Compositions and methods for improved isoprene production using two types of ispg enzymes

Abstract

The invention provides for compositions and methods for producing isoprene by using recombinantly engineered cells that utilize a system of dual IspG enzymes in addition to isoprene synthase.

What is claimed is: 
     
         1 . A method of producing isoprene, the method comprising:
 (a) culturing recombinant cells capable of producing isoprene comprising (i) a nucleic acid encoding a first 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) polypeptide of a first species and a nucleic acid encoding a second IspG polypeptide of a second species, wherein the second species differs from the first species; (ii) a nucleic acid encoding at least one 1-deoxyxylulose-5-phosphate (DXP) pathway enzyme; and (iii) a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells are cultured under conditions suitable for producing isoprene, and wherein the cell produces isoprene at a greater titer as compared to a cell comprising a nucleic acid encoding an IspG polypeptide of a single species; and   (b) producing isoprene.   
     
     
         2 . The method of  claim 1 , further comprising recovering the isoprene. 
     
     
         3 . The method of  claim 1 , wherein the recombinant cell produces greater than about 8.4 g/l of isoprene. 
     
     
         4 . The method of  claim 1 , wherein the recombinant cell is a bacterial, algal, fungal or yeast cell. 
     
     
         5 . The method of  claim 4 , wherein the cell is a bacterial cell. 
     
     
         6 . The method of  claim 5 , wherein the bacterial cell is a gram-positive bacterial cell or gram-negative bacterial cell. 
     
     
         7 . The method of  claim 6 , wherein the bacterial cell is selected from the group consisting of  E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas  sp., and  P. alcaligenes  cells. 
     
     
         8 . The method of  claim 1 , wherein the cell is a yeast cell. 
     
     
         9 . The method of  claim 8 , wherein the yeast cell is selected from the group consisting of  Saccharomyces  sp.,  Schizosaccharomyces  sp.,  Pichia  sp., and  Candida  sp. 
     
     
         10 . The method of  claim 9 , wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         11 . The method of  claim 1 , wherein the cell is a fungal cell. 
     
     
         12 . The method of  claim 11 , wherein the fungal cell is selected from the group consisting of  Trichoderma longibrachiatum, T. viride, T. koningii, T. harzianum, Penicillium  sp.,  Humicola insolens, H. lanuginose, H. grisea, Chrysosporium  sp.,  Gliocladium  sp.,  Aspergillus  sp.,  Fusarium sp, Neurospora  sp.,  Hypocrea  sp.,  Mucor  sp.,  Rhizopus  sp. and  Emericella  sp. cells. 
     
     
         13 . The method of  claim 1 , wherein the recombinant cell further comprises (iv) at least one heterologous nucleic acid encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide or at least one copy of an endogenous nucleic acid encoding an IDI polypeptide; and (v) at least one heterologous nucleic acid encoding a mevalonate (MVA) pathway polypeptide or at least one copy of an endogenous nucleic acid encoding a MVA pathway polypeptide. 
     
     
         14 . The method of  claim 13 , further comprising recovering the isoprene. 
     
     
         15 . The method of  claim 13 , wherein the recombinant cell produces greater than about 8.4 g/l of isoprene. 
     
     
         16 . The method of  claim 13 , wherein the recombinant cell is a bacterial, algal, fungal or yeast cell.