Mutant microorganisms resistant to lactose killing

Abstract

The present invention relates to a method to produce mutated microorganisms which resist the phenomenon of lactose killing and to the microorganisms obtainable via said method. Such engineered microorganisms can be applied for the production of specialty products, such as but not limited to specialty carbohydrates, glycolipids and galactosylated compounds.

1 . A genetically modified yeast producing a human milk oligosaccharide, wherein said yeast is genetically modified by introduction and/or upregulation of a gene encoding an enzyme involved in a pathway for production of said human milk oligosaccharide, and wherein said yeast is further genetically modified to express a lactose transporter. 
     
     
         2 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a GDP-fucose synthase, a gene encoding a GDP-mannose 4,6-dehydratase, a gene encoding a GDP-D-mannose pyrophosphorylase, a gene encoding a phosphomannose isomerase, and/or a gene encoding a phosphomannomutase. 
     
     
         3 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a GDP-D-mannose pyrophosphorylase, a gene encoding a phosphomannose isomerase, and/or a gene encoding a phosphomannomutase; or by upregulation of a gene encoding a mannokinase, and/or a gene encoding a GDP-D-mannose pyrophosphorylase. 
     
     
         4 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a galactokinase, a gene encoding a galactose 1-phosphate uridyl transferase, a gene encoding a UDP-galactose-4-epimerase, a gene encoding a UDP-galactose/glucose pyrophosphorylase, a gene encoding a lactose synthase, a gene encoding a lactose phosphorylase, and/or a gene encoding a sucrose phosphorylase. 
     
     
         5 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a glucokinase, a gene encoding a UDP-glucose pyrophosphorylase, a gene encoding a sucrose phosphorylase, a gene encoding a phosphoglucomutase, and/or a gene encoding a sucrose synthase. 
     
     
         6 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a L-glutamine:D-fructose-6-phosphate aminotransferase, a gene encoding a phosphoglucosamine mutase, a gene encoding a glucosamine-1-phosphate acetyltransferase, a gene encoding a N-acetylglucosamine-1-phosphate uridyltransferase, a gene encoding a UDP-N-acetylglucosamine 2-epimerase, a gene encoding a N-acetylneuraminate synthase, and/or a gene encoding a cytidine 5′-monophosphate N-acetylneuraminate synthetase. 
     
     
         7 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a L-glutamine:D-fructose-6-phosphate aminotransferase, a gene encoding a phosphoglucosamine mutase, a gene encoding a glucosamine-1-phosphate acetyltransferase, a gene encoding a N-acetylglucosamine-1-phosphate uridyltransferase, a gene encoding a glucosamine-6-phosphate N-acetyltransferase, a gene encoding a phosphoacetylglucosamine mutase, and/or a gene encoding a UDP-N-acetylglucosamine pyrophosphorylase. 
     
     
         8 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a L-glutamine:D-fructose-6-phosphate aminotransferase, a gene encoding a phosphoglucosamine mutase, a gene encoding a glucosamine-1-phosphate acetyltransferase, a gene encoding a N-acetylglucosamine-1-phosphate uridyltransferase, a gene encoding a glucosamine-6-phosphate N-acetyltransferase, a gene encoding a phosphoacetylglucosamine mutase, a gene encoding a UDP-N-acetylglucosamine pyrophosphorylase, and/or a gene encoding a UDP-N-acetylglucosamine 2-epimerase. 
     
     
         9 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a L-glutamine:D-fructose-6-phosphate aminotransferase, a gene encoding a phosphoglucosamine mutase, a gene encoding a glucosamine-1-phosphate acetyltransferase, a gene encoding a N-acetylglucosamine-1-phosphate uridyltransferase, a gene encoding a glucosamine-6-phosphate N-acetyltransferase, a gene encoding a phosphoacetylglucosamine mutase, a gene encoding a UDP-N-acetylglucosamine pyrophosphorylase, and/or a gene encoding a UDP-N-acetylglucosamine C4-epimerase. 
     
     
         10 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a glucokinase, a gene encoding a UDP-glucose pyrophosphorylase, a gene encoding a sucrose phosphorylase, a gene encoding a phosphoglucomutase, a gene encoding a sucrose synthase, and/or a gene encoding a UDP-glucose dehydrogenase. 
     
     
         11 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a glucokinase, a gene encoding a UDP-glucose pyrophosphorylase, a gene encoding a sucrose phosphorylase, a gene encoding a phosphoglucomutase, a gene encoding a sucrose synthase, a gene encoding a UDP-glucose dehydrogenase, and/or a gene encoding a UDP-D-xylose synthase. 
     
     
         12 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a glucokinase, a gene encoding a UDP-glucose pyrophosphorylase, a gene encoding a sucrose phosphorylase, a gene encoding a phosphoglucomutase, a gene encoding a sucrose synthase, a gene encoding a UDP-glucose dehydrogenase, and/or a gene encoding aUDP-D-glucuronic acid 4-epimerase. 
     
     
         13 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a glucokinase, a gene encoding a UDP-glucose pyrophosphorylase, a gene encoding a sucrose phosphorylase, a gene encoding a phosphoglucomutase, a gene encoding a sucrose synthase, a gene encoding a UDP-glucose dehydrogenase, a gene encoding a UDP-D-xylose 4-epimerase, a gene encoding an arabinose kinase, and/or a gene encoding an UDP-L-arabinose pyrophosphorylase. 
     
     
         14 . The yeast according to  claim 1 , wherein said yeast is genetically modified by upregulation of a gene encoding a dTDP-glucose pyrophosphorylase, a gene encoding a dTDP-glucose 4,6-dehydratase, a gene encoding a dTDP-4-dehydrorhamnose 3,5-epimerase, a gene encoding a dTDP-4-dehydrorhamnose reductase, a gene encoding a glucose-1-phosphate thymidylyltransferase, and/or a gene encoding a nucleotide rhamnose synthase. 
     
     
         15 . The yeast according to  claim 1 , wherein said yeast is genetically modified by introduction of a heterologous gene encoding a β-1,4-galactosyltransferase and/or a heterologous gene encoding a β-1,3-N-acetylglucosaminyltransferase. 
     
     
         16 . The yeast according to  claim 15 , wherein said human milk oligosaccharide comprises lactoNtetraose. 
     
     
         17 . The yeast according to  claim 1 , wherein said yeast is genetically modified by introduction of a heterologous gene encoding a GDP-fucose synthase, a heterologous gene encoding a GDP-mannose 4,6-dehydratase, and/or a heterologous gene encoding a fucosyltransferase. 
     
     
         18 . The yeast according to  claim 17 , wherein said human milk oligosaccharide comprises 2-fucosyllactose. 
     
     
         19 . The yeast according to  claim 1 , wherein said human milk oligosaccharide is selected from the group consisting of 3-fucosyllactose, 2′-fucosyllactose, 6-fucosyllactose, 2′,3-difucosyllactose, 2′,2-difucosyllactose, 3,4-difucosyllactose, 6′-sialyllactose, 3′-sialyllactose, 3,6-disialyllactose, 6,6′-disialylactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides and sialylated oligosaccharides. 
     
     
         20 . The yeast according to  claim 1 , wherein said yeast resists lactose killing when grown in an environment in which lactose is combined with one or more other carbon sources. 
     
     
         21 . The yeast according to  claim 1 , wherein said yeast comprises a lactose transporter expression cassette comprising: a heterologous promoter in front of an endogenous or exogenous lactose transporter gene, a mutation in the untranslated region in front of the coding sequence that contains the ribosome binding or Kozak sequence, and/or an endogenous lactose transporter gene, wherein the codon usage has been modified. 
     
     
         22 . The yeast according to  claim 1 , wherein said lactose transporter is a lactose permease. 
     
     
         23 . The yeast according to  claim 1 , wherein said yeast comprises a lactose transporter expression cassette comprising a sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         24 . The yeast according to  claim 1 , wherein said yeast is further genetically modified by rendering a gene encoding for an enzyme from the lactose and/or galactose degradation pathway less functional or non-functional. 
     
     
         25 . The yeast according to  claim 1 , wherein said yeast is  Saccharomyces cerevisiae.    
     
     
         26 . The yeast according to  claim 25 , wherein said yeast is  Saccharomyces cerevisiae  strain BY4742. 
     
     
         27 . A method for production of a human milk oligosaccharide, comprising:
 culturing a yeast according to  claim 1  in a medium comprising lactose and at least one other carbon source that is not lactose.   
     
     
         28 . The method according to  claim 27 , wherein said at least one other carbon source that is not lactose is selected from the group consisting of glycerol, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols, organic acids, and pentoses. 
     
     
         29 . The method according to  claim 27 , wherein said carbon source that is not lactose is sucrose. 
     
     
         30 . The method according to  claim 27 , wherein said human milk oligosaccharide is selected from the group consisting of: 3-fucosyllactose, 2′-fucosyllactose, 6-fucosyllactose, 2′,3-difucosyllactose, 2′,2-difucosyllactose, 3,4-difucosyllactose, 6′-sialyllactose, 3′-sialyllactose, 3,6-disialyllactose, 6,6′-disialylactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, sialyllacto-N-tetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-tetraose c, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N-hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a, difucosyllacto-N-hexaose c, galactosylated chitosan, fucosylated oligosaccharides, and sialylated oligosaccharides.