Thermophilic organisms for conversion of lignocellulosic biomass to ethanol
Mutant thermophilic organisms that consume a variety of biomass derived substrates are disclosed herein. Strains of Thermoanaerobacterium saccharolyticum with acetate kinase and phosphotransacetylase expression eliminated are disclosed herein. Further, strain ALK1 has been engineered by site directed homologous recombination to knockout both acetic acid and lactic acid production. Continuous culture involving a substrate concentration challenge lead to evolution of ALK1, and formation of a more robust strain designated ALK2. The organisms may be utilized for example in thermophilic SSF and SSCF reactions performed at temperatures that are optimal for cellulase activity to produce near theoretical ethanol yields without expressing pyruvate decarboxylase.
1. An engineered Gram-positive bacterium that ferments a saccharification product of a cellulolytic substrate to produce ethanol, wherein said bacterium converts pyruvate to acetyl-CoA by means of pyruvate-ferredoxin oxidoreductase, and wherein at least three genes in said bacterium have been disrupted through homologous recombination, said three genes being ldh gene, phosphotransacetylase (pta) gene and acetate kinase (ack) gene, wherein said bacterium has an ethanol yield at least 90% of theoretical yield.
2. A method for producing ethanol, said method comprising:
transforming a native bacterium to produce the engineered bacterium of claim 1 ; and
culturing the engineered Gram-positive bacterium in a medium that contains a substrate including a material selected from the group consisting of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the substrate to produce ethanol.
3. The bacterium of claim 1 , wherein said Gram-positive bacterium belongs to the genus selected from the group consisting of the Thermoanaerobacterium genus and the Thermoanaerobacter genus.
4. The bacterium of claim 1 , wherein said bacterium is a Thermoanaerobacterium saccharolyticum.
5. The bacterium of claim 1 , wherein said bacterium is designated ALK1 and deposited under Patent Deposit Designation No. PTA-7206.
6. A biologically pure culture of a microorganism designated ALK1 and deposited under Patent Deposit Designation No. PTA-7206.
7. A genetic construct designated pSGD9 comprising a polynucleotide having a sequence at least 99% identical to:
(a) the sequence of SEQ ID NO: 10; or
(b) the sequence of SEQ ID NO: 9.
8. The genetic construct of claim 7 , wherein the polynucleotide comprises a sequence 100% identical to the sequence of (a) or (b).
9. A vector comprising the genetic construct of claim 7 .
10. A host cell genetically engineered to express the vector of claim 9 .
11. The host cell of claim 10 , wherein the host cell is a bacterial cell.
12. A method of producing ethanol, comprising the step of: culturing the engineered Gram-positive bacterium of claim 1 in a medium containing a substrate selected from the group consisting of glucose, xylose, mannose, arabinose, galactose, fructose, cellobiose, sucrose, maltose, xylan, mannan, starch, and combinations thereof under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the substrate to produce ethanol.
13. The method of claim 12 , wherein the engineered Gram-positive bacterium is Thermoanaerobacterium saccharolyticum.
14. The method of claim 13 , wherein the engineered Gram-positive bacterium is Thermoanaerobacterium saccharolyticum ALK1 (JW/SL-YS485 ALK1).
15. A recombinant bacterium comprising the genetic construct of claim 7 .
16. The recombinant bacterium of claim 15 , wherein said bacterium is Thermoanaerobacterium saccharolyticum.
17. A method for producing ethanol, said method comprising:
providing within a reaction vessel, a reaction mixture comprising lignocellulosic substrate, cellulase and a fermentation agent, the fermentation agent comprising the engineered Gram-positive bacterium of claim 1 ,
wherein the reaction mixture is reacted under suitable conditions for a period of time sufficient to allow saccharification and fermentation of the lignocellulosic substrate to produce ethanol.
18. The method of claim 17 , wherein the suitable conditions comprise a temperature of at least 50° C.
19. The method of claim 17 , wherein the Gram-positive bacterium is a member of the Thermoanaerobacter genus.
20. The method of claim 17 , wherein the Gram-positive bacterium is a Thermoanaerobacterium saccharolyticum.