Saccharomyces strain with ability to grow on pentose sugars under anaerobic cultivation conditions

Abstract

The invention relates to an improved Saccharomyces strain displaying improved viability and growth during anaerobic fermentation of pentose carbon sources such as xylose and producing fermentation products such as ethanol.

1 . A method of producing a fermentation product and cell mass comprising the steps of:
 a) providing a medium comprising xylose and a  Saccharomyces  sp. strain which grows on pentose sugars under anaerobic cultivation conditions;   b) adding the medium and strain to a fermentation reactor;   c) performing fermentation with said strain under anaerobic conditions; and   d) utilizing the carbon source xylose and producing a fermentation product,   
       wherein the  Saccharomyces  sp. strain comprises a genome comprising
 (i) a xylose reductase (XR) gene encoding an XR having NADH-preference, wherein expression of the XR gene is controlled by a constitutive promoter, and 
 (ii) a xylitol dehydrogenase (XDH) gene which is expressed. 
 
     
     
         2 . The method according to  claim 1 , wherein a gene encoding phosphoglucomutase activity is constitutively overexpressed in the  Saccharomyces  sp. strain. 
     
     
         3 . The method according to  claim 1 , wherein the constitutive promoter is selected from the group consisting of TDH3, truncated HXT7, TEF1 and PGK1. 
     
     
         4 . The method according to  claim 1 , wherein the XR gene is derived from  Pichia stipitis  and the XR encoded by the XR gene comprises the amino acid substitution K270R. 
     
     
         5 . The method according to  claim 1 , wherein the XR gene is derived from  Pichia stipitis  and the XR encoded by the XR gene comprises the amino acid substitutions N272D and/or P275Q. 
     
     
         6 . The method according to  claim 1 , wherein the XDH gene is constitutively overexpressed. 
     
     
         7 . The method according to  claim 1 , wherein the genome comprises a gene encoding a transaldolase, wherein expression of the transaldolase gene is increased as compared to the parental strain of the  Saccharomyces  sp. strain. 
     
     
         8 . The method according to  claim 1 , wherein the genome comprises overexpressed genes encoding transaldolase, transketolase, ribose 5-phosphate ketol-isomerase and/or ribulose 5-phosphate epimerase. 
     
     
         9 . The method according to  claim 1 , wherein the genome comprises a xylulokinase (XK) gene which is overexpressed. 
     
     
         10 . The method according to  claim 1 , wherein the  Saccharomyces  sp. strain is selected from the group consisting of  Saccharomyces cerevisiae, Saccharomyces bayanus  and  Saccharomyces carlsbergensis.    
     
     
         11 . A method of producing a fermentation product and cell mass, the method comprising
 adding to a fermentation reactor a medium comprising xylose and a  Saccharomyces  sp. strain, and   producing a fermentation product and cell mass from the xylose under anaerobic conditions,   
       wherein the  Saccharomyces  sp. strain comprises a genome comprising a xylose reductase (XR) gene encoding an XR having NADH-preference and a xylitol dehydrogenase (XDH) gene, wherein the XR and XDH genes are constitutively overexpressed. 
     
     
         12 . The method of  claim 11 , wherein the XR gene is derived from  Pichia stipitis  and the XR encoded by the XR gene comprises the amino acid substitution K270R. 
     
     
         13 . The method according to  claim 12 , wherein the genome further comprises a constitutively overexpressed phosphoglucomutase gene. 
     
     
         14 . The method according to  claim 12 , wherein the genome further comprises overexpressed XK, transaldolase, transketolase, ribose 5-phosphate ketol-isomerase and ribulose 5-phosphate epimerase genes. 
     
     
         15 . The method according to  claim 14 , wherein at least one gene is under the control of a constitutive promoter is selected from the group consisting of TDH3, truncated HXT7, TEF1 and PGK1. 
     
     
         16 . The method according to  claim 15 , wherein the  Saccharomyces  sp. strain is a  Saccharomyces cerevisiae  strain. 
     
     
         17 . The method according to  claim 16 , wherein the fermentation product is ethanol. 
     
     
         18 . The method according to  claim 17 , wherein the ethanol yield is at least 0.35 g/g consumed sugar, the xylose consumption rate is at least 0.28 g/g biomass/h, or the ethanol production rate is at least 0.1 g/g biomass/h. 
     
     
         19 . A  Saccharomyces  sp. strain comprising a genome comprising
 (i) a xylose reductase (XR) gene encoding an XR having NADH-preference, wherein expression of the XR gene is controlled by a constitutive promoter;   (ii) a xylitol dehydrogenase (XDH) gene which is expressed; and   (iii) a gene encoding phosphoglucomutase activity which is constitutively overexpressed.   
     
     
         20 . The  Saccharomyces  sp. strain according to  claim 19 , wherein the XR gene is derived from  Pichia stipitis  and the XR encoded by the XR gene comprises the amino acid substitution K270R, N272D and/or P275Q.