Host Cells and Methods for Production of Isobutanol

Abstract

Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

1 . A recombinant host cell comprising an engineered isobutanol production pathway and
 a. at least one of
 i. a heterologous polypeptide with ketol-acid reductoisomerase (KARI) activity selected from the group consisting of:
 1. a polypeptide having at least about 90% identity to a KARI enzyme derived from  Bifidobacterium angulatum, Bifidobacterium dentium, Zymomonas mobilis, Clostridium beijerinckii  or  Anaerostipes caccae , or an active fragment thereof, 
 2. a polypeptide having at least about 90% identity or at least about 95% identity to SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, or 65; or 
 
 ii. a heterologous polynucleotide encoding the heterologous polypeptide with KARI activity of a); and 
   b. at least one host cell modification that enhances performance of the engineered isobutanol production pathway.   
     
     
         2 . (canceled) 
     
     
         3 . The recombinant host cell of  claim 1  wherein the host cell modification comprises reduced or eliminated aldehyde dehydrogenase expression or activity and reduced or eliminated acetolactate reductase expression or activity. 
     
     
         4 - 10 . (canceled) 
     
     
         11 . The recombinant host cell of  claim 1 , wherein said host cell modification comprises at least one of a deletion, mutation, or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase activity. 
     
     
         12 - 17 . (canceled) 
     
     
         18 . The recombinant host cell of  claim 1 , wherein said host cell comprises at least one or more of a deletion, mutation, or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde oxidase activity. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The recombinant host cell of  claim 1 , wherein said host cell comprises at least one or more deletion, mutation, or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. 
     
     
         22 . The recombinant host cell of  claim 21 , wherein the endogenous polynucleotide is selected from the group consisting of SEQ ID NO: 676, SEQ ID NO: 678, SEQ ID NO: 680, SEQ ID NO: 682, SEQ ID NO: 684, SEQ ID NO: 686, SEQ ID NO: 688, SEQ ID NO: 690, SEQ ID NO: 692, SEQ ID NO: 694, SEQ ID NO: 696, SEQ ID NO:702, SEQ ID NO: 704, SEQ ID NO: 706, SEQ ID NO: 708, SEQ ID NO: 710, SEQ ID NO: 712, SEQ ID NO: 714, SEQ ID NO: 716, SEQ ID NO: 718, SEQ ID NO: 720, SEQ ID NO: 722, SEQ ID NO: 724, SEQ ID NO:726, SEQ ID NO:728, and SEQ ID NO: 730. 
     
     
         23 . (canceled) 
     
     
         24 . The recombinant host cell of  claim 1 , wherein the host cell is a yeast host cell. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . The recombinant host cell of  claim 1 , wherein the engineered isobutanol production pathway comprises the following substrate to product conversions:
 a. pyruvate to acetolactate   b. acetolactate to 2,3-dihydroxyisovalerate   c. 2,3-dihydroxyisovalerate to 2-ketoisovalerate   d. 2-ketoisovalerate to isobutyraldehyde; and   e. isobutyraldehyde to isobutanol   
       wherein each substrate to product conversions is catalyzed by an enzyme that is recombinantly expressed by the host cell. 
     
     
         28 . The recombinant host cell of  claim 27 , wherein all of the substrate to product conversions are catalyzed by enzymes heterologous to the host cell. 
     
     
         29 . (canceled) 
     
     
         30 . The recombinant host cell of  claim 27 , wherein the substrate to product conversions are catalyzed by enzymes substantially localized to the cytosol. 
     
     
         31 - 32 . (canceled) 
     
     
         33 . The recombinant host cell of  claim 1 , wherein the host cell is yeast and has reduced, disrupted or eliminated pyruvate decarboxylase expression or activity. 
     
     
         34 . (canceled) 
     
     
         35 . The recombinant host cell of  claim 1 , wherein the host cell has reduced or eliminated NAD-dependent glycerol-3-phosphate dehydrogenase expression or activity. 
     
     
         36 . (canceled) 
     
     
         37 . The recombinant host cell of  claim 1 , wherein the host cell has reduced or eliminated FRA2 expression or activity. 
     
     
         38 - 41 . (canceled) 
     
     
         42 . A method for producing isobutanol comprising:
 a. providing the recombinant host cell of  claim 1 , and   b. contacting the host cell of a) with a carbon substrate under conditions whereby isobutanol is produced.   
     
     
         43 . The method of  claim 42 , wherein at least a portion of the contacting occurs under anaerobic conditions. 
     
     
         44 . The method of  claim 42 , wherein the contacting occurs in the presence of an extractant. 
     
     
         45 . The method of  claim 44 , wherein the contacting occurs in the presence of a sufficient quantity of organic extractant to form a two-phase system comprising an aqueous phase and an organic phase. 
     
     
         46 - 50 . (canceled) 
     
     
         51 . A method for the production of butanol comprising:
 a. growing a recombinant yeast comprising a biosynthetic pathway capable of converting pyruvate to acetolactate under conditions whereby butanol is produced; and   b. removing DHMB from the culture.   
     
     
         52 - 74 . (canceled)