The pck (cg3169) gene of Corynebacterium glutamicum encodes a phosphoenolpyruvate carboxykinase (PEPCK). Here, a candidate transcriptional regulator that binds to the promoter region of pck was detected using a DNA affinity purification approach. An isolated protein was identified to be PckR (Cg0196), a GntR family transcriptional regulator which consists of 253 amino acids with a mass of 27 kDa as measured by peptide mass fingerprinting. The results of electrophoretic mobility shift assays verified that PckR specifically binds to the pck promoter. The putative regulator binding region extended from position ؊44 to ؊27 (an 18-bp sequence) relative to the transcriptional start point of the pck gene. We measured the expression of pck in a pckR deletion mutant by using quantitative real-time reverse transcription-PCR. The expression level of pck in the pckR mutant was 7.6 times higher than that in wild-type cells grown in glucose. Comparative DNA microarray hybridizations and bioinformatic searches revealed the gene composition of the transcriptional regulon of C. glutamicum. Based on these results, PckR seemed to play an important role in the regulation of PEPCK in C. glutamicum grown in glucose. In particular, these assays revealed that PckR acts as a repressor of pck expression during glucose metabolism.
Saccharomyces cerevisiae is used for edible purposes, such as human food or as an animal feed supplement. Fatty acids are also beneficial as feed supplements, but S. cerevisiae produces small amounts of fatty acids. In this study, we enhanced fatty acid production of S. cerevisiae by overexpressing acetyl-CoA carboxylase, thioesterase, and malic enzyme associated with fatty acid metabolism. The enhanced strain pAMT showed 2.4-fold higher fatty acids than the wild-type strain. To further increase the fatty acids, various nitrogen sources were analyzed and calcium nitrate was selected as an optimal nitrogen source for fatty acid production. By concentration optimization, 672 mg/L of fatty acids was produced, which was 4.7-fold higher than wild-type strain. These results complement the low level fatty acid production and make it possible to obtain the benefits of fatty acids as an animal feed supplement while, simultaneously, maintaining the advantages of S. cerevisiae.
To construct a self-assembled biosensor with signal amplification, a cellulosome system, comprising type I and type II dockerin-cohesin interactions with different specificities, from the anaerobic Clostridia bacterium was applied. The self-assembled biosensor was highly sensitive and achieved 128.1-fold increase in detection levels compared to the control.
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