Production of isoprene under reduced oxygen inlet levels

Abstract

This invention relates to methods for producing isoprene by culturing recombinant cells (e.g., cells engineered to produce isoprene) under reduced oxygen inlet levels.

1 - 32 . (canceled) 
     
     
         33 . A method for producing isoprene comprising (a) culturing a recombinant fungal host cell comprising a heterologous nucleic acid encoding isoprene synthase under reduced oxygen inlet levels, wherein the reduced oxygen inlet levels comprise between about 4% to about 15% oxygen and wherein the cell is in fermentation or production phase; and (b) producing isoprene. 
     
     
         34 . The method of  claim 33 , further comprising recovering the isoprene. 
     
     
         35 . The method of  claim 33 , wherein the reduced oxygen inlet level is between about 5% to about 15% oxygen. 
     
     
         36 . The method of  claim 35 , wherein the reduced oxygen inlet level is between about 7% to about 10% oxygen. 
     
     
         37 . The method of  claim 36 , wherein the reduced oxygen inlet level is about 7.7% oxygen. 
     
     
         38 . The method of  claim 36 , wherein the reduced oxygen inlet level is about 9.3% oxygen. 
     
     
         39 . The method of  claim 33 , wherein the isoprene synthase is a plant isoprene synthase. 
     
     
         40 . The method of  claim 39 , wherein the plant isoprene synthase is a poplar isoprene synthase, a kudzu isoprene synthase, a willow isoprene synthase, or a  eucalyptus  isoprene synthase. 
     
     
         41 . The method of  claim 39 , wherein the plant isoprene synthase is an isoprene synthase from  Pueraria  or  Populus  or a hybrid,  Populus alba×Populus tremula.   
     
     
         42 . The method of  claim 39 , wherein the plant isoprene synthase polypeptide is selected from the group consisting of  Pueraria montana, Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra , and  Populus trichocarpa.   
     
     
         43 . The method of  claim 33 , wherein the isoprene synthase is an isoprene synthase variant. 
     
     
         44 . The method of  claim 33 , wherein the cell further comprises a heterologous nucleic acid encoding for one or more mevalonate (MVA) pathway polypeptides and/or one or more 1-deoxy-d-xylulose 5-phosphate (DXP) pathway polypeptides. 
     
     
         45 . The method of  claim 44 , wherein the cell further comprises a heterologous nucleic acid encoding for one or more isopentenyl diphosphate isomerase (IDI) polypeptides. 
     
     
         46 . The method of  claim 44 , wherein any one or more copies of a heterologous nucleic acid is overexpressed. 
     
     
         47 . The method of  claim 44 , wherein the heterologous nucleic acid is cloned into a multicopy plasmid. 
     
     
         48 . The method of  claim 44 , wherein the heterologous nucleic acid is placed under an inducible promoter or a constitutive promoter. 
     
     
         49 . The method of  claim 44 , wherein any one or more of the heterologous nucleic acids is integrated into the chromosome of the recombinant host cell. 
     
     
         50 . The method of  claim 33 , wherein the fungal host cells are filamentous fungi cells. 
     
     
         51 . The method of  claim 50 , wherein the filamentous fungi cells are  Aspergillus  or  Trichoderma  cells. 
     
     
         52 . The method of  claim 50 , wherein the filamentous fungi cells are selected from the group consisting of  A. oryzae, A. niger, T. reesei, H. insolens, H. lanuginose, H. grisea, C. lucknowense, A. sojae, A. japonicus, A. nidulans, A. aculeatus, A. awamori, F. roseum, F. graminum F. cerealis, F. oxysporuim, F. venenatum, N. crassa, M. miehei, T. viride, F. oxysporum , and  F. solani  cells. 
     
     
         53 . The method of  claim 33 , wherein the fungal host cells are yeast cells. 
     
     
         54 . The method of  claim 53 , wherein the yeast cells are selected from the group consisting of  Saccharomyces  sp.,  Schizosaccharomyces  sp.,  Pichia  sp., or  Candida  sp. 
     
     
         55 . The method of  claim 54 , wherein the yeast cells are  Saccharomyces cerevisiae  or  Schizosaccharomyces pombe.   
     
     
         56 . A method for producing isoprene comprising (a) culturing a recombinant yeast or fungus host cell comprising a heterologous nucleic acid encoding isoprene synthase under reduced oxygen inlet levels having an inlet airflow rate of between about 8.0 standard liter per minute (SLPM) and about 14 SLPM, wherein the cell is in fermentation or production phase; and (b) producing isoprene. 
     
     
         57 . The method of  claim 56 , further comprising recovering the isoprene. 
     
     
         58 . The method of  claim 56 , wherein the inlet airflow rate is about 10.0 SLPM.