Isolation, evaluation and use of two strong, carbon source-inducible promoters from Corynebacterium glutamicum.

AIMS: To obtain strong, carbon source-inducible promoters useful for industrial applications of Corynebacterium glutamicum. METHODS AND RESULTS: DNA microarray and qRT-PCR enabled identification of the promoters of cgR_2367 (malE1) and cgR_2459 (git1) as strong, maltose- and gluconate-inducible promoters, respectively, in C. glutamicum. Promoter probe assays revealed that in the presence of the inducing sugars, PmalE1 and Pgit1, respectively, facilitated 3.4- and 4.2-fold increased beta-galactosidase activities compared to the same activity induced by glucose. In addition, PmalE1 was not functional in Escherichia coli, in which Pgit1 function was repressible, which enabled the cloning of a hitherto 'difficult-to-clone' heterologous gene of a lignocellulolytic enzyme, whose secretion was consequently induced by the carbon sources. CONCLUSIONS: PmalE1 and Pgit1 are strong, carbon source-inducible promoters of C. glutamicum whose characteristics in E. coli are integral to the secretion ability of C. glutamicum to secrete lignocellulolytic enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: Corynebacterium glutamicum, like its counterpart industrial workhorses E. coli and Bacillus subtilis, does exhibit strong, carbon source-inducible promoters, and the functionality of two of which was demonstrated in this study. While this study may be most relevant in the ongoing efforts to establish technologies of the biorefinery, it should also be of interest to general microbiologists exploring the versatility of industrial micro-organisms. In so doing, the study should impact future advances in industrial microbiology.

Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1.

The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD+-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K(m) values of the enzyme were 6.4 microM for acetaldehyde, 4.2 microM for hexanal, 2.8 microM for octanal, and 0.84 microM for decanal, whereas the kcat values for these substrates were nearly equal (51-64 min(-1)). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.