Characterization and genetic manipulation of thermophiles for ethnanol production

In a search for potential ethanologens, waste compost was screened and 17thermophilic bacterial strains were isolated. Two of these strains (M5EXG andM10EXGT) were further characterized due to their relatively high ethanol tolerancelevels (5 and 10% v/v respectively) and Gram-negative nature. Both isolates werefacultatively anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negativerods that were capable of utilizing a range of carbon sources includingarabinose, galactose, mannose, glucose, and xylose and produced low amounts ofethanol, acetate and lactate.A chromosomal DNA fragment from M10EXGr, which contained adhT and otheroperon reading frames (ORFs) that have deduced peptide sequences similar to3-hexulose-6-phosphate synthase (HPS), 6-phospho-3-hexuloisomerase (PHI) and anuncharacterized oxidoreductase, was sequenced. The adhT gene encoding thethermostable alcohol dehydrogenase (ADH-T) from M10EXGr, that demonstratedenzymatic activities with ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol,1-octanol and 2-propanol but not methanol, was cloned and over-expressed inEscherichia coli.Nineteen putative thermophilic promoter fragments were isolated from M10EXGTwith 6 of them showing elevated promoter activities. Nucleotide sequences of these 6putative promoter fragments indicated that one of them (Pm117) contained a partialORF that has deduced peptide sequence similar to a trigger factor protein in a numberof Bacillus species. Furthermore, it also contained -10, -3 5 and SD regions upstreamof the partial ORF. As such, we believe that promoter fragment Pm117 contains anauthentic thermophilic promoter, probably expressing a trigger-factor-like protein inM10EXGt.Construction of the production-of-ethanol (pet) operon, containing adhT fromM 1 OEXG T and pdc (encoding pyruvate decarboxylase) from Zymomonas mobilis wascarried out. Promoterless pdc was sub-cloned downstream of the previously clonedadhT. Electroporation and conjugation experiments were also carried out in an effort tointroduce the constructed pet operon into Ml OEXGT. However, despite severalattempts, no transformants or transconjugants were obtained. Recombinant E. colistrains harboring the constructed pet operon produced enhanced levels of ethanol(0.5 g/1) and ethanologenic enzyme activities (0 .2 U ADH/mg protein and 0.02 UPDC/mg protein), although these levels were relatively low most likely due to weakactivity of the native adhT promoter in E. coli