Recombinant host cells and media for ethanol production

Abstract

Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.

What is claimed is: 
     
         1 - 65 . (canceled) 
     
     
         66 . A minimal medium that supports growth and ethanol production by a recombinant host cell suitable for degrading a saccharide, comprising:
 a defined nitrogen source;   a complex nitrogen source;   a source of phosphate; and   a source of magnesium.   
     
     
         67 . The minimal medium according to  claim 66 , wherein the defined nitrogen source is a urea-like compound. 
     
     
         68 . The minimal medium according to  claim 67 , wherein the defined nitrogen source is urea. 
     
     
         69 . The minimal medium according to  claim 66 , wherein the complex nitrogen source is selected from the group consisting of corn steep liquor (CSL), yeast autolysate and/or extract, corn processing by-product, soy processing by-product, and spent fermentation broth. 
     
     
         70 . The minimal medium according to  claim 67 , wherein the concentration of urea nitrogen is from about 0.1 mM to about 100 mM. 
     
     
         71 . The minimal medium according to  claim 70 , wherein the concentration of urea nitrogen is from about 2 mM to about 20 mM. 
     
     
         72 . The minimal medium according to  claim 71 , wherein the concentration of urea nitrogen is from about 8 mM to about 12 mM. 
     
     
         73 . The minimal medium according to  claim 69 , wherein the concentration of CSL is from about 0.1 g L −1  to about 100 g L −1 . 
     
     
         74 . The minimal medium according to  claim 73 , wherein the concentration of CSL is from about 1 g L −1  to about 20 g L −1 . 
     
     
         75 . The minimal medium according to  claim 74 , wherein the concentration of CSL is from about 5 g L −1  to about 10 g L −1 . 
     
     
         76 . The minimal medium  claim 66 , wherein the concentration of total phosphate is from about 1 mM to about 100 mM. 
     
     
         77 . The minimal medium according to  claim 76 , wherein the concentration of phosphate is from about 10 mM to about 12 mM. 
     
     
         78 . The minimal medium of  claim 66 , wherein the concentration of magnesium is from about 0.1 mM to about 5.0 mM. 
     
     
         79 . The minimal medium according to  claim 78 , wherein the concentration of magnesium is from about 0.25 mM to about 1.0 mM. 
     
     
         80 . The minimal medium of  claim 66 , optimized to support growth and ethanol production by a recombinant host cell suitable for degrading a saccharide. 
     
     
         81 . The minimal medium according to  claim 80 , optimized to support growth and ethanol production at acidic pH. 
     
     
         82 . The minimal medium of  claim 66 , optimized to support growth and ethanol production by a recombinant host cell suitable for degrading a saccharide comprising: (a) a heterologous polynucleotide sequence that codes for an enzyme that converts sugars to ethanol, wherein said host cell expresses said heterologous polynucleotide sequence at a sufficient functional level so as to facilitate production of ethanol as a primary fermentation product by said cell; and (b) a mutation in at least one polynucleotide sequence that codes for a protein in a metabolic pathway in said cell that produces a product other than ethanol from sugars, wherein said mutation results in increased ethanol production by said cell, as compared to ethanol production by the cell in the absence of said mutation. 
     
     
         83 . The minimal medium according to  claim 80 , optimized to support growth and ethanol production by strains of  Klebsiella oxytoca.    
     
     
         84 . The minimal medium of  claim 66 , further comprising a source of saccharide. 
     
     
         85 . The minimal medium according to  claim 84  wherein the source of saccharide comprises lignocellulose. 
     
     
         86 - 111 . (canceled)