Bioproduction of para-hydroxycinnamic acid

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.

What is claimed is:  
     
         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.    
     
     
         2 . A method according to  claim 1  wherein said fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, carbon dioxide, methanol, formaldehyde, formate, and carbon-containing amines.  
     
     
         3 . A method according to  claim 2  wherein said fermentable carbon substrate is glucose.  
     
     
         4 . A method according to  claim 1  wherein said recombinant host cell is selected from the group consisting of bacteria, yeasts, filamentous fungi, algae and plant cells.  
     
     
         5 . A method according to  claim 4  wherein said recombinant host cell is selected from the group consisting of Aspergillus, Arthrobotrys, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Rhodobacter, Synechocystis, Streptomyces, and Pseudomonas.  
     
     
         6 . A method according to  claim 1  wherein said recombinant host cell is selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, broccoli, cauliflower, cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses.  
     
     
         7 . A method according to  claim 1  wherein said tyrosine ammonia lyase has a catalytic efficiency from about 4.14×10 3  M −1 sec −1  to about 1×10 9  M −1 sec −1 .  
     
     
         8 . A method according to  claim 1  wherein said gene encoding a tyrosine ammonia lyase activity encodes the polypeptide set forth in SEQ ID NO:8 or SEQ ID NO:10.  
     
     
         9 . A method according to  claim 1  wherein the gene encoding a tyrosine ammonia lyase activity is derived from Rhodosporidium sp.  
     
     
         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.  
     
     
         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.  
     
     
         12 . A cell according to  claim 10  wherein said tyrosine ammonia lyase has a catalytic efficiency from about 4.14×10 3  M −1 sec −1  to about 1×10 9  M −1 sec −1 .  
     
     
         13 . A tyrosine ammonia lyase gene encoding the polypeptide set forth in SEQ ID NO:10.  
     
     
         14 . A polypeptide as set forth in SEQ ID NO:10  
     
     
         15 . A method for the production of PHCA comprising: 
 (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.    
     
     
         16 . A method according to  claim 15  wherein said fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, carbon dioxide, methanol, formaldehyde, formate, and carbon-containing amines.  
     
     
         17 . A method according to  claim 16  wherein said fermentable carbon substrate is glucose.  
     
     
         18 . A method according to  claim 15  wherein said recombinant yeast cell is selected from the group consisting of Aspergillus, Arthrobotrys, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, and Penicillium.  
     
     
         19 . A method according to  claim 15  wherein the genes encoding a plant P-450/P-450 reductase system are derived from plants selected from the group consisting of Jerusilum artichoke, soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, broccoli, cauliflower, cabbage, parsnips, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees, and forage grasses.  
     
     
         20 . A method according to  claim 19  wherein the genes encoding a plant P-450/P-450 reductase system are set forth in SEQ ID NO:11 and SEQ ID NO:13.  
     
     
         21 . A method according to  claim 15  wherein the gene encoding a yeast PAL activity is derived from the group consisting of Rhodotorula sp., Rhodosporidium sp. and Sporobolomyces sp.  
     
     
         22 . A method according to  claim 21  wherein the gene encoding a yeast PAL activity encodes a polypeptide as set forth in SEQ ID NO:8.  
     
     
         23 . A recombinant host cell having the ATCC designation, PTA 408.  
     
     
         24 . A method for the production of PHCA comprising: 
 (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.    
     
     
         25 . A method for identifying a gene encoding a TAL activity comprising: 
 (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.    
     
     
         26 . A method for identifying a gene encoding a TAL activity comprising: 
 (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.