Ethanol production

Abstract

The present invention relates to the production of ethanol as a product of bacterial fermentation. In particular this invention relates to a novel method of gene inactivation and gene expression based upon homologous recombination.

1 . A Gram-positive bacterium wherein the ethanol production characteristics have been enhanced through stabilisation of a ldh mutation using a plasmid integration method based on the homologous recombination of a plasmid and insertion element (IE) sequence within the native ldh gene.  
     
     
         2 . A thermophilic Gram-positive bacterium which has been transformed using a method of homologous recombination for stabilising a gene mutation and for inserting an expressible gene.  
     
     
         3 . A Gram-positive bacterium according to  claim 1  which is a thermophilic.  
     
     
         4 . A Gram-positive bacterium according to  claim 1 ,  2  or  3  wherein the bacterium is a Bacillus sp selected from  B. stearothermophilus, B. calvodelox, B. caldotenax, B. thermoglucosidasius, B. coagulans, B. licheniformis, B. thermodenitrificans,  and  B. caldolyticus.    
     
     
         5 . A Gram-positive bacterium according to any preceding claim wherein the Bacillus sp is  B. thermoglucosidasius  strain TN-T9 (NCIMB Accession No. NCIMB 41075).  
     
     
         6 . A Gram-positive bacterium according to any one of  claims 1  to  4  wherein the Bacillus sp is  B. thermoglucosidasius  strain TN-TK (NCIMB Accession No. NCIMB 41115).  
     
     
         7 . A Gram-positive bacterium according to any one of the  claims 1  to  6  wherein the bacterium has been transformed with a shuttle vector comprising the IE sequence, or a functional portion or variant thereof, as set forth in FIG. 1.  
     
     
         8 . A Gram-positive bacterium according to  claim 7  wherein the IE sequence, a functional or portion variant thereof, is stably incorporated into the chromosome of the recombinant bacterium by homologous recombination.  
     
     
         9 . A Gram-positive bacterium according to any preceding claim wherein a native ldh gene has been inactivated by homologous recombination.  
     
     
         10 . A Gram-positive bacterium according to any preceding claim which is sporulation deficient.  
     
     
         11 . A Gram-positive bacterium according to any preceding claim wherein the native ldh gene has been inactivated and which expresses a heterologous pdc gene.  
     
     
         12 . A Gram positive bacterium according to any one of claims  9 ,  10  or  11  wherein the native ldh gene has been irreversibly inactivated.  
     
     
         13 . A method of inactivating a native ldh gene and inserting one or more expressible genes comprising homologous recombination.  
     
     
         14 . The method according to  claim 13  wherein the one or more expressible genes are a pdc gene and a adh gene.  
     
     
         15 . The method according to claim 14  wherein the pdc gene and the adh gene form part of a PDC operon operatively linked to the IE sequence of FIG. 1.  
     
     
         16 . The method according to  claim 13 ,  14  or  15  in which the heterologous pdc gene is from a Zymomonas sp.  
     
     
         17 . The method according to any one of  claims 13  to  16  in which the heterologous pdc gene is from  Zymomonas mobilis.    
     
     
         18 . The method according to any one of  claims 14  to  17  wherein the adh gene is from Bacillus strain LN.  
     
     
         19 . The method according to any one of  claims 13  to  18  wherein an insertion sequence and a PDC operon, or portions thereof, are stably integrated into the chromosome of the recombinant bacterium.  
     
     
         20 . The method according to any one of  claims 13  to  14  comprising using a shuttle vector, as set forth in FIG. 5, which is able to replicate in  E. coli  and Bacillus strains.  
     
     
         21 .A shuttle vector which harbours an IE sequence, or a portion thereof, as set forth in FIG. 1.  
     
     
         22 . The shuttle vector according to  claim 21  which harbours a pdc gene operatively linked to the IE sequence of FIG. 1.  
     
     
         23 . The shuttle vector according  claim 22  wherein the pdc gene forms part of a PDC operon.  
     
     
         24 . A method of selecting for a recombinant bacterium having an ldh gene, wherein plasmid DNA has been stably integrated into the ldh gene of the bacterium by homologous recombination, comprising use of PCR to amplify the ldh gene.  
     
     
         25 . A method according to  claim 23 , wherein failure to amplify a PCR product from the ldh gene of the recombinant bacterium indicates integration of the IE sequence as set forth in FIG. 1, or a portion thereof, into the chromosome of the recombinant bacterium.  
     
     
         26 . A DNA sequence as set forth in FIG. 6, or a functional variant thereof, which codes for a polypeptide having the biological activity of the enzyme lactate dehydrogenase.  
     
     
         27 . A DNA sequence which hybridises to the DNA sequence of FIG. 6 under stringent conditions, or which is degenerate to the DNA sequence of FIG. 6 and which codes for a polypeptide having the activity of the enzyme lactate dehydrogenase.  
     
     
         28 . A DNA sequence as set forth in FIG. 7B, or a functional variant thereof, which codes for a polypeptide having the biological activity of the enzyme lactate permease.  
     
     
         29 .A DNA sequence which hybridises to the DNA sequence of FIG. 7B under stringent conditions, or which is degenerate to the DNA sequence of FIG. 7B and which codes for a polypeptide having the activity of the enzyme lactate permease.  
     
     
         30 . A DNA sequence as set forth in FIG. 8, or a functional variant thereof, which codes for a polypeptide having the biological activity of the enzyme alcohol dehydrogenase.  
     
     
         31 . A DNA sequence which hybridises to the DNA sequence of FIG. 8 under stringent conditions, or which is degenerate to the DNA sequence of FIG. 8 and which codes for a polypeptide having the activity of the enzyme alcohol dehydrogenase.  
     
     
         32 . A DNA sequence as set forth in FIG. 1 or a portion or functional variant thereof which codes for one or more polypeptides having the biological activity of a transposase, and has been classified as an insertion sequence.  
     
     
         33 . An DNA sequence according to  claim 32 , wherein the sequence is from thermophilic Bacillus strain TN.  
     
     
         34 . One or more polypeptides encoded by the sequence show in FIG. 1 from residue 652 to residue 3800 or a functional variant or portion thereof, wherein the one or more polypeptides have the biological activity of a transposase.  
     
     
         35 . A process for the production of ethanol by bacterial fermentation of the Gram-positive bacterium according to any one of  claims 1  to  12  comprising using optimised fermentation conditions wherein the pH of the fermentation medium is within the range of pH 5.5-7.5.  
     
     
         36 . The process according to  claim 35  wherein the pH of the fermentation medium is preferably between pH 6.0-7.0.  
     
     
         37 . The process according to  claim 35  or  36  wherein the pH of the fermentation medium is preferably between pH 6.4-6.9.  
     
     
         38 . A process for the production of ethanol by bacterial fermentation of the Gram-positive bacterium according to any one of  claims 1  to  12  comprising using optimised fermentation conditions wherein the temperature of the fermentation medium is within the range 40-75° C.  
     
     
         39 . The process according to  claim 38  wherein the temperature of the fermentation medium is preferably between 52-70° C.  
     
     
         40 . The process according to  claim 38  or  39  wherein the temperature of the fermentation medium is preferably between 60-68° C.  
     
     
         41 . A process for the production of ethanol by bacterial fermentation comprising using air sparging within the culture such that the redox potential is between −360 and −400 mV.  
     
     
         42 . The process according to  claim 41  wherein the redox values are preferably between 370 and −380 mV.  
     
     
         43 . The process according to  claim 40  or  41  wherein the Gram-positive bacterium according to any one of  claims 1  to  12 .  
     
     
         44 . A process for the continuous production of ethanol by bacterial fermentation of the Gram-positive bacterium according to any one of  claims 1  to  12  in which the feed dilution rates are between 0.3-0.8  −1 .  
     
     
         45 . A process according to  claim 43  wherein the feed dilution rates are preferably between 0.4-0.6 h −1 .  
     
     
         46 . A process according to any one of  claims 35  to  44  in which the bacterium used is Bacillus strain TN.