Microorganisms and methods for enhancing the availability of reducing equivalents in the presence of methanol, and for producing 3-hydroxyisobutyrate or methacrylic acid related thereto

Abstract

Provided herein is a non-naturally occurring microbial organism having a methanol metabolic pathway that can enhance the availability of reducing equivalents in the presence of methanol. Such reducing equivalents can be used to increase the product yield of organic compounds produced by the microbial organism, such as 3-hydroxyisobutyrate or MAA. Also provided herein are methods for using such an organism to produce 3-hydroxyisobutyrate or MAA.

1 .- 71 . (canceled) 
     
     
         72 . A method of producing methyl ester of methacrylic acid (MMA), comprising culturing a non-naturally occurring microorganism in the presence of methanol under conditions and for a sufficient period of time to produce methacrylic acid (MAA), and esterifying the MAA with methanol to produce MMA. 
     
     
         73 . The method of  claim 72 , wherein culturing the microorganism is in a substantially anaerobic culture medium. 
     
     
         74 . The method of  claim 73 , wherein the culture medium contains methanol. 
     
     
         75 . The method of  claim 74 , wherein esterifying the MAA with methanol occurs in the culture medium. 
     
     
         76 . The method of  claim 72 , wherein said microorganism comprises:
 (A) a methanol metabolic pathway, wherein said organism comprises at least one exogenous nucleic acid encoding a methanol metabolic pathway enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of methanol, wherein said methanol metabolic pathway comprises:
 (i) a methanol methyltransferase and a methylenetetrahydrofolate reductase; 
 (ii) a methanol dehydrogenase; or 
 (iii) a methanol dehydrogenase and a formaldehyde activating enzyme; and 
   (B) a MAA pathway.   
     
     
         77 . The method of  claim 76 , wherein said organism comprises at least one exogenous nucleic acid encoding a MAA pathway enzyme expressed in a sufficient amount to produce MAA, wherein said MAA pathway comprises
 (1) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA epimerase; (iv) a methylmalonyl-CoA reductase (aldehyde forming); (v) a methylmalonate semialdehyde reductase; and (vi) a 3-hydroxyisobutyrate dehydratase;   (2) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA reductase (aldehyde forming); (iv) a methylmalonate semialdehyde reductase; and (v) a 3-hydroxyisobutyrate dehydratase; or   (3) (i) a succinyl-CoA transferase, ligase, or synthetase; (ii) a methylmalonyl-CoA mutase; (iii) a methylmalonyl-CoA reductase (alcohol forming); and (iv) a 3-hydroxyisobutyrate dehydratase.   
     
     
         78 . The method of  claim 76 , wherein the microorganism comprises two, three, four, five or six exogenous nucleic acids, each encoding a MAA pathway enzyme. 
     
     
         79 . The method of  claim 76 , wherein said at least one exogenous nucleic acid encoding a MAA pathway enzyme is a heterologous nucleic acid. 
     
     
         80 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol methyltransferase, a methylenetetrahydrofolate reductase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase, and a formyltetrahydrofolate deformylase. 
     
     
         81 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol methyltransferase, a methylenetetrahydrofolate reductase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase. 
     
     
         82 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase. 
     
     
         83 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase. 
     
     
         84 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase and a formaldehyde dehydrogenase. 
     
     
         85 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase, a S-(hydroxymethyl)glutathione synthase, a glutathione-dependent formaldehyde dehydrogenase and a S-formylglutathione hydrolase. 
     
     
         86 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase, a glutathione-dependent formaldehyde dehydrogenase and a S-formylglutathione hydrolase. 
     
     
         87 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate deformylase. 
     
     
         88 . The method of  claim 76 , wherein the methanol metabolic pathway comprises a methanol dehydrogenase, a formaldehyde activating enzyme, a methylenetetrahydrofolate dehydrogenase, a methenyltetrahydrofolate cyclohydrolase and a formyltetrahydrofolate synthetase. 
     
     
         89 . The method of  claim 76 , wherein the methanol metabolic pathway further comprises a formate dehydrogenase. 
     
     
         90 . The method of  claim 76 , wherein the methanol metabolic pathway further comprises a formate hydrogen lyase. 
     
     
         91 . The method of  claim 76 , wherein the methanol metabolic pathway further comprises a hydrogenase.