Gene modification in clostridium for increased alcohol production
The present disclosure is directed to genetically engineered bacteria strains with enhanced biofuel-producing capabilities from cellulosic substrates. The bacteria strains of the present disclosure comprise an inactivated Type I glutamine synthetase gene. The present disclosure is also directed to methods of producing biofuels from cellulosic biomass using the genetically engineered bacteria strains.
1. A genetically engineered bacteria strain with enhanced alcohol production from cellulosic substrates, wherein said bacteria strain comprises an inactivated Type I glutamine synthetase (glnA) gene.
2. The bacteria strain of claim 1 , wherein said bacteria strain is a strain of Clostridium.
3. The bacteria strain of claim 1 , wherein said bacteria strain is a strain of Clostridium thermocellum ( C. thermocellum ).
4. The bacteria strain of claim 1 , wherein the inactivation of the Type I glutamine synthetase (glnA) gene in said bacteria strain is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system, and homologous recombination.
5. The bacteria strain of claim 1 , wherein said bacteria strain further comprises at least one inactivated gene selected from the list consisting of spoOA encoding a master regulator of sporulation, NfnAB complex encoding the ferredoxin-NADH:NADP+ oxidoreductase, ppdK encoding pyruvate:phosphate dikinase, mdh encoding malate dehydrogenase, mae encoding malic enzyme, pta encoding phosphotransacetlylase, pfl encoding pyruvate:formate lyase, ldh encoding lactate dehydrogenase, hydG encoding [FeFe] hydrogenase maturation factor, and ech endocing [NiFe] Ech-type hydrogenase.
6. The bacteria strain of claim 1 , wherein said bacteria strain further comprises at least one overexpressed gene selected from the list consisting of Rnf encoding Ferredoxin:NADH oxidoreductase, PDC encoding pyruvate decarboxylase, adhE encoding bifunctional aldehyde/alcohol dehydrogenase, and pryK encoding pyruvate kinase.
7. A method of producing alcohol from a cellulosic substrate, comprising adding cells of the genetically modified bacteria strain of claim 1 to a fermentation mixture, allowing said cells to ferment and produce alcohol, and recovering the alcohol produced.
8. The method of claim 7 , wherein the produced alcohol is ethanol.
9. The method of claim 7 , wherein the produced alcohol is butanol.
10. The method of claim 7 , wherein the produced alcohol is isobutanol.
11. The method of claim 7 , wherein fermentation is carried out at a temperature of about 50-65° C.
12. The method of claim 7 , wherein the efficiency of alcohol production is at least 70% of a theoretical maximum of 2 moles alcohol per mole hexose.
13. The method of claim 7 , wherein said bacteria strain is a strain of Clostridium.
14. The method of claim 7 , wherein said bacteria strain is a strain of Clostridium thermocellum ( C. thermocellum ).
15. The method of claim 7 , wherein said bacteria strain further comprises at least one inactivated gene selected from the list consisting of spoOA encoding a master regulator of sporulation, NfnAB complex encoding the ferredoxin-NADH:NADP+ oxidoreductase, ppdK encoding pyruvate:phosphate dikinase, mdh encoding malate dehydrogenase, mae encoding malic enzyme, pta encoding phosphotransacetlylase, pfl encoding pyruvate:formate lyase, ldh encoding lactate dehydrogenase, hydG encoding [FeFe] hydrogenase maturation factor, and ech endocing [NiFe] Ech-type hydrogenase.
16. The method of claim 7 , wherein said bacteria strain further comprises at least one overexpressed gene selected from the list consisting of Rnf encoding Ferredoxin:NADH oxidoreductase, PDC encoding pyruvate decarboxylase, adhE encoding bifunctional aldehyde/alcohol dehydrogenase, and pryK encoding pyruvate kinase.
17. A method of enhancing alcohol production of a bacteria strain from cellulosic substrates, comprising inactivating in said bacteria strain the endogenous gene coding for Type I glutamine synthetase (glnA).
18. The method of claim 17 , wherein the inactivation of the Type I glutamine synthetase (glnA) gene in the bacteria is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system, and homologous recombination.