This disclosure describes recombinant Caldicellulosiruptor bescii microbes designed to produce greater amounts of acetate, H 2 , and/or ethanol than a comparable wild type control. this disclosure also describes methods that generally include growing such recombinant microbes under conditions effective for the recombinant microbes to produce acetate, H 2 , and/or ethanol.
1. A recombinant Caldicellulosiruptor microbe genetically modified to produce a greater amount of ethanol than a comparable wild type control, wherein the Caldicellulosiruptor microbe:
comprises a knock-out of the coding region of a lactate dehydrogenase and produces lactate in an amount less than a comparable wild type control;
comprises at least one heterologous polynucleotide that encodes an enzyme in a metabolic pathway that converts acetyl-CoA to ethanol, wherein the enzyme is an acetaldehyde dehydrogenase that can convert acetyl-CoA to acetaldehyde, an aldehyde dehydrogenase that can convert acetyl-CoA to acetaldehyde, or a combination thereof; and wherein the Caldicellulosiruptor microbe is C. bescii, C. kristjansonii, C. obsidiansis, C. hydrothermalis, C. kronotskvensis, C. lactoaceticus , or C. saccharolvticus.
2. The recombinant Caldicellulosiruptor microbe of claim 1 wherein the Caldicellulosiruptor microbe is Caldicellulosiruptor bescii.
3. The recombinant Caldicellulosiruptor microbe of claim 1 comprising a deletion of at least a portion of a lactate dehydrogenase coding region.
4. The recombinant Caldicellulosiruptor microbe of claim 3 wherein the lactate dehydrogenase coding region encodes an NADH-dependent lactate dehydrogenase (Cbes 1918).
5. The recombinant Caldicellulosiruptor microbe of claim 1 further genetically modified to exhibit decreased activity of an endonuclease that digests unmethylated DNA in a Caldicellulosiruptor spp. (CbeI) compared to a comparable wild-type control.
6. The recombinant Caldicellulosiruptor microbe of claim 1 further genetically modified to exhibit increased efficiency of transformation with unmethylated heterologous DNA.
7. A method comprising:
growing the recombinant Caldicellulosiruptor microbe of claim 1 , wherein the Caldicellulosiruptor microbe is genetically modified to produce a greater amount of ethanol than a comparable wild type control under conditions effective for the recombinant Caldicellulosiruptor microbe to produce ethanol, wherein the Caldicellulosiruptor microbe:
comprises a knock-out of the coding region of a lactate dehydrogenase and produces lactate in an amount less than a comparable wild type control;
comprises at least one heterologous polynucleotide that encodes an enzyme in a metabolic pathway that converts acetyl-CoA to ethanol, wherein the enzyme is an acetaldehyde dehydrogenase that can convert acetate to acetaldehyde, an aldehyde dehydrogenase that can convert acetyl-CoA to acetaldehyde, or a combination thereof; and wherein the
Caldicellulosiruptor microbe is C. bescii, C. kristjansonii, C. obsidiansis, C. hydrothermalis, C. kronotskvensis, C. lactoaceticus , or C. saccharolvticus.
8. The method of claim 7 further comprising collecting at least a portion of the ethanol.
9. The method of claim 7 wherein the conditions comprise a carbon source that comprises napier grass, Bermuda grass, switchgrass, or a hardwood.
10. The method of claim 9 wherein the carbon source comprises unpretreated switchgrass.
11. The recombinant Caldicellulosiruptor microbe of claim 1 further comprising a knock-out of the coding region of an acetate kinase where the microbe further comprises an alcohol dehydrogenase that converts acetaldehyde to ethanol.