Selection Method and Recombinant Microorganisms and uses Therefor

Abstract

One or more genes in a biosynthesis pathway for a vitamin or other essential nutrient which is needed for the survival of a microorganism can be used as an effective selective marker to identify cells transformed with an exogenous nucleic acid. The microorganism does not naturally contain or express the one or more gene. This permits genetic manipulations to be performed. It permits lower cost fermentations to be performed. It permits production of the essential nutrient for subsequent commodity use.

We claim: 
     
         1 . A process for converting CO in a gaseous CO-containing substrate into higher molecular weight products, the process comprising:
 a) passing the gaseous CO-containing substrate to a bioreactor containing a culture of carboxydotrophic acetogenic bacteria in a culture medium such that the bacteria convert the CO to higher molecule weight products, and   b) recovering the higher molecular weight products from the bioreactor,   
       wherein the carboxydotrophic acetogenic bacteria are genetically engineered to express an enzyme in a biosynthetic pathway of an essential nutrient that is absent from the culture medium, and wherein the carboxydotrophic acetogenic bacteria are prototrophic for an essential nutrient selected from the group consisting of thiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, and cyanocobalamine, by virtue of an exogenous gene encoding the enzyme. 
     
     
         2 . The process of  claim 1  wherein the bacteria are prototrophic for thiamine and/or pantothenate by virtue of an heterologous thiC gene and/or an heterologous panBCD gene cluster. 
     
     
         3 . An isolated, genetically engineered carboxydotrophic acetogenic bacterium which is prototrophic for a vitamin selected from the group consisting of thiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, and cyanocobalamine, by virtue of an expressed exogenous gene encoding an enzyme in a biosynthetic pathway that makes the vitamin. 
     
     
         4 . The bacterium of  claim 3  which is prototrophic for thiamine and/or pantothenate by virtue of an heterologous thiC gene and/or an heterologous panBCD gene cluster. 
     
     
         5 . The bacterium of  claim 3  which is selected from the group consisting of  Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii,  and  Thermoanaerobacter kiuvi.    
     
     
         6 . The bacterium of  claim 3  which is unable to convert 4-aminoimidazole ribonucleotide to 4-amino-5-hydroxymethyl-2-methylpyrimidine in the absence of said heterologous thiC gene. 
     
     
         7 . The bacterium of  claim 3  which is auxotrophic for thiamine when cured of a plasmid. 
     
     
         8 . The bacterium of  claim 3  which is auxotrophic for pantothenate when cured of a plasmid. 
     
     
         9 . The bacterium of  claim 4  wherein the heterologous panBCD gene cluster is from  C. beijerenckei.    
     
     
         10 . A method of culturing an isolated, genetically engineered carboxydotrophic acetogenic bacterium which is prototrophic for a vitamin selected from the group consisting of thiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, and cyanocobalamine, by virtue of an expressed exogenous gene encoding an enzyme in a biosynthetic pathway that makes the vitamin, the method comprising, growing the bacterium in a medium comprising a gaseous carbon source, wherein the carbon source comprises CO. 
     
     
         11 . A method of culturing an isolated, genetically engineered carboxydotrophic acetogenic bacterium which is prototrophic for a vitamin selected from the group consisting of thiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, and cyanocobalamine, by virtue of an expressed exogenous gene encoding an enzyme in a biosynthetic pathway that makes the vitamin, the method comprising, growing the bacterium in a medium comprising an energy source, wherein the energy source comprises CO. 
     
     
         12 . The method of  claim 10  wherein the bacterium comprises an heterologous thiC gene and the medium is devoid of thiamine. 
     
     
         13 . The method of  claim 11  wherein the bacterium comprises an heterologous thiC gene and the medium is devoid of thiamine. 
     
     
         14 . The method of  claim 10  wherein the bacterium comprises an heterologous panBCD gene cluster and the medium is devoid of pantothenate. 
     
     
         15 . The method of  claim 11  wherein the bacterium comprises an heterologous panBCD gene cluster and the medium is devoid of pantothenate. 
     
     
         16 . The method of  claim 10  wherein the gaseous carbon source comprises a product selected from the group consisting of automobile exhaust fumes, waste gas from ferrous metal products manufacturing, waste gas from non-ferrous products manufacturing, waste gas from petroleum refining processes, waste gas from gasification of coal, waste gas from electric power production, waste gas from carbon black production, waste gas from ammonia production, waste gas from methanol production, waste gas from coke manufacturing, and syngas. 
     
     
         17 . The method of  claim 11  wherein the gaseous carbon source comprises a product selected from the group consisting of automobile exhaust fumes, waste gas from ferrous metal products manufacturing, waste gas from non-ferrous products manufacturing, waste gas from petroleum refining processes, waste gas from gasification of coal, waste gas from electric power production, waste gas from carbon black production, waste gas from ammonia production, waste gas from methanol production, waste gas from coke manufacturing, and syngas 
     
     
         18 . A method for transferring an heterologous nucleic acid into a population of carboxydotrophic acetogenic bacteria which are auxotrophic for an essential nutrient, the method comprising:
 a) transforming the bacteria with a first nucleic acid which comprises an exogenous gene in a biosynthetic pathway of an essential nutrient, said gene operably linked to a promoter; and   b) selecting for bacteria which are prototrophic for the essential nutrient among the transformed bacteria.   
     
     
         19 . The method of  claim 18  wherein the step of transforming comprises co-transforming the bacteria with a second nucleic acid which comprises an heterologous or endogenous gene conferring a desired property when expressed in the bacterium. 
     
     
         20 . The method of  claim 18  wherein the essential nutrient is selected from the group consisting of thiamine, pantothenate, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid, and cyanocobalamine. 
     
     
         21 . The method of  claim 20  wherein the essential nutrient is thiamine or pantothenate, and the heterologous nucleic acid is a thiC gene or a panBCD gene cluster, respectively. 
     
     
         22 . The method of  claim 18  further comprising the step of screening prototrophic, transformed bacteria for the presence of the first nucleic acid or second nucleic acid. 
     
     
         23 . The method of  claim 19  wherein the first and second nucleic acids are treated to form methylated first and second nucleic acids prior to the step of co-transforming.