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Carbon monoxide dehydrogenase derived from Carboxydothermus hydrogenoformans (ChCODH) is a representative anaerobic enzyme that contains [4Fe–4S] clusters. To functionally express ChCODH under aerobic conditions in genetically engineered E. coli, co-expression with the SUF system that facilitates the assembly of iron–sulfur clusters under oxidative stress and iron limitation was performed. Results showed that compared to the expression of ChCODH alone under aerobic conditions, a 10-fold and 2-fold increase in the specific activity and protein yield, respectively, resulting in a total activity of up to 29,185 U/L was observed in E. coli co-expressed with ChCODH and the SUF system. Co-expression of Tig, a chaperone protein, and the addition of 2 mM FeSO4, which constitutes the iron-sulfur cluster, further increased the total activity up to 97,758 U/L. Moreover, the amount of Fe incorporated into ChCODH was proportional to the specific activity, while the amount of Ni was not correlated with the specific activity. Finally, high cell density cultivation under optimized aerobic conditions in the SUF system and the chaperone protein Tig co-expressed in recombinant E. coli resulted in a total activity of up to 235,689 U/L. [ABSTRACT FROM AUTHOR]

Two putative methylglyoxal synthases, which catalyze the conversion of dihydroxyacetone phosphate to methylglyoxal, from Oceanithermus profundus DSM 14,977 and Clostridium difficile 630 have been characterized for activity and thermal stability. The enzyme from O. profundus was found to be hyperthermophilic, with the optimum activity at 80 °C and the residual activity up to 59% after incubation of 15 min at 95 °C, whereas the enzyme from C. difficile was mesophilic with the optimum activity at 40 °C and the residual activity less than 50% after the incubation at 55 °C or higher temperatures for 15 min. The structural analysis of the enzymes with molecular dynamics simulation indicated that the hyperthermophilic methylglyoxal synthase has a rigid protein structure with a lower overall root-mean-square-deviation value compared with the mesophilic or thermophilic counterparts. In addition, the simulation results identified distinct regions with high fluctuations throughout those of the mesophilic or thermophilic counterparts via root-mean-square-fluctuation analysis. Specific molecular interactions focusing on the hydrogen bonds and salt bridges in the distinct regions were analyzed in terms of interatomic distances and positions of the individual residues with respect to the secondary structures of the enzyme. Key interactions including specific salt bridges and hydrogen bonds between a rigid beta-sheet core and surrounding alpha helices were found to contribute to the stabilisation of the hyperthermophilic enzyme by reducing the regional fluctuations in the protein structure. The structural information and analysis approach in this study can be further exploited for the engineering and industrial application of the enzyme. [ABSTRACT FROM AUTHOR]

Several bioprocessing technologies, such as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP), have been highlighted to produce bio-based fuels and chemicals from lignocellulosic biomass. Successful CBP, an efficient and economical lignocellulosic biorefinery process compared with other processes, requires microorganisms with sufficient cellulolytic activity and biofuel/chemical-producing ability. Here, we report the complete genome of Paenibacillus sp. CAA11, a newly isolated promising microbial host for CBP-producing ethanol and organic acids from cellulose. The genome of Paenibacillus sp. CAA11 comprises one 4,888,410 bp chromosome with a G + C content of 48.68% containing 4418 protein-coding genes, 102 tRNA genes, and 39 rRNA genes. The functionally active cellulase, encoded by CAA_GH5 was identified to belong to glycosyl hydrolase family 5 (GH5) and consisted of a catalytic domain and a cellulose-binding domain 3 (CBM3). When cellulolytic activity of CAA_GH5 was assayed through Congo red method by measuring the size of halo zone, the recombinant Bacillus subtilis RIK1285 expressing CAA_GH5 showed a comparable cellulolytic activity to B. subtilis RIK1285 expressing Cel5, a previously verified powerful bacterial cellulase. This study demonstrates the potential of Paenibacillus sp. CAA11 as a CBP-enabling microbe for cost-effective biofuels/chemicals production from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Carbon dioxide (CO2) emissions are related to global warming. However, CO2 can be used as an abundant and cheap carbon source for production of valuable chemicals using carbon capture and storage technology. Here, the genes related to carbon flux toward pyruvate biosynthesis in E. coli were deleted to enhance the incorporation of CO2 for succinate production. The codonoptimized carbonic anhydrase gene (SP(-)HCCA) derived from Hahella chejuensis KCTC 2396 and the phosphoenolpyruvate carboxylase gene (ppc) of E. coli W3110 were co-overexpressed to enhance carbon flux toward oxaloacetate synthesis in E. coli. Finally, we constructed SGJS134, which shows the highest production of succinate derived from CO2 compared with other strains. SGJS134 produced approximately 6.5 mM succinate from CO2 and yielded approximately 13.0 mM succinate per dry cell weight. These results may be useful for enhancing the incorporation of CO2 for succinate production in E. coli. Additionally, the metabolic engineering method used in this study will propose the potential of E. coli to convert CO2 to valuable chemicals. [ABSTRACT FROM AUTHOR]

Conversion of crude glycerol derived from biodiesel processes to value-added chemicals has attracted much attention. Herein, Raoultella ornithinolytica B6 was investigated for the high production of 2,3-butanediol (2,3-BD) from glycerol without 1,3-propanediol (1,3-PD) formation, a by-product hindering 2,3-BD purification. By evaluating the effects of temperature, agitation speed, and pH control strategy, the fermentation conditions favoring 2,3-BD production were found to be 25 °C, 400 rpm, and pH control with a lower limit of 5.5, respectively. Notably, significant pH fluctuations which positively affect 2,3-BD production were generated by simply controlling the lower pH limit at 5.5. In fed-batch fermentation under those conditions, R. ornithinolytica B6 produced 2,3-BD up to 79.25 g/L, and further enhancement of 2,3-BD production (89.45 g/L) was achieved by overexpressing homologous 2,3-BD synthesis genes (the budABC). When pretreated crude glycerol was used as a sole carbon source, R. ornithinolytica B6 overexpressing budABC produced 78.10 g/L of 2,3-BD with the yield of 0.42 g/g and the productivity of 0.62 g/L/h. The 2,3-BD titer, yield, and productivity values obtained in this study are the highest 2,3-BD production from glycerol among 1,3-PD synthesis-deficient 2,3-BD producers, demonstrating R. ornithinolytica B6 as a promising 2,3-BD producer from glycerol. [ABSTRACT FROM AUTHOR]

A newly isolated bacterium, designated as Klebsiella oxytoca M1, produced 2,3-butanediol (2,3-BDO) or acetoin selectively as a major product depending on temperature in a defined medium. K. oxytoca M1 produced 2,3-BDO mainly (0.32~0.34 g/g glucose) at 30 °C while acetoin was a major product (0.32~0.38 g/g glucose) at 37 °C. To investigate factors affecting product profiles according to temperature, the expression level of acetoin reductase (AR) that catalyzes the conversion of acetoin to 2,3-BDO was analyzed using crude protein extracted from K. oxytoca M1 grown at 30 and 37 °C. The AR expression at 37 °C was 12.8-fold lower than that at 30 °C at the stationary phase and reverse transcription PCR (RT-PCR) analysis of the budC (encoding AR) was also in agreement with the AR expression results. When AR was overexpressed using K. oxytoca M1 harboring pUC18CM- budC, 2,3-BDO became a major product at 37 °C, indicating that the AR expression level was a key factor determining the major product of K. oxytoca M1 at 37 °C. The results in this study demonstrate the feasibility of using K. oxytoca M1 for the production of not only 2,3-BDO but also acetoin as a major product. [ABSTRACT FROM AUTHOR]