Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone-butanol-ethanol production.
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SUBMITTER: Lin L
PROVIDER: S-EPMC8456130 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundFurfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis.ResultsA total of 5,003 C. beijerinckii NCIMB 8052 genes capturing about 99.7% of the genome were examined. About 111 genes were differentially expressed (up- or down-regulated) by C. beijerinckii when it was challenged with furfural at acidogenic growth phase compared with 721 genes that were differentially expressed (up- or down-regulated) when C. beijerinckii was challenged with furfural at solventogenic growth phase. The differentially expressed genes include genes related to redox and cofactors, membrane transporters, carbohydrate, amino sugar and nucleotide sugar metabolisms, heat shock proteins, DNA repair, and two-component signal transduction system. While C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control, ABE production by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase.ConclusionGenome-wide transcriptional response of C. beijerinckii to furfural stress was investigated for the first time using microarray analysis. Stresses emanating from ABE accumulation in the fermentation medium; redox balance perturbations; and repression of genes that code for the phosphotransferase system, cell motility and flagellar proteins (and combinations thereof) may have caused the premature termination of C. beijerinckii 8052 growth and ABE production following furfural challenge at the solventogenic phase.This study provides insights into basis for metabolic engineering of C. beijerinckii NCIMB 8052 for enhanced tolerance of lignocellulose-derived microbial inhibitory compounds, thereby improving bioconversion of lignocellulose biomass hydrolysates to biofuels and chemicals. Indeed, two enzymes encoded by Cbei_3974 and Cbei_3904 belonging to aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) families have been identified to be involved in furfural detoxification and tolerance.
| S-EPMC3681630 | biostudies-literature
Project description:Calcium carbonate increases growth, substrate utilization, and acetone-butanol-ethanol (ABE) fermentation by Clostridium beijerinckii NCIMB 8052. Toward an understanding of the basis for these pleiotropic effects, we profiled changes in the C. beijerinckii NCIMB 8052 proteome that occur in response to the addition of CaCO(3). We observed increases in the levels of different heat shock proteins (GrpE and DnaK), sugar transporters, and proteins involved in DNA synthesis, repair, recombination, and replication. We also noted significant decreases in the levels of proteins involved in metabolism, nucleic acid stabilization, sporulation, oxidative and antibiotic stress responses, and signal transduction. We determined that CaCO(3) enhances ABE fermentation due to both its buffering effects and its ability to influence key cellular processes, such as sugar transport, butanol tolerance, and solventogenesis. Moreover, activity assays in vitro for select solventogenic enzymes revealed that part of the underpinning for the CaCO(3)-mediated increase in the level of ABE fermentation stems from the enhanced activity of these catalysts in the presence of Ca(2+). Collectively, these proteomic and biochemical studies provide new insights into the multifactorial basis for the stimulation of ABE fermentation and butanol tolerance in the presence of CaCO(3).
| S-EPMC3536099 | biostudies-literature
Project description:Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the single-cell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels.
| S-EPMC4500237 | biostudies-literature
Project description:Acetone-butanol-ethanol (ABE) fermentation is a traditional way for solvents production through bioconversion by Clostridium species. It is still a challenge to obtain metabolic engineering strains with high ABE yield. Screening strains with remarkable characteristics from nature and improving ABE yield by mutation are viable approaches. Clostridium beijerinckii XH 0906, a newly isolated strain, produces butanol and isopropanol (BI) as the main end-products (9.1 g/L BI) during fermentation with glucose as the sole carbon source. The screening process for this strain was performed under aerobic conditions rather than anaerobic environment. Thus, it is a robust stain capable of oxygen-tolerant BI fermentation. Furthermore, C. beijerinckii XH 0906 fermented xylose and glucose simultaneously to produce BI. A mutant strain obtained by ultraviolet (UV) mutagenesis, C. beijerinckii XH 29, had improved BI production capacity and could produce 17.0 g/L BI and 18.4 g/L BI using glucose or corn stover hydrolysate, respectively as the carbon source. Interestingly, C. beijerinckii XH 29 also produced up to 19.3 g/L isopropanol through fermentation of a glucose-acetone mix. These results indicate that C. beijerinckii XH 29 is an excellent BI producer with great potential for industrial applications.
| S-EPMC9107568 | biostudies-literature
Project description:An engineered B. subtilis 1A1 strain (BsADH2) expressing a secondary alcohol dehydrogenase (CpSADH) was co-cultured with C. beijerinckii G117 under an aerobic condition. During the fermentation on glucose, B. subtilis BsADH2 depleted oxygen in culture media completely and created an anaerobic environment for C. beijerinckii G117, an obligate anaerobe, to grow. Meanwhile, lactate produced by B. subtilis BsADH2 was re-assimilated by C. beijerinckii G117. In return, acetone produced by C. beijerinckii G117 was reduced into isopropanol by B. subtilis BsADH2 via expressing the CpSADH, which helped maintain the redox balance of the engineered B. subtilis. In the symbiotic system consisting of two strains, 1.7 ?g/L of acetone, 4.8 ?g/L of butanol, and 0.9 ?g/L of isopropanol (with an isopropanol/acetone ratio of 0.53) was produced from 60 ?g/L of glucose. This symbiotic system also worked when oxygen was supplied to the culture, although less isopropanol was produced (0.9 ?g/L of acetone, 4.9 ?g/L of butanol, and 0.2 ?g/L of isopropanol). The isopropanol titer was increased substantially to 2.5 ?g/L when we increased the inoculum size of B. subtilis BsADH2 and optimized other process parameters. With the Bacillus-Clostridium co-culture, switching from the original acetone-butanol (AB) fermentation to an aerobic acetone-butanol-isopropanol (ABI) fermentation can be easily achieved without genetic engineering of Clostridium. This strategy of employing a recombinant Bacillus to co-culture with Clostridium should be potentially useful to modify traditional acetone-butanol-ethanol fermentation for the production of other value-added chemicals.
| S-EPMC7322341 | biostudies-literature
Project description:Producing biobutanol from lignocellulosic biomass has shown promise to ultimately reduce greenhouse gases and alleviate the global energy crisis. However, because of the recalcitrance of a lignocellulosic biomass, a pretreatment of the substrate is needed which in many cases releases soluble lignin compounds (SLCs), which inhibit growth of butanol-producing clostridia. In this study, we found that SLCs changed the acetone/butanol ratio (A/B ratio) during butanol fermentation. The typical A/B molar ratio during Clostridium beijerinckii NCIMB 8052 batch fermentation with glucose as the carbon source is about 0.5. In the present study, the A/B molar ratio during batch fermentation with a lignocellulosic hydrolysate as the carbon source was 0.95 at the end of fermentation. Structural and redox potential changes of the SLCs were characterized before and after fermentation by using gas chromatography/mass spectrometry and electrochemical analyses, which indicated that some exogenous SLCs were involved in distributing electron flow to C. beijerinckii, leading to modulation of the redox balance. This was further demonstrated by the NADH/NAD+ ratio and trxB gene expression profile assays at the onset of solventogenic growth. As a result, the A/B ratio of end products changed significantly during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source compared to glucose as the carbon source. These results revealed that SLCs not only inhibited cell growth but also modulated the A/B ratio during C. beijerinckii butanol fermentation.IMPORTANCE Bioconversion of lignocellulosic feedstocks to butanol involves pretreatment, during which hundreds of soluble lignin compounds (SLCs) form. Most of these SLCs inhibit growth of solvent-producing clostridia. However, the mechanism by which these compounds modulate electron flow in clostridia remains elusive. In this study, the results revealed that SLCs changed redox balance by producing oxidative stress and modulating electron flow as electron donors. Production of H2 and acetone was stimulated, while butanol production remained unchanged, which led to a high A/B ratio during C. beijerinckii fermentation using corn stover-derived hydrolysate as the carbon source. These observations provide insight into utilizing C. beijerinckii to produce butanol from a lignocellulosic biomass.
| S-EPMC5359496 | biostudies-literature
Project description:Acetone-butanol-ethanol (ABE) fermentation by clostridia has shown promise for industrial-scale production of biobutanol. However, the continuous ABE fermentation suffers from low product yield, titer, and productivity. Systems analysis of the continuous ABE fermentation will offer insights into its metabolic pathway as well as into optimal fermentation design and operation. For the ABE fermentation in continuous Clostridium acetobutylicum culture, this paper presents a kinetic model that includes the effects of key metabolic intermediates and enzymes as well as culture pH, product inhibition, and glucose inhibition. The kinetic model is used for elucidating the behavior of the ABE fermentation under the conditions that are most relevant to continuous cultures. To this end, dynamic sensitivity analysis is performed to systematically investigate the effects of culture conditions, reaction kinetics, and enzymes on the dynamics of the ABE production pathway. The analysis provides guidance for future metabolic engineering and fermentation optimization studies.
| S-EPMC4972440 | biostudies-literature
Project description:In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH?5.7 and pH?4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH?5.3 and pH?5.0 with an intermediate state at pH?5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone-butanol-ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C.?acetobutylicum.
| S-EPMC3918155 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2009-06-02 | GSE12365 | GEO
Project description:Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.
| S-EPMC3294493 | biostudies-literature