./min, where the temperature was held for 3 min. The mass spectrometer detector was scanned at 30 to 800 amu in the electron impact mode. To aid peak identification, authentic references (C9-C19 terminal alkenes, Tokyo Chemical Industry) were used, and their retention times and fragmentation patterns were compared with those from the extracted alkenes.

(34) Bioreactor Conditions

(35) Selected strain was used for production of alkenes through fed-batch fermentation. YPD+G418 containing 3% glucose was used for both seed preparation and fermentation. Seed culture was prepared by inoculating colonies into a 250 mL flask containing 50 mL culture medium, and incubating at 30 C. and 250 rpm for 24 h. The seed was then transferred to a 5 L bioreactor (BIOSTAT B-DCU II, Sartorius) containing 1 L medium with an initial OD.sub.600 0.4. The fermentation was carried out at 30 C. The dissolved oxygen concentration in the bioreactor was maintained at around 60% by controlling the air flow rate and agitation speed. 150 ml 200 g/L glucose was fed to the fermenter every 24 h and samples were withdrawn at the indicated time intervals. All of the fermentation experiments were performed in triplicate.

(36) Results

(37) Screening Enzymes for Alkene Biosynthesis in S. cerevisiae

(38) To enable terminal alkene production in S. cerevisiae, the inventors attempted to use the cytochrome P450 fatty acid decarboxylase OleT.sub.JE from Jeotgalicoccus sp. ATCC 8456, which reportedly decarboxylates fatty acids to terminal alkenes [1] (FIG. 1B). The inventors also used its codon-optimized version oleT.sub.JE-CO (SEQ ID NO 8) and six of its homologous genes, based on high sequence identity to OleT.sub.JE Table 2. Native oleT.sub.JE and synthesized codon-optimized homologous genes were cloned into the high copy plasmid pESC-URA (Table 3) and transformed into S. cerevisiae. The induced protein expression in S. cerevisiae was confirmed by western blot (data not shown). The inventors evaluated the performance of the abovementioned enzymes by quantifying the alkene profiles and measuring the alkene concentrations from the cell cultures grown for 48 h. The inventors found that the cells carrying the empty plasmid and OleT.sub.MP from Methylobacterium populi BJ001 produced no detectable alkenes (data not shown), whilst the transformants expressing the other OleT enzymes produced a range of alkenes. As shown in FIG. 2, OleT.sub.SM, OleT.sub.SP, OleT.sub.BS and OleT.sub.CE produced alkenes with the chain lengths of C13, C15 and C17, whereas OleT.sub.MC exhibited a narrower alkene profile, producing C13 and C15 alkenes. OleT.sub.JE and its codon-optimized version OleT.sub.JE-CO exhibited the broadest product profile range, producing odd chain terminal alkenes from C11 to C19. The inventors observed lower alkene titers for shorter chain lengths possibly because longer chain fatty acids are more abundant than shorter chain fatty acids in yeast cells [32].

(39) Aside from the varying alkene profiles, the total titers of the produced alkenes varied among the tested OleT enzymes. FIG. 2 shows that OleT.sub.SM led to the lowest total alkene titer (1.4 g/L), whereas OleT.sub.JE-CO gave the highest total alkene titer (54.5 g/L), which served as the baseline titer for this study.

(40) Increase in Free Fatty Acid Production Improved Alkene Production

(41) As a first step in improving the alkene production, the inventors attempted to increase the production of free fatty acids, which are precursors to alkenes (FIG. 1A). The de novo fatty acid biosynthesis in S. cerevisiae requires acetyl-CoA carboxylase (ACC1; encoded by the ACC1) and fatty acid synthase complex (FAS; encoded by FAS1 and FAS2) [33-36]. ACC1 converts acetyl-CoA into malonyl-CoA, and the overexpression of ACC1 results in increase in final fatty acid level [33, 34]. The FAS complex produces fatty acyl-CoAs by condensation of one acetyl-CoA to 7-8 malonyl-CoAs [37]. The de novo produced fatty acyl-CoAs are further hydrolyzed to free fatty acids; however, free fatty acids are converted back to fatty acyl-CoAs by endogenous fatty acyl-CoA synthetase (FAA1-4, FAT1). As an active form of free fatty acids, fatty acyl-CoAs are further degraded mainly through (3-oxidation pathway (PDX1, FOX2, POT1). Hence, in order to enhance the metabolic flux towards free fatty acid, the inventors attempted to overexpress ACC1 and disrupt FAA1 and FAA4, the two main fatty acyl-CoA synthetases [38-40].

(42) First, the inventors expressed oleT.sub.JE-CO with ACC1 under the control of the strong inducible promoters P.sub.GAL1 and P.sub.GAL10, respectively, generating the strain BY11 (ACC1, oleT.sub.JE-CO). Second, the inventors deleted FAA1 and/or FAA4, and expressed oleT.sub.JE-CO, resulting in three different strains BY12 (faa1, oleT.sub.JE-CO), BY13 (faa4, oleT.sub.JE-CO), and BY14 (faa1faa4, oleT.sub.JE-CO). As shown in FIG. 3A, the co-expression of ACC1 and oleT.sub.JE-CO in S. cerevisiae led to lower alkene levels compared with the singularly expressed oleT.sub.JE-CO (BY10, control strain). Moreover, increased alkene production levels were observed in both BY12 (6.2-fold) and BY14 (7-fold). In particular, the double-deletion strain BY14 produced the highest alkene titer of 382.8 g/L. However, for an unknown reason, the single-deletion of FAA4 (BY13) led to 2.5-fold lower alkene production. These results suggest that the deletion of FAA1 in tandem with FAA4 has a synergic effect on fatty acid accumulation, where FAA1 accounts for most of this effect. In addition to the total alkene titers, changes in alkene profiles were also studied (Table 4).

(43) TABLE-US-00004 TABLE 4 Comparison of alkene production obtained by engineered S. cerevisiae strains Alkene (fractional abundance %) Total alkene Strain C11 C13 C15 C17 C19 (g/L) BYSM 17.3 10.3 72.4 1.4 0.3 BYMC 14.0 86.0 4.6 0.1 BYSP 13.7 39.3 46.9 24.4 0.3 BYBS 6.5 44.2 49.3 7.2 0.4 BYCE 16.3 36.8 46.8 19.7 0.3 BYJE 1.2 1.0 4.2 48.0 45.6 47.6 0.8 BYFSM 2.6 13.1 84.3 75.0 5.2 BYFMC 7.3 92.7 25.5 0.3 BYFSP 3.1 41.1 55.8 121.2 7.6 BYFBS 3.3 33.4 63.3 85.4 4.7 BYFCE 2.3 36.8 61.0 129.6 13.7 BYFJE 0.2 0.5 5.0 88.2 6.1 362.1 3.0 BY10 1.4 1.3 5.4 52.4 39.5 54.5 2.2 BY11 2.6 44.4 52.9 21.0 2.3 BY12 0.3 2.2 94.1 3.5 339.2 10.8 BY13 2.7 4.9 46.5 45.8 21.8 2.0 BY14 0.2 0.4 4.1 89.5 5.9 382.8 12.6 BY14.sup.a 0.3 0.8 7.0 87.6 4.2 716.9 30.0 BY14.sup.b 1.0 1.6 14.0 79.1 4.3 684.0 27.5 BY14.sup.c 0.4 1.2 8.5 87.0 3.0 1387.4 48.9 BY15 0.1 0.3 3.7 92.2 3.7 403.8 5.4 BY16 0.1 0.4 4.3 91.1 4.2 402.0 13.9 BY17 0.2 0.3 3.1 92.5 3.8 472.7 8.6 BY18 0.2 1.1 8.3 77.2 13.2 1720.8 156.9 BY19 1.0 1.4 9.0 69.6 19.0 453.2 29.8 BY20 0.6 1.0 10.7 71.9 15.8 409.9 25.9 BY21 0.3 0.5 10.2 77.9 11.1 882.2 195.4 BY22 0.1 0.4 8.3 85.0 6.2 2088.7 66.4 BY23 0.6 0.9 9.5 59.6 29.4 551.9 16.3 BY24 0.6 0.8 8.7 70.2 19.7 450.8 3.8 BY22.sup.d 0.4 0.3 5.8 52.1 41.4 763.9 32.4 BY22.sup.e 1.0 0.3 4.4 82.0 12.2 2243.5 117.3 BY22.sup.f 0.2 0.6 3.9 74.5 20.8 3289.1 217.9 BY22.sup.g 0.6 0.7 5.6 58.6 34.5 3675.5 218.4 .sup.aHeme supplementation in medium .sup.bH.sub.2O.sub.2 supplementation in medium .sup.cHeme and H.sub.2O.sub.2 supplementation in medium .sup.d24 h growth in bioreactor .sup.e48 h growth in bioreactor .sup.f72 h growth in bioreactor .sup.g144 h growth in bioreactor : not detected

(44) As shown in the gas chromatography (GC) profile, BY14 showed a significant improvement in the production of C15 and C17 alkenes compared to BY10, but a lower improvement for other alkenes (FIG. 3B). This increase in the production of C15 and C17 alkenes could be attributed to that BY14 accumulated more C16 and C18 free fatty acids (data not shown). The inventors then expressed all eight OleT enzymes in the double-deletion strain (faa1faa4), respectively, and evaluated the alkene titers. The inventors found that the overexpression of oleT.sub.JE-CO showed the highest total alkene titer in the double-deletion strain (faa1faa4) (FIG. 3C), in line with the result from the overexpression of oleT.sub.JE-CO in the wild-type strain. Thus, the inventors selected BY14 (faa1faa4, oleT.sub.JE-CO) for further engineering, which showed a 7-fold improvement in the titer to the control alkene-producing strain BY10 (oleT.sub.JE-CO).

(45) Cofactor Engineering Further Increased Alkene Production Level

(46) 1) Supplementation of Cofactors: Heme and Hydrogen Peroxide

(47) The inventors then improved the enzyme cofactor availability to further increase the associated metabolic flux towards alkene production. OleT.sub.JE is a cytochrome P450 enzyme in the cyp152 family, which contains heme as a cofactor [1], and the overexpression of cytochrome P450 enzymes can lead to heme depletion [41]. Further, OleT.sub.JE is highly active in the presence of hydrogen peroxide which serves as the sole electron and oxygen donor [1]. Therefore, the inventors hypothesized that cellular depletion of heme and hydrogen peroxide resulting from the overexpression of the P450 enzyme OleT.sub.JE could be a limiting factor, and thus, increasing the availability of the two cofactors heme and hydrogen peroxide might improve alkene synthesis.

(48) To test this hypothesis, the inventors supplemented BY14 (faa1faa4, oleT.sub.JE-CO) with heme, hydrogen peroxide, or both. As shown in FIG. 4, the supplementation with heme, hydrogen peroxide or both increased the titer by 87%, 79%, and 3.6-fold respectively, with the highest production at 1.4 mg/L. The improved alkene production demonstrated that cofactors supplementation during OleT enzyme expression could be employed to boost the alkene titers.

(49) 2) Overexpression of HEM3, and Triple-Deletion of CTT1, CTA1 and CCP1

(50) Based on the abovementioned result from the cofactor supplementation, the inventors attempted to increase the alkene titer using genetic cofactor engineering to eliminate the need for cofactor supplementation, which could be costly. The inventors first aimed to improve cellular heme production, which could be achieved by overexpression of rate-limiting enzymes responsible for heme biosynthesis. Multiple enzymes are involved in the heme biosynthesis pathway including three rate-limiting enzymes, HEM2, HEM3 and HEM12 [42]; however, the co-expression of these three HEM enzymes could be stressful to the host cells [41]. For example, the strains expressing only HEM3 exhibited no growth defect, and in combination with expression of P450 enzyme, showed high theophylline titers [41]. Therefore, in this study, HEM3 was integrated into genome and constitutively expressed under the control of TEF1 promoter, referred to as strain BY15 (faa1faa4, P.sub.TEF1-HEM3, oleT.sub.JE-CO). Secondly, the inventors aimed to accumulate endogenous hydrogen peroxide by deleting its utilization enzymes, catalase T (CTT1) located in cytoplasm, catalase A (CTA1) located in peroxisomes [43], and the antioxidant enzyme cytochrome c peroxidase (CCP1) located in mitochondria [44]. Previous studies showed that increased levels of hydrogen peroxide were detected in catalase mutants and cells with chemically inactivated catalases [45, 46]. Hence, the inventors further deleted CTT1, CTA1 and CCP1 genes to generate a series of deletion strains that could improve cofactor availability (Table 3).

(51) As shown in FIG. 4, HEM3 expression (BY15) brought a slight improvement in the total alkene titer compared to BY14 (without HEM3 overexpression). However, among all the deletion mutants, only BY16 (faa1faa4ctt1cta1ccp1, oleT.sub.JE-CO) showed a slightly higher titer compared to BY14 (faa1faa4, oleT.sub.JE-CO), while the rest deletion mutants showed no improved alkene titers (data not shown). To examine the potential synergistic effect of the aforementioned two approaches, the inventors integrated HEM3 into the genome of BY16, resulting in BY17 (faa1faa4ctt1cta1ccp1, P.sub.TEF1-HEM3, oleT.sub.JE-CO). As shown in FIG. 4, BY17 (faa1faa4ctt1cta1ccp1, P.sub.TEF1-HEM3, oleT.sub.JE-CO) produced a total alkene tilter of 472.7 g/L, 23% improvement to the fatty acid-overproducing strain BY14 (faa1faa4, oleT.sub.JE-CO) and 8.7-fold improvement to the control strain BY10 (oleT.sub.JE-CO).

(52) Gene Expression Tuning for Alkene Production in Rich Medium

(53) The inventors then enhanced the cell growth in rich medium and tuned the expression level of the heterologous genes. In the highest producing strain so far BY17, the oleT.sub.JE-CO was placed under the control of the galactose inducible promoter P.sub.GAL1 on the high-copy plasmid pESC-URA containing the auxotrophic URA marker. Rich medium frequently increase cell growth and final cell amount, resulting in higher product titers [47]. Thus, here the inventors replaced the auxotrophic pESC-URA plasmid with pRS plasmids containing the KanMX resistance marker. Moreover, to optimize the expression level of the heterologous genes, the inventors used pRS41K (low copy) and pRS42K (high copy) as cloning vectors [48]. P.sub.GAL1 (a strong inducible promoter), Ppm (a weak constitutive promoter) and P.sub.TEF1 (a strong constitutive promoter) were employed in both vectors to modulate the oleT.sub.JE-CO transcription. A total of six engineered strains were constructed and tested for alkene production (Table 3).

(54) All the engineered oleT.sub.JE-CO containing strains were cultivated in rich medium supplied with 2% galactose or glucose for alkene production. The inventors found that all the engineered strains exhibited increased cell growth and much higher final cell amount, where OD.sub.60030 was achieved in the rich medium while OD.sub.6008 in the minimal medium). As shown in FIG. 5, among the abovementioned six constructed strains, BY22 (faa1faa4ctt1cta1ccp1, P.sub.TEF1-HEM3, P.sub.TEF1-oleT.sub.JE-CO (pRS41K)), which contains the strong constitutive promoter P.sub.TEF1 on the low copy plasmid pRS41K, showed the highest alkene production, 2.1 mg/L, 4.4-fold higher than BY17 and 38.3-fold higher than the control strain BY10. The strains containing oleT.sub.JE-CO under the control of the weak promoter Ppm showed 2.2-fold higher alkene production on the high copy plasmid pRS42K (BY21) than that on the low copy plasmid pRS41K (BY20). This result indicates that sufficient expression of oleT.sub.JE-CO is needed for relatively higher alkene production. In contrast, with the strong promoter P.sub.GAL1 or P.sub.TEF1, the strains with the high copy plasmid (BY19 and BY23) showed 3.8-fold lower alkene production compared with the strains with the low copy plasmid (BY18 and BY22). These results suggest that in our study, i) the use of a strong promoter on a low copy plasmid provided sufficient enzyme levels for alkene production and ii) the use of a strong promoter on a high copy plasmid might cause metabolic burden on the cell, making the overall process non-beneficial [49]. To further address the plasmid burden [50] and to avoid the antibiotics cost, oleT.sub.JE-CO was chromosomally integrated and constitutively expressed under the TEF1 promoter. This constructed strain BY24 (faa1faa4ctt1cta1ccp1, P.sub.TEF1-HEM3, P.sub.TEF1-oleT.sub.JE-CO) produced about 4.6-fold less alkene than BY22 harboring oleT.sub.JE-CO on a low-copy plasmid, suggesting that a single copy of oleT.sub.JE-CO likely brought about insufficient gene expression level.

(55) Bioreactor Process Optimization for Higher Alkene Production

(56) The inventors then conducted fed-batch fermentation and optimized the fermentation conditions to achieve higher alkene production. The inventors used BY22 (faa1faa4ctt1cta1ccp1, P.sub.TEF1-HEM3, P.sub.TEF1-oleT.sub.JE-CO (pRS41K)), the highest alkene production strain so far in shake flask culture, to test in fed-batch bioreactors. Three parameters, temperature, pH and dissolved oxygen concentration (pO2), were controlled and monitored. Three different operation temperatures, 25 C., 30 C. and 35 C. gave comparable alkene titers (data not shown). pH 5, pH 7 and pH off were tested, where pH off showed a higher alkene titer (data not shown). Since heme biosynthesis requires oxygen [42] and an aerobic condition could give higher cell growth, the pO2 level was maintained at around 60% saturation, a general aerobic condition for yeast growth. Thus, the inventors chose temperature 30 C., pH off and pO.sub.2 60% as our operation condition.

(57) As shown in FIG. 6A, during the first 48 h, BY22 grew steadily and the levels of the produced alkene were increased to 2.2 mg/L. After 48 h, strain went through the stationary phase, and the alkene levels were further increased from 2.2 mg/L to 3.3 mg/L at 72 h; however, longer incubations only marginally increased alkene levels. These growth conditions gave rise to the highest level of production at 144 h, resulting in the alkene titer of 3.7 mg/L, 1.8-fold increase to the shake flask condition and 67.4-fold increase to the control strain BY10. Finally, FIG. 6B and Table 4 summarize the abovementioned sequential improvements in the alkene production through enzyme screening, precursor boosting, cofactor engineering, gene expression tuning and process optimization.

CONCLUSIONS

(58) In this study, the inventors engineered S. cerevisiae to produce terminal alkene and further improved the alkene production 67.4-fold by combinatorial engineering strategies. First, OleT.sub.JE and its homologous enzymes were characterized to convert free fatty acids into alkenes. In particular, OleT.sub.JE-CO (codon optimized OleT from Jeotgalicoccus sp.) showed the broadest alkene profiles and the highest production level. Second, the deletion of both FAA1 and FAA4 significantly improved the alkene titer, likely due to increased free fatty acid pool. Third, genetic cofactor engineering involving the overexpression of HEM3 and the triple-deletion of CTT1, CTA1 and CCP1 further improved the alkene titer. Fourth, the tuning of the heterologous gene expression in the rich medium enabled a further improvement in the titer (i.e. BY22 (faa1faa4ctt1cta1ccp1, P.sub.TEF1-HEM3, P.sub.TEF1-oleT.sub.JE-CO (pRS41K)). Finally, the optimization of the culturing conditions in fed-batch bioreactors further improved the alkene production in BY22. This study represents the first report of terminal alkene biosynthesis in the yeast S. cerevisiae, and taken together, the abovementioned combinatorial engineering approaches increased the titer of the alkene production of S. cerevisiae 67.4-fold. The inventors envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

(59) The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

(60) While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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(62) TABLE-US-00005 SEQUENCES OleT.sub.SM(SEQIDNO1): ATGTTCGTCGATTCCATCTTGGTCTTGAGATTGAACTTGTTGAAAACCGGTATACAAT TGGAAATGAAGAACGGTGGTATTAAGGTTGCTAAGAAATTGCCAAAGGTTAAGGGT TTGGATAACACCGTTGATATCATTAAGGGTGGTTACACTTACGTTCCAGGTAAGTTG GAAGAATTCGATTCTAAGGCTTTCGAAGTTAGAGCTTTGGGTGGTAAAAAGATTGCT GTCATGTCTGGTAAAGAAGCCGCTGAAATTTTCTACGACAACGAAAAGATGGAAAG ACAAGGTACTTTGCCAAAGAGAATCGTTAACACTTTGTTTGGTAAGGGTGCTATTCA TACCACTGCTGGTAAAAAACACGTTGATAGAAAGGCCTTGTTCATGTCTTTGATGAC TGACGAAAACTTGAACTACTTGAGAGAATTGACCAGAAACTACTGGTTTATGAACA CCGAAAGAATGCAATCCATGGACAAGGTTAACGTCTACAACGAATCTATCTACATGT TGACCAAGATCGGTTTTAGATGGGCCGGTATTATTCAAACTCCTGAAGAAGCTGAAC AAAACGCTAAAGATATGGACACCATGATCAACTCATTCGTCAGTTTGGGTTCTGCTT ACAAAGGTTACAAAAAGGCTAAGAAGGCCAGAAAGAGAGTCGAAGATTTTTTGGAA AAGCAAATCATCGACGTCAGAAAGGGTAAATTGCATCCAGAAGAAGGTACTGCCTT GTACGAATTTGCTCATTGGGAAGATTTGAACGATAACCCAATGGATTCTCATTTGTG CGCTGTTGATTTGATGAACGTTGTTAGACCATTGGCTGCCATTAACAGATTCATTTCT TACGGTGTTAAGGTCTTGATTGAATTCGACCAAGAAAAAGAAAAGTTGAGATTGGA AAACAACGAAGATTACGCCTACAAGTTCGCTCAAGAAGTTAGAAGAATCTTTCCATT CGTTCCATACTTGCCAGGTAGAGCTGCAGTTGATTTGGAATATGATGGTTACAAGAT TCCAGCTGGTATGATGACTGCTTTGGATGTTTATGGTACTACCCACGATGAAGATTT GTGGGAAAATCCAGATCAATTCAACCCAAACAGATTCGATAATTGGGATGGTTCTCC ATTCGATTTGATTCCACAAGGTGGTGGTGATTTCTACACTAATCATAGATGTGCTGG TGAATGGATCACCGTTATTATCATGGAAGAAACCATGAAGTATTTCGCCAACAAGAT CGAATTTGACGTCCCATCTCAAGATTTGTCTGTTAAGTTGGATAAGTTGCCTGGTAAT GTTACCTCCGGTACTATTATTTCTAACGTCAGACCAAGAGTTGCCAGAAAGTAA OleT.sub.MC(SEQIDNO2): ATGAGAGTCGAATTCACCATCAACTACATTAACGTCGAAGGTATCTCCATGTCTAAG AGAGTTCCAAAGGATAGAGGTATCGACAACTCCTTGAAGATTATGAAGGAAGGTTA CGAATACGTTCCAGCCAGAATGAAGAAGTTCAACACCAACATTTTCGAAACCAGAG TTTTGGGTGGTAAGACCGCTGTTGTTATTTCTGGTAAAGATGCTGCCGAATTATTCTA CGATAACGACAAGACTGAAAGAAAGGGTACTTTGCCAAAGAGAGTTGTTAAGACTT TGTTTGGTAAGGGTGCTATTCATACCACTACCGGTAAGAAACATATTGACAGAAAGG CCTTGTTCATGTCTTTGATGACTGACGAAAATTTGGCCTACTTGAGAAAGTTGACTA GAATCTACTGGTTCCAAAACATCGAACACATGCAATACAAGCAAAAGGTCAACGTT TACGAAGAAGCCACTGAATTATTGACCAAGGTTGGTTTGAGATGGGCTGGTATAGTT GATCATCCAGAAAACATTCAAAAGATGGCCGACGATATGAACAAGATGATCGATTC TTTTTCCGCCATCGGTTCATTATATGGTGGTTACAGAGAAGCTAAAAAGGCTAGAGC TAGAGTCGAACAATTCTTGGAAGATCAAATTACCGCTGTCAGAAAAGGTAAGATTC ACCCAGAAAAAGGTACTGCCTTGTACGAATTTTCTCACTGGGAAGATATGAACGGTA AACCTATGGATGCTAGATTGTGTGCTGTTGATTTGATGAACGTTATCAGACCATTGG TTGCCATCAACAAGTTTGTTTCTTTTGGTGTTTTGGCCTTGCATGAATTTCCAGGTGA AAGAGTTAGAGTTGCTTTGAACGAAGGTGATTACGCTTACAAGTTCGTTCAAGAAGT CAGAAGATATTACCCATTCGTTCCATTTTTGCCAGGTAAGGCTAAAGAAAACATCAC TTTCGATGGTTACAAGATCCAAAAGGACACCATGATGTTGTTGGATATCTACGGTAC ATTGCACAGAGATGACTTGTTTTCTGAACCAGAAAGATTCAACCCATACAGATTCGA TAATTGGGATGGTTCTCCATTCGATTTGATTCCACAAGGTGGTGGTGATTACTACACT AATCATAGATGTGCTGGTGAATGGATGACCATCATTATTATGGAAGAAACCATGAA GTTCTTCGCCAACGAAATCTCTTATGATGTTCCACCACAAGATTTCACTGTTGATACC ACTAAGTTCCCAGGTAAAGTTGCTTCTGGTATGGATATCGAAAACATTAGAGTCAAC ATCGACAGAACTAAGTAA OleT.sub.SP(SEQIDNO3): ATGGCTAAGAAGTTGCCAAAGGATACTGGTTTGGATAACACCTTGAAGATGATTAA CGAAGCCTACACTTACGTCCCAAAGAGATTGGAAAAATTCGGTACTAAGGCTTTCGA AACTAGAGCTTTGGGTATGAAGCCAATCGTTGTTATTTCTGGTAAAGCTGCTGCCGA ATTATTCTACGATAACGACAAAATCTCCAGAAAGGGTACTTTGCCAAAGAGAATCGT TCATACTTTGTTTGGTAAGGGTGCTATTCATACCACTGAAGGTAAAGTTCACGTTGA TAGAAAGGCCTTGTTCATGTCTTTGATGACCGAAAAGAACTTGAAGTACTTGAGAGA ATTGACCAGAAACTACTGGTTCATGCATACCGAAAGAATGCAAAACAAGGATGAAG TCAACGTTTACCAAGAAGCCGGTTTGATTTTGACTAAGGTTGGTTTTAGATGGGCTG GTTTGAAGCAAACTGATGAACAAGCTGCTCAAAACGCTGAAGATATGAACACCATG ATCGATTCTTTTTCCGGTTTGGGTCAATCTTTGAAGGGTTACAGAGAAGCTAAAAAG GCTAGAGCTAGAGTCGAACAATTCTTACAAGAACAAATCGAAGCCGTTAGAGTCGG TCAACAATACGCTGAACCAGGTACTGCATTATACGAATTTGCTCATTGGAAGGACTT GAACGATCAACCTATGGATCCACATTTGTGTGCTGTTGATTTGATGAACATCGTTAG ACCATTGGTTGCCGTTAACAGATTTGTTTCTTATGGTGTTAAGGCCTTGATTGAATTC GACCAAGAAAGAAAAAAGTTGCAAGTTACCAACGATCCAAACTACGCTTACAAGTT CGCTCAAGAAGTTAGAAGAATCTTCCCATTCGTTCCATTTTTGCCAGGTAGATTGAA AAAGACCGTTGAATTTGACGGTTTCAAGTTGAAGAAGGGTACATTGACCGTTTTGGA TATTTTCGGTACAACCCACGATCCAGAATTATTCGAAAATCCATACCAATTCAACCC AGACAGATTCGATAATTGGGATGGTTCTCCATTCGATTTGATTCCACAAGGTGGTGG TGATTTCTACACTAATCATAGATGTGCTGGTGAATGGATGACCGTTATAGTTATGGA AGAAACCATTCAATACTTCGCCAACAAGATCGATTTCGTTGTTCCAGCTCAAGATTT GTCCGTTAAGTTGTCTCAATTTCCAGGTAAGGTTACCTCTGGTACTATGATCAAAAA TGTCTACCCAAGAATTTGA OleT.sub.BS(SEQIDNO4): ATGAACGAACAAATCCCACACGATAAGTCCTTGGATAACTCTTTGACCTTGTTGAAA GAAGGTTACTTGTTCATCAAGAACAGAACCGAAAGATACAACTCCGATTTGTTCCAA GCTAGATTATTGGGTAAGAACTTCATCTGTATGACTGGTGCTGAAGCTGCTAAGGTT TTTTACGATACTGACAGATTCCAAAGACAAAACGCTTTGCCAAAGAGAGTCCAAAA GTCTTTGTTTGGTGTTAACGCCATTCAAGGTATGGATGGTTCTGCTCATATTCACAGA AAGATGTTGTTCTTGTCTTTGATGACTCCACCACATCAAAAAAGATTGGCTGAATTG ATGACCGAAGAATGGAAAGCTGCTGTTACTAGATGGGAAAAAGCTGATGAAGTTGT CTTGTTCGAAGAAGCCAAAGAAATCTTGTGTAGAGTTGCTTGTTATTGGGCTGGTGT TCCATTGAAAGAAACCGAAGTAAAAGAAAGAGCCGACGATTTCATCGATATGGTTG ATGCTTTTGGTGCTGTTGGTCCAAGACATTGGAAAGGTAGAAGAGCTAGACCAAGA GCTGAAGAATGGATTGAAGTTATGATTGAAGATGCTAGAGCCGGTTTGTTGAAAACT ACTTCTGGTACTGCTTTACACGAAATGGCTTTCCATACTCAAGAAGATGGTTCCCAA TTGGATTCAAGAATGGCTGCTATTGAATTGATCAACGTTTTAAGACCAATCGTCGCT ATCTCCTACTTCTTGGTTTTTTCTGCTTTGGCCTTGCATGAACACCCAAAGTACAAAG AATGGTTGAGATCTGGTAACTCCAGAGAAAGAGAAATGTTCGTCCAAGAAGTCAGA AGATATTACCCATTTGGTCCATTTTTGGGTGCCTTGGTTAAGAAGGATTTTGTTTGGA ACAACTGCGAATTCAAGAAGGGTACTTCTGTTTTGTTGGACTTGTACGGTACTAATC ACGATCCAAGATTGTGGGATCATCCAGATGAATTCAGACCAGAAAGATTCGCCGAA AGAGAAGAAAACTTGTTCGACATGATTCCACAAGGTGGTGGTCATGCTGAAAAAGG TCATAGATGTCCAGGTGAAGGTATTACCATTGAAGTAATGAAGGCCTCCTTGGATTT TTTGGTTCACCAAATCGAATACGACGTCCCAGAACAATCATTGCATTATTCATTGGC TAGAATGCCATCCTTGCCAGAATCTGGTTTTGTTATGTCTGGTATCAGAAGAAAGTC TTAA OleT.sub.MP(SEQIDNO5): ATGCCAGCTGCTATTGCTACTCATAGATTCAGAAAAGCTAGAACCTTGCCAAGAGAA CCAGCTCCAGATTCTACTTTGGCTTTGTTGAGAGAAGGTTACGGTTTCATTAGAAAC AGATGCAGAAGACACGATTCCGATTTGTTTGCTGCTAGATTGTTGTTGTCTCCAGTTA TCTGTATGTCTGGTGCTGAAGCTGCTAGACATTTTTATGATGGTCACAGATTCACCA GAAGACATGCTTTGCCACCAACATCTTTTGCCTTGATTCAAGATCATGGTTCCGTTAT GGTTTTGGATGGTGCTGCTCATTTGGCTAGAAAAGCAATGTTTTTGTCCTTGGTTGGT GAAGAAGCCTTGCAAAGATTGGCTGGTTTGGCTGAAAGACATTGGAGAGAAGCTGT TTCTGGTTGGGCAAGAAAAGATACTGTTGTTTTGTTGGATGAAGCCCACAGAGTTTT GACTGCTGCTGTTTGTGAATGGGTTGGTTTGCCATTGGGTCCAACTGAAGTTGATGC TAGAGCTAGAGAATTTGCTGCAATGATTGATGGTACTGGTGCTGTTGGTCCAAGAAA TTGGAGAGGTCACTTGTATAGAGCAAGAACTGAAAGATGGGTTAGAAAGGTTATCG ACGAAATCAGATCTGGTAGAAGAGATGTTCCACCAGGTGCTGCAAGAACTATTGCT GAACATCAAGATGCTGACGGTCAAAGATTAGATAGAACTGTTGCTGGTGTCGAATT GATCAACGTTTTAAGACCAACAGTTGCCAACGCCAGATATATCGTTTTCGCTGCTAT GGCTTTACATGATCATCCACATCAAAGAGCTGCTTTAGCTGACGGTGGTGAAGCAGC TGAAAGATTCACTGATGAAGTTAGAAGATTCTACCCATTCATCCCTTTCATTGGTGG TAGAGTTAGAGCCCCATTTCATTTTGGTGGTCATGATTTTAGAGAAGGTGAATGGGT CTTGATGGACTTGTATGGTACTAATAGAGATCCAAGATTGTGGCACGAACCAGAAA GATTTGATCCAGATAGATTCGCCAGAGAAACCATTGATCCATTCAACATGGTTTCAC ATGGTGCTGGTTCTGCTAGAGATGGTCATAGATGTCCAGGTGAAGGTATTACCAGAA TCTTGTTGAGAACCTTGAGTAGACAATTGGCTGCTACTAGATATACAGTTCCACCAC AAGATTTGACTTTGGATTTGGCTCATGTTCCAGCTAGACCAAGATCTGGTTTTGTTAT GAGAGCTGTTCATGCTCCATGA OleT.sub.CE(SEQIDNO6): ATGGAAGAAGTTCCTCCAATGACTCAAACTTCTTCTTGTCCATTTGCTCCAGGTGAA CAAGCTCCAAATTTGTTGAGACATGGTTACTTGTTCTTGTCTAGATTGAGAAGAAAG GCCGGTATTTCTCCAGATGCTAATACTCCATTGAGATCCAGAATGTTGTTCAAGCCA GTTACTATCGTTAGAGGTTCTGCTGGTGTTGAATTATTCTACGATAACGACAGAATG AAGAGAGATGGTGCTATGCCAGCTGTTATTAGAATTCCTTTGTTTGGTGAAGGTGCC GTTCATTCTTTGGATGGTGAAGAACATAGATTAAGAAAAAGACAATTGGCCGATGTT GCCTACGATGATGATAAGGTTGCTGAATTTGATGCCTTGGTTAGAAGAGAAGTTGAT AGAGTTGTACAAGATTGGGCTAGAGAACCAGGTACTGTTTATGATGGTGCTGCTTTG GCTTTTGGTAGAGCTGCTTATAGATGGGCAGGTATTGAATTGTCTCAAAAAGAAGCT AGTAGAAGAGCCCATCAAATGGCTGAATTGGTTTACCAATTTGGTCATCCATTGAAG GGTCATGCTTTGGGTTGGATTAACAGAGCTAGATTGAACAGATGGGCCTTGAAGTTG ATTAGACAAGCTAGAGCTGGTGAAAGACATGTTGCACCAGGTTCAGCTTTGGAAGC TATGTCAAGATTGGTTGGTCCAGATGGTGAATTAGTTGATGCTTCTATTGCTGGTATC GAATTGCAAAACTTGACTAGACCAACTGTTGCCGTTTCTTTGTTTGCTTCATTTGCTG GTTCTGCATTGGTTGAACATCCTGAATGGGTTGAAAAGATTAGAGAAGGTGGTCAAC CAGTTGCATTTGCTTTTGCTCAAGAAGTCAGAAGAGTTTACCCATTCGTTCCAATGTT GCCAGCTATTGCTACTACTGATACTGAAATTCAAGGTTGCCCAGTTCATGAAGGTGA AAGAGTTATTATCGACATCTACGGTACTAATACCGATCCAAATGAATGGGAAAATCC ATCTGCATTCCAACCAGAAAGATTTTTGTCCAGAGAAGATTTGGGTACTCAAGAAGA TTACGAAAGATTGACCTCTTTCGTTCCACAAGGTGGTGCTGGTGTCTATACTGGTCAT AGATGTCCTGGTGAAAAAATTGCTATGGCTGCTTTGACTGCTATGGTTGAAGCTTTG TGTAGACCAGGTGTTGTTTTGTCTACTGATCCAGCTGATACAAGATTTCCATGGACTC AAATGTTGACCAGATCTGAAACTGGTATGAGAGTTAGAGTCGAAAGATAA OleT.sub.JE(SEQIDNO7): ATGGCAACACTTAAGAGGGATAAGGGCTTAGATAATACTTTGAAAGTATTAAAGCA AGGTTATCTTTACACAACAAATCAGAGAAATCGTCTAAACACATCAGTTTTCCAAAC TAAAGCACTCGGTGGTAAACCATTCGTAGTTGTGACTGGTAAGGAAGGCGCTGAAA TGTTCTACAACAATGATGTTGTTCAACGTGAAGGCATGTTACCAAAACGTATCGTTA ATACGCTTTTTGGTAAAGGTGCAATCCATACGGTAGATGGTAAAAAACACGTAGAC AGAAAAGCATTGTTCATGAGCTTGATGACTGAAGGTAACTTGAATTATGTACGAGA ATTAACGCGTACATTATGGCATGCGAACACACAACGTATGGAAAGTATGGATGAGG TAAATATTTACCGTGAATCTATCGTACTACTTACAAAAGTAGGAACACGTTGGGCAG GCGTTCAAGCACCACCTGAAGATATCGAAAGAATCGCAACAGACATGGACATCATG ATCGATTCATTTAGAGCACTTGGTGGTGCCTTTAAAGGTTACAAGGCATCAAAAGAA GCACGTCGTCGTGTTGAAGATTGGTTAGAAGAACAAATTATTGAGACTCGTAAAGG GAATATTCATCCACCAGAAGGTACAGCACTTTACGAATTTGCACATTGGGAAGACTA CTTAGGTAACCCAATGGACTCAAGAACTTGTGCGATTGACTTAATGAACACATTCCG CCCATTAATCGCAATCAACAGATTCGTTTCATTCGGTTTACACGCGATGAACGAAAA CCCAATCACACGTGAAAAAATTAAATCAGAACCTGACTATGCATATAAATTCGCTCA AGAAGTTCGTCGTTACTATCCATTCGTTCCATTCCTTCCAGGTAAAGCGAAAGTAGA CATCGACTTCCAAGGCGTTACAATTCCTGCAGGTGTAGGTCTTGCATTAGATGTTTAT GGTACAACGCATGATGAATCACTTTGGGACGATCCAAATGAATTCCGCCCAGAAAG ATTCGAAACTTGGGACGGATCACCATTTGACCTTATTCCACAAGGTGGTGGAGATTA CTGGACAAATCACCGTTGTGCAGGTGAATGGATCACAGTAATCATCATGGAAGAAA CAATGAAATACTTTGCAGAAAAAATAACTTATGATGTTCCAGAACAAGATTTAGAA GTGGACTTAAACAGTATCCCAGGATACGTTAAGAGTGGCTTTGTAATCAAAAATGTT CGCGAAGTTGTAGACAGAACATAA OleT.sub.JE-CO(SEQIDNO8): ATGGCTACTTTGAAGAGAGATAAGGGTTTGGATAACACCTTGAAGGTTTTGAAGCA AGGTTACTTGTACACCACCAATCAAAGAAACAGATTGAACACCTCCGTTTTCCAAAC AAAAGCTTTGGGTGGTAAGCCATTCGTTGTTGTTACTGGTAAAGAAGGTGCTGAAAT GTTCTACAACAATGACGTTGTTCAAAGAGAAGGTATGTTGCCAAAGAGAATTGTCA ACACTTTGTTTGGTAAGGGTGCCATTCATACTGTTGATGGTAAGAAACACGTTGACA GAAAGGCTTTGTTCATGTCTTTGATGACTGAAGGTAACTTGAACTACGTCAGAGAAT TGACTAGAACTTTGTGGCATGCTAACACCCAAAGAATGGAATCTATGGATGAAGTC AACATCTACAGAGAATCCATCGTTTTGTTGACCAAGGTTGGTACTAGATGGGCTGGT GTTCAAGCTCCACCAGAAGATATTGAAAGAATTGCTACCGATATGGACATCATGATC GATTCTTTTAGAGCTTTAGGTGGTGCTTTCAAAGGTTACAAGGCTTCTAAAGAAGCC AGAAGAAGAGTTGAAGATTGGTTGGAAGAACAAATCATCGAAACCAGAAAGGGTA ACATTCATCCACCTGAAGGTACTGCCTTGTATGAATTTGCTCATTGGGAAGATTACTT GGGTAACCCAATGGATTCTAGAACCTGTGCTATTGATTTGATGAACACCTTCAGACC ATTGATCGCCATTAACAGATTTGTTTCTTTCGGTTTACACGCCATGAACGAAAACCC AATTACCAGAGAAAAGATCAAGTCCGAACCAGATTACGCTTACAAGTTTGCTCAAG AAGTTAGAAGATATTACCCATTCGTCCCATTTTTGCCAGGTAAAGCTAAGGTTGATA TCGATTTCCAAGGTGTCACTATTCCAGCTGGTGTTGGTTTGGCTTTGGATGTTTATGG TACTACCCATGATGAATCCTTGTGGGATGATCCAAATGAATTCAGACCAGAAAGATT CGAAACTTGGGATGGTTCTCCATTCGATTTGATTCCACAAGGTGGTGGTGATTACTG GACTAATCATAGATGTGCCGGTGAATGGATTACCGTTATTATCATGGAAGAAACCAT GAAGTACTTTGCCGAAAAGATTACCTACGATGTTCCAGAACAAGATTTGGAAGTTGA CTTGAACTCTATTCCAGGTTACGTTAAGTCCGGTTTCGTTATTAAGAACGTTAGAGA AGTTGTCGACAGAACTTAA