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• The cell growth was enhanced by addition of W with 5.10-fold increase of biomass comparing W-free cultivation using methane. • The maximum biomass in the W-added medium was 5.90-fold higher than that in the W-free cultivation using methanol. • Biomass yield and total product yield increased up to 11.50-fold and 1.28-fold, respectively compared with W-free cultivation using methanol. It is carried out for researches to convert methane, the second most potent greenhouse gas, to high-value chemicals and fuels by using methanotrophs. In this study, we observed that cell growth of Methylomicrobium alcaliphilum 20Z in the batch cultures on methane or methanol was stimulated by the addition of tungsten (W) without formate accumulation. Not only biomass yield but also the total products yield (biomass and formate) on carbon basis increased up to 11.50-fold and 1.28-fold respectively in W-added medium. Furthermore, a significant decrease in CO 2 yield from formate was observed in the W-added cells, which indicates that W might have affected the activity of certain enzymes involved in carbon assimilation as well as formate dehydrogenase (FDH). The results of this study suggest that M. alcaliphilum 20Z is a promising model system for studying the physiology of the aerobic methanotroph and for enabling its industrial use for methane conversion through high cell density cultivation. [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]

In this study, a gas chromatography–mass spectrometry (GC–MS) analysis method was developed and validated for the quantitative analysis of limonene that was attempted to be industrially mass‐produced from genetically engineered Saccharomyces cerevisiae (S. cerevisiae). Owing to the high potential economic value of limonene, (e.g., for biofuels, fragrant additives, pharmaceuticals, or organic solvents), it is important to develop and validate a readily available quantitative analysis method for limonene produced from genetically engineered yeast or microorganisms. The engineered S. cerevisiae was cultured using two‐phase extractive fermentation with n‐dodecane, which is known to alleviate monoterpene toxicity, used as an extractive solvent. The quantitative GC–MS analysis of the n‐dodecane overlay that contained limonene produced from the engineered S. cerevisiae was validated to be in accord with the international standards set forth by the Food and Drug Administration. [ABSTRACT FROM AUTHOR]

Biological production of 2,3-butandiol (2,3-BD) has received great attention as an alternative to the petroleum-based 2,3-BD production. In this study, a high production of 2,3-BD in fed-batch fermentation was investigated with a newly isolated bacterium designated as Raoultella ornithinolytica B6. The isolate produced 2,3-BD as the main product using hexoses (glucose, galactose, and fructose), pentose (xylose) and disaccharide (sucrose). The effects of temperature, pH-control schemes, and agitation speeds on 2,3-BD production were explored to optimize the fermentation conditions. Notably, cell growth and 2,3-BD production by R. ornithinolytica B6 were higher at 25°C than at 30°C. When three pH control schemes (no pH control, pH control at 7, and pH control at 5.5 after the pH was decreased to 5.5 during fermentation) were tested, the best 2,3-BD titer and productivity along with reduced by-product formation were achieved with pH control at 5.5. Among different agitation speeds (300, 400, and 500 rpm), the optimum agitation speed was 400 rpm with 2,3-BD titer of 68.27 g/L, but acetic acid was accumulated up to 23.32 g/L. Further enhancement of the 2,3-BD titer (112.19 g/L), yield (0.38 g/g), and productivity (1.35 g/L/h) as well as a significant reduction of acetic acid accumulation (9.71 g/L) was achieved by the overexpression of homologous budABC genes, the 2,3-BD-synthesis genes involved in the conversion of pyruvate to 2,3-BD. This is the first report presenting a high 2,3-BD production by R.ornithinolytica which has attracted little attention with respect to 2,3-BD production, extending the microbial spectrum of 2,3-BD producers. [ABSTRACT FROM AUTHOR]

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase. [ABSTRACT FROM AUTHOR]

Both ThyA and ThyX proteins catalyze the transfer of the methyl group from methylenetetrahydrofolate ( CH2H4-folate) to d UMP, forming d TMP. To estimate the relative steady state expression levels of ThyA and ThyX, Western blot analysis was performed using ThyA or ThyX antiserum on total protein from the wild-type, ΔthyX, and thyX-complemented strains of Corynebacterium glutamicum. The level of ThyA decreased gradually during the stationary growth phase but that of ThyX was maintained steadily. Whereas the expression level of ThyA in a ΔsigB strain was comparable to that of the wild-type, the level of ThyX was significantly diminished in the deletion mutant and was restored to that of the wild-type in the complemented strain, indicating that the level of ThyX was regulated by Sig B. Growth of the C. glutamicum ΔsigB strain was dependent upon coupling activity of dihydrofolate reductase ( DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210- HCl. These results suggested that the relative levels of ThyA and ThyX differ in response to different growth phases and that Sig B is necessary for maintenance of the level of ThyX during transition into the stationary growth phase. [ABSTRACT FROM AUTHOR]

Methane is an abundant, inexpensive one-carbon feedstock and one of the most powerful greenhouse gases. Because it does not compete with food demand, it is considered a promising carbon feedstock for the production of valuable products using methanotrophic bacteria. Here, we isolated a novel methanotrophic bacterium, Methylomonas sp. SW1, from a sewage sample obtained from Wonju City Water Supply Drainage Center, Republic of Korea. The conditions for uracil production by Methylomonas sp. SW1, such as Cu 2+ concentration and temperature were investigated and optimized. As a result, Methylomonas sp. SW1 produced uracil from methane as a sole carbon source with a titer of 2.1 mg/L in 84 h without genetic engineering under the optimized condition. The results in this study demonstrate the feasibility of using Methylomonas sp. SW1 for the production of uracil from methane. This is the first report of uracil production from gas feedstock by methanotrophic bacteria. [ABSTRACT FROM AUTHOR]