Dataset Information

High Production of 2,3-Butanediol (2,3-BD) by Raoultella ornithinolytica B6 via Optimizing Fermentation Conditions and Overexpressing 2,3-BD Synthesis Genes.

ABSTRACT: 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.

SUBMITTER: Kim T

PROVIDER: S-EPMC5070830 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

High Production of 2,3-Butanediol (2,3-BD) by Raoultella ornithinolytica B6 via Optimizing Fermentation Conditions and Overexpressing 2,3-BD Synthesis Genes.

Kim Taeyeon T Cho Sukhyeong S Lee Sun-Mi SM Woo Han Min HM Lee Jinwon J Um Youngsoon Y Seo Jin-Ho JH

PloS one 20161019 10

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 agita ...[more]

PMID: 27760200

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Project description:UNLABELLED: BACKGROUND:Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination. RESULTS:This work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7?g/L of acetoin and 14.5?g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography-mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. ?-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C. CONCLUSIONS:Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work.

Project description:Background:2,3-butanediol (2,3-BD) is a bulk platform chemical with various potential applications such as aviation fuel. 2,3-BD has three optical isomers: (2R, 3R)-, (2S, 3S)- and meso-2,3-BD. Optically pure 2,3-BD is a crucial precursor for the chiral synthesis and it can also be used as anti-freeze agent due to its low freezing point. 2,3-BD has been produced in both native and non-native hosts. Several pathogenic bacteria were reported to produce 2,3-BD in mixture of its optical isomers including Klebsiella pneumoniae and Klebsiella oxytoca. Engineered hosts based on episomal plasmid expression such as Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis are not ideal for industrial fermentation due to plasmid instability. Results:Pichia pastoris is generally regarded as safe and a well-established host for high-level heterologous protein production. To produce pure (2R, 3R)-2,3-BD enantiomer, we developed a P. pastoris strain by introducing a synthetic pathway. The alsS and alsD genes from B. subtilis were codon-optimized and synthesized. The BDH1 gene from S. cerevisiae was cloned. These three pathway genes were integrated into the genome of P. pastoris and expressed under the control of GAP promoter. Production of (2R, 3R)-2,3-BD was achieved using glucose as feedstock. The optical purity of (2R, 3R)-2,3-BD was more than 99%. The titer of (2R, 3R)-2,3-BD reached 12 g/L with 40 g/L glucose as carbon source in shake flask fermentation. The fermentation conditions including pH, agitation speeds and aeration rates were optimized in batch cultivations. The highest titer of (2R, 3R)-2,3-BD achieved in fed-batch fermentation using YPD media was 45 g/L. The titer of 2,3-BD was enhanced to 74.5 g/L through statistical medium optimization. Conclusions:The potential of engineering P. pastoris into a microbial cell factory for biofuel production was evaluated in this work using (2R, 3R)-2,3-BD as an example. Engineered P. pastoris could be a promising workhorse for the production of optically pure (2R, 3R)-2,3-BD.

Dataset Information

High Production of 2,3-Butanediol (2,3-BD) by Raoultella ornithinolytica B6 via Optimizing Fermentation Conditions and Overexpressing 2,3-BD Synthesis Genes.

Publications

High Production of 2,3-Butanediol (2,3-BD) by Raoultella ornithinolytica B6 via Optimizing Fermentation Conditions and Overexpressing 2,3-BD Synthesis Genes.

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