Seung Kyou You

GntR-Type Transcriptional Regulator PckR Negatively Regulates the Expression of Phosphoenolpyruvate Carboxykinase in Corynebacterium glutamicum

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.

Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass.

Carrageenan is a generic name for a family of polysaccharides obtained from certain species of red algae. New methods to produce useful cost-efficiently materials from red algae are needed to convert enzymatic processes into fermentable sugars. In this study, we constructed chimeric genes cCgkA and cCglA containing the catalytic domain of κ-carrageenase CgkA and λ-carrageenase CglA from Pseudoalteromonas carrageenovora fused with a dockerin domain. Recombinant strains expressing the chimeric carrageenase resulted in a halo formation on the carrageenan plate by alcian blue staining. The recombinant cCgkA and cCglA were assembled with scaffoldin miniCbpA via cohesin and dockerin interaction. Carbohydrate binding module (CBM) in scaffoldin was used as a tag for cellulose affinity purification using cellulose as a support. The hydrolysis process was monitored by the amount of reducing sugar released from carrageenan. Interestingly, these results indicated that miniCbpA, cCgkA and cCglA assembled into a complex and that the dockerin-fused enzymes on the scaffoldin had synergistic activity in the degradation of carrageenan. The observed enhancement of activity by carrageenolytic complex was 3.1-fold-higher compared with the corresponding enzymes alone. Thus, the assemblies of advancement of active enzyme complexes will facilitate the commercial production of useful products from red algae biomass which represents inexpensive and sustainable feed-stocks.

Bio-based Production of Dimethyl Itaconate from Rice Wine Waste-derived Itaconic Acid†

Dimethyl itaconate is an important raw material for copolymerization, but it is not synthesized from itaconic acid by organisms. Moreover, Corynebacterium glutamicum is used as an important industrial host for the production of organic acids, but it does not metabolize itaconic acid. Therefore, the biosynthetic route toward dimethyl itaconate from itaconic acid is highly needed. In this study, a biological procedure for dimethyl itaconate production is developed from rice wine waste-derived itaconic acid using the engineered C. glutamicum strain. The first step is to investigate the effect of the co-overexpression of the codon-optimized cis-aconitic acid decarboxylase (CadA*) and a transcriptional regulator of genes involved in acetic acid metabolism (RamA) on itaconic acid production. The second step is to convert itaconic acid into dimethyl itaconate by lipase-catalyzed esterification. The CadA* and RamA-overexpressing CG4 strain increases the itaconic acid concentration under N-starvation with glucose and acetic acid compared with the concentration produced in the base mCGXII medium with glucose. Furthermore, the rice wine waste-derived itaconic acid is successfully converted into dimethyl itaconate using lipase from Rhizomucor miehei and a methanol substrate. This study is the first trial for bio-based production of dimethyl itaconate from rice wine waste-derived itaconic acid.

Synergistic effect of the enzyme complexes comprising agarase, carrageenase and neoagarobiose hydrolase on degradation of the red algae

In the practice of converting red algae biomass into biofuel or valuable biomaterials, the critical step is the decomposition process of the agarose to give fermentable monomeric sugars. In this study, we selected three enzymes such as agarase, carrageenase and neoagarobiose hydrolase to inducible the simultaneous hydrolysis of the major substrates such as agar and carrageenan constituting the pretreated red algae, and expressed the chimeric enzymes and formed a complexes through optimization of addition ratio. As a result, hydrolysis by enzyme complexes showed a maximum sugar release of 679 mg L-1 with 67.9% saccharification yield from G. verrucosa natural substrate. The difference in the reducing sugar by the enzyme complexes was 3.6-fold higher than that of the monomer enzyme (cAgaB yield 188.6 mg L-1). The synergistic effect of producing sugars from red algae biomass through these enzyme complexes can be a very important biological tools aimed at bioenergy production.

Enhancing Fatty Acid Production of Saccharomyces cerevisiae as an Animal Feed Supplement

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.