Enzymes and methods for styrene synthesis

Abstract

The subject technology generally relates to biosynthesis of styrene. Certain embodiments of the subject technology is based, in part, on the recognition that phenylalanine can be converted to styrene by a two-step pathway of deamination and decarboxylation, with trans-cinnamic acid (tCA) as the intermediate. Two types of enzymes are directly involved in this process, phenylalanine ammonia lyase (PAL), which converts phenylalanine to tCA, and cinnamic acid decarboxylase, which coverts tCA to styrene. Host cells expressing these two types of enzymes can be cultured in bioreactor to produce styrene from renewable substrates such as glucose.

1 . A fusion protein comprising: (a) a first domain comprising a phenylalanine ammonia lyase, and (b) a second domain comprising a cinnamic acid decarboxylase. 
     
     
         2 . The fusion protein of  claim 1 , wherein the phenylalanine ammonia lyase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and a functional fragment or variant thereof. 
     
     
         3 . The fusion protein of  claim 1 , wherein the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:8; SEQ ID NO: 10; SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, and a functional fragment or variant thereof. 
     
     
         4 . The fusion protein of  claim 1 , wherein the cinnamic acid decarboxylase is a mutant cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to a position selected from the group consisting of: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and combinations thereof. 
     
     
         5 - 8 . (canceled) 
     
     
         9 . A nucleic acid encoding the fusion protein of  claim 1 . 
     
     
         10 . A vector comprising the nucleic acid of  claim 9 . 
     
     
         11 . A host cell comprising the vector of  claim 10 . 
     
     
         12 . A method for producing styrene, the method comprising:
 (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising a fusion protein of  claim 1 ; and   (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.   
     
     
         13 . A cinnamic acid decarboxylase comprising a mutation at an amino acid residue position corresponding to a position selected from the group consisting of: 155, 156, 159, 162, 163, 164, 172, 173, 174, 175, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 226, 227, 280, 285, 286, 287, 291, 326, 331, 360, 361, 395, 396, 398, 440, 441 of SEQ ID NO:8, and combinations thereof. 
     
     
         14 . (canceled) 
     
     
         15 . The cinnamic acid decarboxylase of  claim 13 , wherein the cinnamic acid decarboxylase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38, and a functional fragment thereof. 
     
     
         16 . An isolated nucleic acid encoding the cinnamic acid decarboxylase of  claim 13 . 
     
     
         17 - 18 . (canceled) 
     
     
         19 . A method for the production of styrene, the method comprising:
 (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) the cinnamic acid decarboxylase of  claim 13 ; and   (b) culturing the host cell in a culture medium for a time sufficient to produce styrene.   
     
     
         20 - 29 . (canceled) 
     
     
         30 . A method of crystallizing a cinnamic acid decarboxylase, the method comprising
 (a) providing a cinnamic acid decarboxylase solution at a concentration of from about 1 mg/ml to about 50 mg/ml;   (b) mixing the cinnamic acid decarboxylase solution with a reservoir solution at a volume ratio of from about 1:10 to about 10:1; and   (c) maintaining the mixture of step (b) at a temperature suitable for the formation of the cinnamic acid decarboxylase crystal.   
     
     
         31 - 32 . (canceled) 
     
     
         33 . The method of  claim 30 , wherein the cinnamic acid decarboxylase is in a complex with a small molecule selected from the group consisting of trans-cinnamic acid, 3-hydroxyl cinnamic acid, ferulic acid, 2-methylcinnamic acid, 4-hydroxy-cinnamic acid, 3,4-dimethoxycinnamic acid, 2,5-dimethoxy-cinnamic acid, and combinations thereof. 
     
     
         34 - 36 . (canceled) 
     
     
         37 . The method of  claim 30 , wherein the cinnamic acid decarboxylase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:8. 
     
     
         38 . A crystal of cinnamic acid decarboxylase, wherein the cinnamic acid decarboxylase is in a complex with 3-hydroxyl cinnamic acid. 
     
     
         39 . The crystal of  claim 38 , wherein the cinnamic acid decarboxylase comprises an amino acid sequence as set forth in SEQ ID NO:16. 
     
     
         40 . The crystal of  claim 39 , wherein the crystal has unit cell parameters a=249.51 Å, b=120.67 Å, c=158.49 Å, and β=94.9°. 
     
     
         41 . A method for producing styrene, the method comprising:
 (a) contacting a host cell with a fermentable carbon substrate, the host cell comprising (i) a phenylalanine ammonia lyase; and (ii) a cinnamic acid decarboxylase; and   (b) culturing the host cell in a culture medium for a time sufficient to produce styrene, wherein the vapor of the styrene product is absorbed by an absorbing material.   
     
     
         42 . The method of  claim 41 , wherein the absorbing material is selected from the group consisting of polymeric resin, activated carbon, cellulosic material, and combination thereof. 
     
     
         43 - 46 . (canceled)