Process for the biological production of 1,3-propanediol with high titer

Abstract

The present invention provides an improved method for the biological production of 1,3-propanediol from a fermentable carbon source in a single microorganism. In one aspect of the present invention, an improved process for the conversion of glucose to 1,3-propanediol is achieved by the use of an E. coli transformed with the Klebsiella pneumoniae dha regulon genes dhaR, orfY, dhaT, orfX, orfW, dhaB1, dhaB2, dhaB3, and orfZ, all these genes arranged in the same genetic organization as found in wild type Klebsiella pneumoniae . In another aspect of the present invention, an improved process for the production of 1,3-propanediol from glucose using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, and a dehydratase reactivation factor compared to an identical process using a recombinant E. coli containing genes encoding a G3PDH, a G3P phosphatase, a dehydratase, a dehydratase reactivation factor and a 1,3-propanediol oxidoreductase (dhaT). The dramatically improved process relies on the presence in E. coli of a gene encoding a non-specific catalytic activity sufficient to convert 3-hydroxypropionaldehyde to 1,3-propanediol.

1 - 6 . (canceled) 
     
     
         7 . A recombinant microorganism comprising genes encoding a G3PDH, a G3P phosphtase, a dehydratase, and a dehydratase reactivation factor
 wherein no functional dhaT gene encoding a 1,3 propanediol oxidoreductase activity is present in the recombinant microorganism and the microorganism is selected from the group consisting of  Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Bacillus, Aerobacter, Streptomyces  and  Pseudomonas.      
     
     
         8 - 10 . (canceled) 
     
     
         11 . The recombinant microorganism of  claim 7 , further comprising a set of endogenous genes, each having a mutation inactivating the gene, the set consisting of:
 (a) a first gene encoding a polypeptide having glycerol kinase activity;   (b) a second gene encoding a polypeptide having glycerol dehydrogenase activity; and   (c) a third gene encoding a polypeptide having triosephosphate isomerase activity.   
     
     
         12 - 17 . (canceled) 
     
     
         18 . A recombinant  E. coli  comprising:
 a set of exogenous genes consisting of:   (i) at least one gene encoding a polypeptide having a dehydratase activity;   (ii) at least one gene encoding a polypeptide having glycerol 3-phosphate dehydrogenase activity;   (iii) at least one gene encoding a polypeptide having glycerol 3-phosphatase activity; and   (iv) at least one gene encoding a dehydratase reactivation factor;   wherein no functional dhaT gene encoding a 1,3 propanediol oxidoreductase activity is present in the recombinant  E. coli.      
     
     
         19 . A recombinant  E. coli  comprising:
 a set of exogenous genes consisting of   (i) at least one gene encoding a polypeptide having glycerol 3-phosphate dehydrogenase activity;   (ii) at least one gene encoding a polypeptide having glycerol 3-phosphatase activity; and   (iii) at least one subset of genes encoding the gene products of dhaR, orfY, orfX, orfW, dhaB1, dhaB2, dhaB3 and orfZ,   wherein no functional dhaT gene encoding a 1,3 propanediol oxidoreductase activity is present in the recombinant  E. coli.      
     
     
         20 . The recombinant  E. coli  of  claim 19  further comprising a set of endogenous genes, each gene having a mutation inactivating the gene, the set consisting of:
 (a) a gene encoding a polypeptide having glycerol kinase activity;   (b) a gene encoding a polypeptide having glycerol dehydrogenase activity; and   (c) a gene encoding a polypeptide having triosephosphate isomerase activity.   
     
     
         21 - 29 . (canceled)