A Genome-Scale Metabolic Model of 2,3-Butanediol Production by Thermophilic Bacteria Geobacillus icigianus.
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SUBMITTER: Kulyashov M
PROVIDER: S-EPMC7409357 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC3538569 | biostudies-literature
Project description:BackgroundKlebsiella oxytoca, a Gram-negative, rod-shaped, and facultative anaerobic bacterium, is one of the most promising 2,3-butanediol (2,3-BD) producers. In order to improve the metabolic performance of K. oxytoca as an efficient biofactory, it is necessary to assess its metabolic characteristics with a system-wide scope, and to optimize the metabolic pathways at a systems level. Provision of the complete genome sequence of K. oxytoca enabled the construction of genome-scale metabolic model of K. oxytoca and its in silico analyses.ResultsThe genome-scale metabolic model of K. oxytoca was constructed using the annotated genome with biochemical and physiological information. The stoichiometric model, KoxGSC1457, is composed of 1,457 reactions and 1,099 metabolites. The model was further refined by applying biomass composition equations and comparing in silico results with experimental data based on constraints-based flux analyses. Then, the model was applied to in silico analyses to understand the properties of K. oxytoca and also to improve its capabilities for 2,3-BD production according to genetic and environmental perturbations. Firstly, in silico analysis, which tested the effect of augmenting the metabolic flux pool of 2,3-BD precursors, elucidated that increasing the pyruvate pool is primarily important for 2,3-BD synthesis. Secondly, we performed in silico single gene knockout simulation for 2,3-BD overproduction, and investigated the changes of the in silico flux solution space of a ldhA gene knockout mutant in comparison with that of the wild-type strain. Finally, the KoxGSC1457 model was used to optimize the oxygen levels during fermentation for 2,3-BD production.ConclusionsThe genome-scale metabolic model, KoxGSC1457, constructed in this study successfully investigated metabolic characteristics of K. oxytoca at systems level. The KoxGSC1457 model could be employed as an useful tool to analyze its metabolic capabilities, to predict its physiological responses according to environmental and genetic perturbations, and to design metabolic engineering strategies to improve its metabolic performance.
| S-EPMC3602198 | biostudies-literature
Project description:BACKGROUND:2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(-)-2,3-BD (purity >99 %) under low oxygen conditions. RESULTS:In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(-)-2,3-BD production was abolished by deleting d-(-)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (?acoA?bdhA?pta?ldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L. CONCLUSION:This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.
| S-EPMC4837526 | biostudies-literature
Project description:Background2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production.ResultsWe first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH) pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 . l-1) and yield (0.113 g . g-1) were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition.ConclusionsWith the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.
| S-EPMC3442981 | biostudies-literature
Project description:Myceliophthora thermophila is a thermophilic fungus with great biotechnological characteristics for industrial applications, which can degrade and utilize all major polysaccharides in plant biomass. Nowadays, it has been developing into a platform for production of enzyme, commodity chemicals and biofuels. Therefore, an accurate genome-scale metabolic model would be an accelerator for this fungus becoming a universal chassis for biomanufacturing. Here we present a genome-scale metabolic model for M. thermophila constructed using an auto-generating pipeline with consequent thorough manual curation. Temperature plays a basic and critical role for the microbe growth. we are particularly interested in the genome wide response at metabolic layer of M. thermophilia as it is a thermophlic fungus. To study the effects of temperature on metabolic characteristics of M. thermophila growth, the fungus was cultivated under different temperature. The metabolic rearrangement predicted using context-specific GEMs integrating transcriptome data.The developed model provides new insights into thermophilic fungi metabolism and highlights model-driven strain design to improve biotechnological applications of this thermophilic lignocellulosic fungus.
2022-09-14 | GSE184074 | GEO
Project description:Klebsiella pneumoniae KCTC2242 has high potential in the production of a high-value chemical, 2,3-butanediol (2,3-BDO). However, accumulation of metabolites such as lactate during cell growth prevent large-scale production of 2,3-BDO. Consequently, we engineered K. pneumoniae to redistribute its carbon flux toward 2,3-BDO production. The ldhA gene deletion and gene overexpression (budA and budB) were conducted to block a pathway that competitively consumes reduced nicotinamide adenine dinucleotide and to redirect carbon flux toward 2,3-BDO biosynthesis, respectively. These steps allowed efficient glucose conversion to 2,3-BDO under slightly acidic conditions (pH 5.5). The engineered strain SGSB105 showed a 40% increase in 2,3-BDO production from glucose compared with that of the host strain, SGSB100. Genes closely related to 2,3-BDO biosynthesis were observed at the gene transcription level by cultivating the SGSB100, SGSB103, SGSB104, and SGSB105 strains under identical growth conditions. Transcription levels for budA, budB, and budC increased approximately 10% during the log phase of cell growth relative to that of SGSB100. Transcription levels of 2,3-BDO genes in SGSB105 remained high during the log and stationary phases. Thus, the carbon flux was redirected toward 2,3-BDO production. Data on batch culture and gene transcription provide insight into improving the metabolic network for 2,3-BDO biosynthesis for industrial applications.
| S-EPMC4203659 | biostudies-literature
Project description:BACKGROUND:D-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis. RESULTS:We developed a genetically modified strain of B. licheniformis (WX-02 ?budC) as a D-2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the budC gene in B. licheniformis WX-02. The budC knock-out strain successfully abolished meso-2,3-butanediol production with enhanced D-2,3-butanediol production. No meso-BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced meso-2,3-butanediol and possessed meso-BDH activity. All of these data suggested that budC encoded the major meso-BDH catalyzing the reversible reaction from acetoin to meso-2,3-butanediol in B. licheniformis. The budC knock-out strain produced D-2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h. CONCLUSIONS:We confirmed the hypothesis that budC gene is responsible to reversibly transfer acetoin to meso-2,3-butanediol in B. licheniformis. A mutant strain of B. licheniformis with depleted budC gene was successfully developed and produced high level of the D-2,3-butanediol with high optimal purity.
| S-EPMC3909405 | biostudies-literature
Project description:(3S)-Acetoin and (2S,3S)-2,3-butanediol are important platform chemicals widely applied in the asymmetric synthesis of valuable chiral chemicals. However, their production by fermentative methods is difficult to perform. This study aimed to develop a whole-cell biocatalysis strategy for the production of (3S)-acetoin and (2S,3S)-2,3-butanediol from meso-2,3-butanediol. First, E. coli co-expressing (2R,3R)-2,3-butanediol dehydrogenase, NADH oxidase and Vitreoscilla hemoglobin was developed for (3S)-acetoin production from meso-2,3-butanediol. Maximum (3S)-acetoin concentration of 72.38 g/L with the stereoisomeric purity of 94.65% was achieved at 24 h under optimal conditions. Subsequently, we developed another biocatalyst co-expressing (2S,3S)-2,3-butanediol dehydrogenase and formate dehydrogenase for (2S,3S)-2,3-butanediol production from (3S)-acetoin. Synchronous catalysis together with two biocatalysts afforded 38.41 g/L of (2S,3S)-butanediol with stereoisomeric purity of 98.03% from 40 g/L meso-2,3-butanediol. These results exhibited the potential for (3S)-acetoin and (2S,3S)-butanediol production from meso-2,3-butanediol as a substrate via whole-cell biocatalysis.
| S-EPMC6017632 | biostudies-literature
Project description:Background2,3-butanediol is an important platform compound which has a wide range of applications, involving in medicine, chemical industry, food and other fields. Especially the optically pure (2R,3R)-2,3-butanediol can be employed as an antifreeze agent and as the precursor for producing chiral compounds. However, some (2R,3R)-2,3-butanediol overproducing strains are pathogenic such as Enterobacter cloacae and Klebsiella oxytoca.ResultsIn this study, a (3R)-acetoin overproducing C. glutamicum strain, CGS9, was engineered to produce optically pure (2R,3R)-2,3-butanediol efficiently. Firstly, the gene bdhA from B. subtilis 168 was integrated into strain CGS9 and its expression level was further enhanced by using a strong promoter Psod and ribosome binding site (RBS) with high translation initiation rate, and the (2R,3R)-2,3-butanediol titer of the resulting strain was increased by 33.9%. Then the transhydrogenase gene udhA from E. coli was expressed to provide more NADH for 2,3-butanediol synthesis, which reduced the accumulation of the main byproduct acetoin by 57.2%. Next, a mutant atpG was integrated into strain CGK3, which increased the glucose consumption rate by 10.5% and the 2,3-butanediol productivity by 10.9% in shake-flask fermentation. Through fermentation engineering, the most promising strain CGK4 produced a titer of 144.9 g/L (2R,3R)-2,3-butanediol with a yield of 0.429 g/g glucose and a productivity of 1.10 g/L/h in fed-batch fermentation. The optical purity of the resulting (2R,3R)-2,3-butanediol surpassed 98%.ConclusionsTo the best of our knowledge, this is the highest titer of optically pure (2R,3R)-2,3-butanediol achieved by GRAS strains, and the result has demonstrated that C. glutamicum is a competitive candidate for (2R,3R)-2,3-butanediol production.
| S-EPMC9310479 | biostudies-literature
Project description:2,3-Butanediol (23BD) is a high-value chemical usually produced petrochemically but which can also be synthesized by some bacteria. To date, the best microbial 23BD production rates have been observed using pathogenic bacteria in fermentation systems that depend on sugars as the carbon and energy sources for product synthesis. Here we present evidence of 23BD production by three nonpathogenic acetogenic Clostridium species-Clostridium autoethanogenum, C. ljungdahlii, and C. ragsdalei-using carbon monoxide-containing industrial waste gases or syngas as the sole source of carbon and energy. Through an analysis of the C. ljungdahlii genome, the complete pathway from carbon monoxide to 23BD has been proposed. Homologues of the genes involved in this pathway were also confirmed for the other two species investigated. A gene expression study demonstrates a correlation between mRNA accumulation from 23BD biosynthetic genes and the onset of 23BD production, while a broader expression study of Wood-Ljungdahl pathway genes provides a transcription-level view of one of the oldest existing biochemical pathways.
| S-EPMC3147483 | biostudies-literature