Increased Heterologous Fe-S Enzyme Activity in Yeast

Abstract

Yeast strains were engineered that have increased activity of heterologous proteins that require binding of an Fe—S cluster for their activity. The yeast strains have reduced activity of an endogenous Fe—S protein. Activities of heterologous fungal or plant 2Fe-2S dihydroxy-acid dehydratases and Fe—S propanediol dehydratase reactivase were increased for increased production of products made using biosynthetic pathways including these enzymes, such as valine, isoleucine, leucine, pantothenic acid (vitamin B5), isobutanol, 2-butanone and 2-butanol.

1 - 24 . (canceled) 
     
     
         25 . A recombinant yeast host cell comprising at least two engineered modifications comprising:
 (i) a heterologous dihydroxy-acid dehydratase Fe—S cluster protein expressed in the cytosol of the recombinant yeast host cell, wherein the heterologous dihydroxy-acid dehydratase Fe—S cluster protein is a polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 183 or 185; and   (ii) an endogenous Fe—S cluster protein with reduced expression, wherein the endogenous Fe—S cluster protein is dihydroxy-acid dehydratase.   
     
     
         26 . The recombinant yeast host cell of  claim 25 , wherein the yeast is selected from the group consisting of  Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia , and  Pichia.    
     
     
         27 . The recombinant yeast host cell of  claim 25 , wherein the endogenous Fe—S cluster protein is expressed in the mitochondria. 
     
     
         28 . The recombinant yeast host cell of  claim 25 , wherein the host cell is  Saccharomyces  that expresses a gene encoding a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 183 or 185. 
     
     
         29 . The recombinant yeast host cell of  claim 25 , wherein the cell comprises an isobutanol biosynthetic pathway. 
     
     
         30 . The recombinant yeast host cell of  claim 29 , wherein the cell produces isobutanol. 
     
     
         31 . A method for the production of isobutanol comprising growing the recombinant yeast host cell of  claim 30 , under conditions wherein isobutanol is produced. 
     
     
         32 . A method for the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate comprising:
 a) providing the recombinant yeast host cell of  claim 25  and a source of 2,3-dihydroxyisovalerate; and 
 b) growing the recombinant host cell of a) with said source of 2,3-dihydroxyisovalerate under conditions where the 2,3-dihydroxyisovalerate is converted by the host cell to α-ketoisovalerate.