Cell-free system for converting methane into fuel and chemical compounds

Abstract

The present disclosure relates, in some aspects, to cell-free methods and systems for large-scale conversion of methane to isobutanol, comprising combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to isobutanol, to form a cell-free reaction mixture, and incubating under suitable conditions the cell-free reaction to convert methane to isobutanol.

1 - 75 . (canceled) 
     
     
         76 . A composition comprising at least two cell lysates and the following enzymes: a methane monooxygenase, a methanol dehydrogenase, a hexulose-6-phosphate synthase, a 6-phospho-3-hexuloisomerase, a 6-phosphofructokinase, a fructose bisphosphate aldolase, a triose phosphate isomerase, a transketolase, a ribose-5-phosphate isomerase or a ribulose-5-phosphate 3-epimerase, a glyceraldehyde 3-phosphate dehydrogenase, a phosphoglycerate kinase, a phosphoglycerate mutase, an enolase, and a pyruvate kinase, wherein at least one of the cell lysates is obtained from recombinant cells that overexpress at least one of the foregoing enzymes. 
     
     
         77 . The composition of  claim 76 , wherein
 (i) the methane monooxygenase has EC number 1.14.13.25 or 1.14.18.3,   (ii) the methanol dehydrogenase has EC number 1.1.1.244, 1.1.2.7, or 1.1.99.37,   (iii) the hexulose-6-phosphate synthase has EC number 4.1.2.43,   (iv) the 6-phospho-3-hexuloisomerase has EC number 5.3.1.27,   (v) the 6-phosphofructokinase has EC number 2.7.1.11,   (vi) the fructose bisphosphate aldolase has EC number 4.1.2.13,   (vii) the triose phosphate isomerase has EC number 5.3.1.1,   (viii) the transketolase has EC number 2.2.1.1,   (xi) the ribulose-5-phosphate 3-isomerase has EC number 5.3.1.6, or the ribulose-5-phosphate 3-epimerase has EC number 5.1.3.1,   (xii) the glyceraldehyde 3-phosphate dehydrogenase has EC number 1.2.1.12,   (xiii) the phosphoglycerate kinase has EC number 2.7.2.3,   (xiv) the phosphoglycerate mutase has EC number 5.4.2.11 or 5.4.2.12,   (xv) the enolase has EC number 4.2.1.11, and   (xvi) the pyruvate kinase has EC number 2.7.1.40.   
     
     
         78 . The composition of  claim 76 , wherein the methane monooxygenase is obtained from a methanotroph. 
     
     
         79 . The composition of  claim 78 , wherein the methanotroph is  Methylococcus capsulatus  (Bath). 
     
     
         80 . The composition of  claim 76 , wherein the methanol dehydrogenase is a NAD-linked methanol dehydrogenase. 
     
     
         81 . The composition of  claim 80 , wherein the NAD-linked methanol dehydrogenase is a  Bacillus methanolicus  NAD-linked methanol dehydrogenase. 
     
     
         82 . The composition of  claim 76 , wherein the recombinant cells are recombinant bacteria. 
     
     
         83 . The composition of  claim 82 , wherein the recombinant bacteria are recombinant  Escherichia coli.    
     
     
         84 . The composition of  claim 76 , wherein the composition further comprises an acetolactate synthase, an acetohydroxy acid isomeroreductase, a dihydroxy acid dehydratase, an alpha-ketoisovalerate decarboxylase, and an isobutanol dehydrogenase. 
     
     
         85 . The composition of  claim 84 , wherein
 (i) the methane monooxygenase has EC number 1.14.13.25 or 1.14.18.3,   (ii) the methanol dehydrogenase has EC number 1.1.1.244, 1.1.2.7, or 1.1.99.37,   (iii) the hexulose-6-phosphate synthase has EC number 4.1.2.43,   (iv) the 6-phospho-3-hexuloisomerase has EC number 5.3.1.27,   (v) the 6-phosphofructokinase has EC number 2.7.1.11,   (vi) the fructose bisphosphate aldolase has EC number 4.1.2.13,   (vii) the triose phosphate isomerase has EC number 5.3.1.1,   (viii) the transketolase has EC number 2.2.1.1,   (xi) the ribose-5-phosphate isomerase has EC number 5.3.1.6, or the ribulose-5-phosphate 3-epimerase has EC number 5.1.3.1,   (xii) the glyceraldehyde 3-phosphate dehydrogenase has EC number 1.2.1.12,   (xiii) the phosphoglycerate kinase has EC number 2.7.2.3,   (xiv) the phosphoglycerate mutase has EC number 5.4.2.11 or 5.4.2.12,   (xv) the enolase has EC number 4.2.1.11,   (xvi) the pyruvate kinase has EC number 2.7.1.40,   (xvii) the acetolactate synthase has EC number 2.2.1.6,   (xviii) the acetohydroxy acid isomeroreductase has EC number 1.1.1.86,   (xix) the dihydroxy acid dehydratase has EC number 4.2.1.9,   (xx) the alpha-ketoisovalerate decarboxylase has EC number 4.1.1.72, and   (xxi) the isobutanol dehydrogenase has EC number 1.1.1.1.   
     
     
         86 . The composition of  claim 84 , wherein the methane monooxygenase is obtained from a methanotroph. 
     
     
         87 . The composition of  claim 86 , wherein the methanotroph is  Methylococcus capsulatus  (Bath). 
     
     
         88 . The composition of  claim 84 , wherein the methanol dehydrogenase is a NAD-linked methanol dehydrogenase. 
     
     
         89 . The composition of  claim 88 , wherein the NAD-linked methanol dehydrogenase is a  Bacillus methanolicus  NAD-linked methanol dehydrogenase. 
     
     
         90 . The composition of  claim 84 , wherein the recombinant cells are recombinant bacteria. 
     
     
         91 . The composition of  claim 90 , wherein the recombinant bacteria are recombinant  Escherichia coli.    
     
     
         92 . The composition of  claim 76  further comprising methane. 
     
     
         93 . The composition of  claim 76  further comprising an organic solvent. 
     
     
         94 . A bioreactor comprising the composition of  claim 76 . 
     
     
         95 . The bioreactor of  claim 94  further comprising methane. 
     
     
         96 . The bioreactor of  claim 95  further comprising oxygen, wherein the bioreactor is pressurized to drive the transformation of methane to pyruvate at a production rate of at least 1 g/L-h.