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Abstract
In a search for potential ethanologens, waste compost was screened and 17
thermophilic bacterial strains were isolated. Two of these strains (M5EXG and
M10EXGT) were further characterized due to their relatively high ethanol tolerance
levels (5 and 10% v/v respectively) and Gram-negative nature. Both isolates were
facultatively anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative
rods that were capable of utilizing a range of carbon sources including
arabinose, galactose, mannose, glucose, and xylose and produced low amounts of
ethanol, acetate and lactate.
A chromosomal DNA fragment from M10EXGr, which contained adhT and other
operon reading frames (ORFs) that have deduced peptide sequences similar to
3-hexulose-6-phosphate synthase (HPS), 6-phospho-3-hexuloisomerase (PHI) and an
uncharacterized oxidoreductase, was sequenced. The adhT gene encoding the
thermostable alcohol dehydrogenase (ADH-T) from M10EXGr, that demonstrated
enzymatic activities with ethanol, 1-butanol, 1-pentanol, 1-heptanol, 1-hexanol,
1-octanol and 2-propanol but not methanol, was cloned and over-expressed in
Escherichia coli.
Nineteen putative thermophilic promoter fragments were isolated from M10EXGT
with 6 of them showing elevated promoter activities. Nucleotide sequences of these 6
putative promoter fragments indicated that one of them (Pm117) contained a partial
ORF that has deduced peptide sequence similar to a trigger factor protein in a number
of Bacillus species. Furthermore, it also contained -10, -3 5 and SD regions upstream
of the partial ORF. As such, we believe that promoter fragment Pm117 contains an
authentic thermophilic promoter, probably expressing a trigger-factor-like protein in
M10EXGt.
Construction of the production-of-ethanol (pet) operon, containing adhT from
M 1 OEXG T and pdc (encoding pyruvate decarboxylase) from Zymomonas mobilis was
carried out. Promoterless pdc was sub-cloned downstream of the previously cloned
adhT. Electroporation and conjugation experiments were also carried out in an effort to
introduce the constructed pet operon into Ml OEXGT. However, despite several
attempts, no transformants or transconjugants were obtained. Recombinant E. coli
strains 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 U
PDC/mg protein), although these levels were relatively low most likely due to weak
activity of the native adhT promoter in E. coli.