Production of polyhydroxyalkanoates from polyols

Abstract

Recombinant processes are provided whereby additional genes are introduced into E. coli which have been genetically engineered to produce PHA so that the improved strains produce PHA homopolymers and copolymers directly from diols. In preferred embodiments, PHAs containing 4-hydroxybutyrate monomers are produced directly from 1,4-butanediol; PHAs containing 5-hydroxyvalerate are produced from 1,5-pentanediol; PHAs containing 6-hydroxyhexanoate (6HH) are produced from 1,6-hexanediol; PHAs containing 3-hydroxypropionate are produced from 1,3-propanediol; PHAs containing 2-hydroxypropionate (lactate) are produced from 1,2-propanediol (propylene glycol); PHAs containing 2-hydroxyethanoate (glycolate) are produced from 1,2-ethanediol (ethylene glycol). Genes encoding these same enzyme activities can be introduced or their expression amplified in wild type PHA producers to improve the production of PHA homopolymers and copolymers directly from diol and other alcohol feedstocks. The PHA polymers are readily recovered and industrially useful as polymers or as starting materials for a range of chemical intermediates.

We claim:  
     
         1 . A method for producing polyhydroxyalkanoates comprising 
 providing genetically engineered organisms which express enzymes selected from the group consisting of diol oxidoreductase, aldehyde dehydrogenase, acyl-CoA transferase, acyl-CoA synthetase, β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase,    providing diols which can be converted into hydroxyalkanoate monomers by enzymes expressed by the organisms, and    culturing the organisms under conditions wherein the hydroxyalkanoate monomers are polymerized to form polyhydroxyalkanoates.    
     
     
         2 . The method of  claim 1  wherein the diol is 1,6-hexanediol and the hydroxyalkanoate monomer is 6-hydroxyhexanoate.  
     
     
         3 . The method of  claim 1  wherein the diol is 1,5-pentanediol and the hydroxyalkanoate monomer is 5-hydroxyvalerate.  
     
     
         4 . The method of  claim 1  wherein the diol is 1,4-butanediol and the hydroxyalkanoate is 4-hydroxybutyrate.  
     
     
         5 . The method of  claim 1  wherein the diol is 1,3-propanediol and the hydroxyalkanoate monomer is 3-hydroxypropionate.  
     
     
         6 . The method of  claim 1  wherein the diol is 1,2-ethanediol and the hydroxyalkanoate is 2-hydroxyethanoate.  
     
     
         7 . The method of  claim 1  wherein the diol is 1,2-propanediol and the hydroxyalkanoate is 2-hydroxypropionate.  
     
     
         8 . A genetically engineered organism for use in the method of  claim 1  comprising an organism which expresses the aldH and dhaT genes.  
     
     
         9 . The organism of  claim 8  wherein the organism is selected from the group consisting of  Escherichia coli, Ralstonia eutropha,  Klebsiella spp.,  Alcaligenes latus,  Azotobacter spp., and Comamonas spp.  
     
     
         10 . A system for making polyhydroxyalkanoates comprising an organism genetically engineered to express enzymes selected from the group consisting of a diol oxidoreductase, aldehyde dehydrogenase, acyl-CoA transferase, acyl-CoA synthetase, β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase, 
 wherein the organism can convert diols into hydroxyalkanoate monomers which are polymerized to form polyhydroxyalkanoates.  
 
     
     
         11 . A composition comprising a polyhydroxyalkanoate copolymer which includes 
 2-hydroxypropionate or 2-hydroxyethanoate or both, and    at least one comonomer selected from the group consisting of 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxypropionate, 2-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate, 6-hydroxyhexanoate, and 3-hydroxyhexanoate, having a weight-average molecular weight (Mw) of at least 300,000.    
     
     
         12 . The composition of example 11 where the comonomer is 3-hydroxybutyrate.  
     
     
         13 . The composition of example 11 where the comonomer is 4-hydroxybutyrate.  
     
     
         14 . The composition of example 11 where the comonomer is 3-hydroxypropionate.  
     
     
         15 . The composition of example 11 where the comonomer is 2-hydroxybutyrate.  
     
     
         16 . The composition of example 11 where the comonomer is 4-hydroxyvalerate.  
     
     
         17 . The composition of example 11 where the comonomer is 5-hydroxyvalerate.  
     
     
         18 . The composition of example 11 where the comonomer is 6-hydroxyhexanoate.  
     
     
         19 . The composition of example 11 where the comonomer is 3-hydroxyhexanoate.  
     
     
         20 . A method for improving a biological system for making polyhydroxyalkanoates with an organism genetically engineered to express enzymes selected from the group consisting of a diol oxidoreductase, aldehyde dehydrogenase, acyl-CoA transferase, acyl-CoA synthetase, β-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase, wherein the organism can convert diols into hydroxyalkanoate monomers which are polymerized to form polyhydroxyalkanoates, the method comprising selecting for mutants with increased enzyme activities by 
 i) introducing mutations into a specific host, and  
 ii) screening pools of the mutants generated for increased ability to synthesize PHA from a selected diol or diols.  
 
     
     
         21 . A DNA fragment encoding a diol oxidoreductase and an aldehyde dehydrogenase, wherein the expressed enzymes can produce hydroxyalkanoate monomer selected from the group consisting of 3-hydroxybutyrate, 4-hydroxybutyrate, 3-hydroxypropionate, 2-hydroxybutyrate, 4-hydroxyvalerate, 5-hydroxyvalerate, 6-hydroxyhexanoate, 3-hydroxyhexanoate, 2-hydroxypropionate, and 2-hydroxyethanoate from diol.