Bioproduction of para-hydroxycinnamic acid
First Claim
1. A method for the production of PHCA comprising:
- (i) contacting a recombinant host cell with a fermentable carbon substrate, said recombinant cell lacking a cinnamate hydroxylase activity and comprising a gene encoding a tyrosine ammonia lyase activity operably linked to suitable regulatory sequences;
(ii) growing said recombinant cell for a time sufficient to produce PHCA; and
(iii) optionally recovering said PHCA.
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Abstract
The present invention provides several methods for biological production of para-hydroxycinnamic acid (PHCA). The invention is also directed to the discovery of new fungi and bacteria that possess the ability to convert cinnamate to PHCA. The invention relates to developing of a new biocatalyst for conversion of glucose to PHCA by incorporation of the wild type PAL from the yeast Rhodotorula glutinis into E. coli underlining the ability of the wildtype PAL to convert tyrosine to PHCA. The invention is also directed to developing a new biocatalyst for conversion of glucose to PHCA by incorporation of the wildtype PAL from the yeast Rhodotorula glutinis plus the plant cytochrome P-450 and the cytochrome P-450 reductase into E. coli. In yet another embodiment, the present invention provides for the developing of a new biocatalyst through mutagenesis of the wild type yeast PAL which possesses enhanced tyrosine ammonia-lyase (TAL) activity.
24 Citations
26 Claims
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1. A method for the production of PHCA comprising:
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(i) contacting a recombinant host cell with a fermentable carbon substrate, said recombinant cell lacking a cinnamate hydroxylase activity and comprising a gene encoding a tyrosine ammonia lyase activity operably linked to suitable regulatory sequences;
(ii) growing said recombinant cell for a time sufficient to produce PHCA; and
(iii) optionally recovering said PHCA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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- 10. A recombinant host cell lacking a cinnamate hydroxylase activity and comprising a gene encoding a tyrosine ammonia lyase activity operably linked to suitable regulatory sequences.
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11. A recombinant host cell comprising a gene encoding a tyrosine ammonia lyase activity operably linked to suitable regulatory sequences selected from the group consisting of cells having the ATCC designation PTA 407 and PTA 409.
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13. A tyrosine ammonia lyase gene encoding the polypeptide set forth in SEQ ID NO:
- 10.
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14. A polypeptide as set forth in SEQ ID NO:
- 10
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15. A method for the production of PHCA comprising:
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(i) contacting a recombinant yeast cell with a fermentable carbon substrate , said recombinant cell comprising;
a) genes encoding a plant P-450/P-450 reductase system; and
b) a gene encoding a yeast PAL activity operably linked to suitable regulatory sequences;
(ii) growing said recombinant cell for a time sufficient to produce PHCA; and
(iii) optionally recovering said PHCA. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22)
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23. A recombinant host cell having the ATCC designation, PTA 408.
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24. A method for the production of PHCA comprising:
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(i) contacting a microbial cell selected from the group consisting of Streptomyces griseus (ATCC 13273, ATCC 13968, TU6), Rhodococcus erythropolis (ATCC
4277), Aspergillus petrakii (ATCC
12337), Aspergillus niger (ATCC
10549) and Arthrobotrys robusta (ATCC
11856) with cinnamate;
(ii) growing said microbial cell for a time sufficient to produce PHCA; and
(iii) optionally recovering said PHCA.
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25. A method for identifying a gene encoding a TAL activity comprising:
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(i) contacting a recombinant microorganism comprising a foreign gene suspected of encoding a TAL activity with PHCA for a time sufficient to metabolize PHCA; and
(ii) monitoring the growth of the recombinant microorganism whereby growth of the organism indicates the presence of a gene encoding a TAL activity.
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26. A method for identifying a gene encoding a TAL activity comprising:
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(i) transforming a host cell which uses PHCA as a sole carbon source with a gene suspected of encoding a TAL activity to create a transformant;
(ii) comparing the rate of growth of the transformant with an un-transformed host cell capable of using PHCA as a sole carbon source wherein an accelerated rate of growth by the transformant indicates the presence of a gene encoding a TAL activity.
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Specification