Isoprenoid production
AssigneeRoche Vitamins Inc.
InventorsAlan Berry, Werner Bretzel, Markus Humbelin, Rual Lopez-Ulibarri, Anne Francoise Mayer, Alexei A. Yeliseev
Life of this patent
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Abstract
Isolated polynucleotides encoding polypeptides having the activity of enzymes in the mevalonate pathway are provided. These sequences are useful for recombinantly producing isoprenoid compounds, such as carotenoids, in particular zeaxanthin. Expression vectors, cultured cells, and methods of making isoprenoid compounds are also provided.
Claims146
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
- (a) an amino acid sequence shown as residues 1 to 340 of SEQ ID NO:
- 43;
- (b) an amino acid sequence shown as residues 1 to 349 of SEQ ID NO:
- 45;
- (c) an amino acid sequence shown as residues 1 to 388 of SEQ ID NO:
- 47;
- (d) an amino acid sequence shown as residues 1 to 378 of SEQ ID NO:
- 49;
- (e) an amino acid sequence shown as residues 1 to 305 of SEQ ID NO:
- 51;
- (f) an amino acid sequence shown as residues 1 to 332 of SEQ ID NO:
- 53;
- (g) at least 30 contiguous amino acid residues from a polypeptide selected from the group consisting of SEQ ID NOs:
- 43, 45, 47, 49, 51, and 53;
- (h) an amino acid sequence of a fragment of a polypeptide selected from the group consisting of SEQ ID NOs:
- 43, 45, 47, 49, 51, and 53, the fragment having the activity of hydroxymethylglutaryl-CoA reductase (HMG-COA reductase), isopentenyl diphosphate isomerase, hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase), mevalonate kinase, phosphomevalonate kinase, or diphosphomevalonate decarboxylase;
- (i) an amino acid sequence of a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a hybridization probe comprising at least 30 consecutive nucleotides of SEQ ID NO:
- 42 or a complement of SEQ ID NO:
- 42, wherein the polypeptide has the activity of HMG-CoA reductase, isopentenyl diphosphate isomerase, HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase, or diphosphomevalonate decarboxylase;
- and
- (j) a conservatively modified variant of SEQ ID NO:
- 43, 45, 47, 49, 51 or 53.
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
- (a) an amino acid sequence shown as residues 1 to 287 of SEQ ID NO:
- 159;
- (b) at least 30 contiguous amino acid residues of SEQ ID NO:
- 159;
- (c) an amino acid sequence of a fragment of SEQ ID NO:
- 159, the fragment having the activity of farnesyl diphosphate synthase (FPP synthase);
- (d) an amino acid sequence of a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a hybridization probe comprising at least 30 consecutive nucleotides spanning positions 295-1158 of SEQ ID NO:
- 157 or a complement thereof, wherein the polypeptide has the activity of FPP synthase;
- and
- (e) a conservatively modified variant of SEQ ID NO:
- 159.
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
- (a) an amino acid sequence shown as residues 1 to 142 of SEQ ID NO:
- 160;
- (b) at least 30 contiguous amino acid residues of SEQ ID NO:
- 160;
- (c) an amino acid sequence of a fragment of SEQ ID NO:
- 160, the fragment having the activity of 1-deoxyxylulose-5-phosphate synthase (DXPS);
- (d) an amino acid sequence of a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a hybridization probe comprising at least 30 consecutive nucleotides spanning positions 1185-1610 of SEQ ID NO:
- 157 or a complement thereof, wherein the polypeptide has the activity of DXPS;
- (e) a conservatively modified variant of SEQ ID NO:
- 160.
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
- (a) an amino acid sequence shown as residues 1 to 390 of SEQ ID NO:
- 178;
- (b) at least 30 contiguous amino acid residues of SEQ ID NO:
- 178;
- (c) an amino acid sequence of a fragment of a polypeptide of SEQ ID NO:
- 178, the fragment having the activity of acetyl-CoA acetyltransferase;
- (d) an amino acid sequence of a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a hybridization probe comprising at least 30 consecutive nucleotides spanning positions 1-1170 of SEQ ID NO:
- 177 or a complement thereof, wherein the polypeptide has the activity of acetyl-CoA acetyltransferase;
- and
- (e) a conservatively modified variant of SEQ ID NO:
- 178.
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
- (a) an amino acid sequence shown as residues 1 to 240 of SEQ ID NO:
- 179;
- (b) at least 30 contiguous amino acid residues of SEQ ID NO:
- 179;
- (c) an amino acid sequence of a fragment of a polypeptide of SEQ ID NO:
- 179, the fragment having the activity of acetoacetyl-CoA reductase;
- (d) an amino acid sequence of a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a hybridization probe comprising at least 30 consecutive nucleotides spanning positions 1258-1980 of SEQ ID NO:
- 177 or a complement thereof, wherein the polyp eptide has the activity of acetoacetyl-CoA reductase;
- and
- (e) a conservatively modified variant of SEQ ID NO:
- 179.
An isolated polynucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:
- 42, variants of SEQ ID NO:
- 42 containing one or more substitutions according to the Paracoccus sp. strain R1534 codon usage table (Table 14), fragments of SEQ ID NO:
- 42 that encode a polypeptide having an activity selected from the group consisting of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), isopentenyl diphosphate isomerase, hydroxymethylglutaryl-CoA synthase (HMG-CoA synthase), mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase, and polynucleotide sequences that hybridize under stringent conditions to a hybridization probe the nucleotide sequence of which consists of at least 30 contiguous nucleotides of SEQ ID NO:
- 42, or the complement of SEQ ID NO:
- 42, which hybrid encodes a polypeptide having an activity selected from the group consisting of HMG-CoA reductase, isopentenyl diphosphate isomerase, HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase.
An isolated polynucleotide sequence comprising a polynucleotide
An isolated polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of the nucleotide sequence spanning positions 295-1158 of SEQ ID NO:
- 157, variants of the nucleotide sequence spanning positions 295-1158 of SEQ ID NO:
- 157 containing one or more substitutions according to the Paracoccus sp. strain R1534 codon usage table (Table 14), fragments of the nucleotide sequence spanning positions 295-1158 of SEQ ID NO:
- 157 that encode a FPP synthase activity, and polynucleotide sequences that hybridize under stringent conditions to a hybridization probe the nucleotide sequence of which consists of at least 30 contiguous nucleotides spanning positions 295-1158 of SEQ ID NO:
- 157, or the complement of such a sequence, wherein the hybrid encodes a polypeptide having FPP synthase activity.
An isolated polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of the nucleotide sequence spanning positions 1185-1610 of SEQ ID NO:
- 157, variants of the nucleotide sequence spanning positions 1185-1610 of SEQ ID NO:
- 157 containing one or more substitutions according to the Paracoccus sp. strain R1534 codon usage table (Table 14), fragments of the nucleotide sequence spanning positions 1185-1610 of SEQ ID NO:
- 157 that encode a polypeptide having 1-deoxyxylulose-5-phosphate synthase activity, and polynucleotide sequences that hybridize under stringent conditions to a hybridization probe the nucleotide sequence of which consists of at least 30 contiguous nucleotides spanning positions 1185-1610 of SEQ ID NO:
- 157, or the complement of such a sequence, wherein the hybrid encodes a polypeptide having 1-deoxyxylulose-5-phosphate synthase activity.
An isolated polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO:
- 177, variants of SEQ ID NO:
- 177 containing one or more substitutions according to the Paracoccus sp. strain R1534 codon usage table (Table 14), fragments of SEQ ID NO:
- 177 that encode a polypeptide having an activity selected from the group consisting of acetyl-CoA acetyltransferase and acetoacetyl-CoA reductase, and polynucleotide sequences that hybridize under stringent conditions to a hybridization probe the nucleotide sequence of which consists of at least 30 contiguous nucleotides of SEQ ID NO:
- 177, or the complement of SEQ ID NO:
- 177, which hybrid encodes a polypeptide having an activity selected from the group consisting of acetyl-CoA acetyltransferase and acetoacetyl-CoA reductase.
An isolated polynucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:
- 42, SEQ ID NO:
- 157, SEQ ID NO:
- 177, and combinations thereof.
An expression vector selected from the group consisting of pBBR-K-mev-op16-1 and pBBR-K-mev-op16-2.
An expression vector selected from the group consisting of pBBR-K-mev-op16-1, pBBR-K-mev-op16-2, pDS-mvaA, pDS-idi, pDS-hcs, pDS-mvk, pDS-pmk, pDS-mvd, pDS-His-mvaA, pDS-His-idi, pDS-His-hcs, pDS-His-mvk, pDS-His-pmk, pDS-His-mvd, pBBR-K-Zea4, pBBR-K-Zea4-up, pBBR-K-Zea4-down, pBBR-K-PcrtE-crtE-3, pBBR-tK-PcrtE-mvaA, pBBR-tK-PcrtE-idi, pBBR-tK-PcrtE-hcs, pBBR-tK-PcrtE-mvk, pBBR-tK-PcrtE-pmk, pBBR-tK-PcrtE-mvd, pBBR-K-PcrtE-mvaA-crtE-3, pDS-His-phaA, pBBR-K-PcrtE-crtW, pBBR-K-PcrtE-crtWZ, pBBR-K-PcrtE-crtZW, and combinations thereof.
An expression vector selected from the group consisting of pBBR-K-Zea4, pBBR-K-Zea4-up, and pBBR-K-Zea4-down.
An expression vector selected from the group consisting of pBBR-K-PcrtE-crtE-3, pBBR-tK-PcrtE-mvaA, pBBR-tK-PcrtE-idi, pBBR-tK-PcrtE-hcs, pBBR-tK-PcrtE-mvk, pBBR-tK-PcrtE-pmk, pBBR-tK-PcrtE-mvd, and combinations thereof.
An expression vector which is pBBR-K-PcrtE-mvaA-crtE-3.
An expression vector which is pDS-His-phaA.
An expression vector selected from the group consisting of pBBR-K-PcrtE-crtW, pBBR-K-PcrtE-crtWZ, and pBBR-K-PcrtE-crtZW.
A method of making a carotenoid-producing cell comprising:
- (a) introducing into a cell a polynucleotide sequence encoding an enzyme in the mevalonate pathway, which enzyme is expressed in the cell;
- and
- (b) selecting a cell containing the polynucleotide sequence of step (a) that produces a carotenoid at a level that is about 1.1-1,000 times the level of the carotenoid produced by the cell before introduction of the polynucleotide sequence.
A method for engineering a bacterium to produce an isoprenoid compound comprising:
- (a) culturing a parent bacterium in a medium under conditions permitting expression of an isoprenoid compound, and selecting a mutant bacterium from the culture medium that produces about 1.1-1,000 times more of an isoprenoid compound than the parent bacterium;
- (b) introducing into the mutant bacterium an expression vector comprising a polynucleotide sequence represented by SEQ ID NO:
- 42 operably linked to an expression control sequence;
- and
- (c) selecting a bacterium that contains the expression vector and produces at least about 1.1 times more of an isoprenoid compound than the mutant in step (a).
A microorganism of the genus Paracoccus, which microorganism has the following characteristics:
- (a) a sequence similiarity to SEQ ID NO:
- 12 of >97% using a similarity matrix obtained from a homology calculation using GeneCompar v. 2.0 software with a gap penalty of 0%;
- (b) a homology to strain R-1512, R1534, R114 or R-1506 of >70% using DNA:DNA hybridization at 81.5° C.;
- (c) a G+C content of its genomic DNA that varies less than 1% from the G+C content of the genomic DNA of R114, R-1512, R1534, and R-1506;
- and
- (d) an average DNA fingerprint that clusters at about 58% similarity to strains R-1512, R1534, R114 and R-1506 using the AFLP procedure of Example 2, with the proviso that the microorganism is not Paracoccus sp. (MBIC3966).
A microorganism of the genus Paracoccus, which microorganism has the following characteristics:
- (a) 18:1w7c comprising at least about 75% of the total fatty acids of the cell membranes;
- (b) an inability to use adonitol, i-erythritol, gentiobiose, β-methylglucoside, D-sorbitol, xylitol and quinic acid as carbon sources for growth;
- and
- (c) an ability to use L-asparagine and L-aspartic acid as carbon sources for growth, with the proviso that the microorganism is not Paracoccus sp. (MBIC3966).
A microorganism of the genus Paracoccus, which microorganism has the following characteristics:
- (a) an ability to grow at 40° C.;
- (b) an ability to grow in a medium having 8% NaCl;
- (c) an ability to grow in a medium having a pH of 9.1;
- and
- (d) a yellow-orange colony pigmentation, with the proviso that the microorganism is not Paracoccus sp. (MBIC3966).
Dependencies are inferred from the claim text.