Enhanced robustness in acetone-butanol-ethanol fermentation with engineered Clostridium beijerinckii overexpressing adhE2 and ctfAB

Lu, Congcong; Yu, Lean; Varghese, Saju; Yu, Mingrui; Yang, Shang‐Tian

doi:10.1016/j.biortech.2017.07.043

Cited by 48 publications

(15 citation statements)

References 45 publications

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“…To identify a candidate enzyme for producing crotonol from crotonyl-CoA involved in the EMC pathway, we tested aldehyde/alcohol dehydrogenase (ADHE2) from C. acetobutylicum and fatty acyl-CoA reductase (FAR) from H. chejuensis and M. manganoxydans. ADHE2 has been used to convert short chain acyl-CoA to alcohols in either engineered M. extorquens AM1 or Clostridium species [16, 44]. FAR is another promiscuous enzyme which has good rates of reduction for longer (C20) and shorter (C8) fatty acyl-CoA groups or longer (C8) and shorter (C2) aldehyde groups to produce various alcohols [27].…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

et al. 2018

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BackgroundButadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis.ResultsIn this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The Km and kcat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s−1, 1.28 mM and 153.14 s−1, and 2.34 mM and 1.15 s−1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase.ConclusionsThese results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1042-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

et al. 2018

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“…Based on the understanding of regulation of alcohol synthesis, we overexpressed SigL in C. beijerinckii, which resulted in significantly improved solvent production. Genetic engineering has been applied to enhance solvent production by C. beijerinckii; however, most of the attempts did not result in a desired solvent producer (Lu et al, 2017;Wen et al, 2017). Although a few transcriptional factors including Spo0A, CcpA, AbrB and Rex have been found to regulate solvent production in C. acetobutylicum (Yang et al, 2018), little is known about the regulatory mechanisms of solvent production in other clostridia.…”

Section: Discussionmentioning

confidence: 99%

Control of solvent production by sigma‐54 factor and the transcriptional activator AdhR in Clostridium beijerinckii

Yang

Nie

et al. 2019

Microbial Biotechnology

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Summary Clostridia are obligate anaerobic bacteria that can produce solvents such as acetone, butanol and ethanol. Alcohol dehydrogenases (ADHs) play a key role in solvent production; however, their regulatory mechanisms remain largely unknown. In this study, we characterized the regulatory mechanisms of two ADH‐encoding genes in C. beijerinckii. SigL (sigma factor σ54) was found to be required for transcription of adhA1 and adhA2 genes. Moreover, a novel transcriptional activator AdhR was identified, which binds to the σ54 promoter and activates σ54‐dependent transcription of adhA1 and adhA2. Clostridia beijerinckii mutants deficient in SigL or AdhR showed severely impaired butanol and ethanol production as well as altered acetone and butyrate synthesis. Overexpression of SigL resulted in significantly improved solvent production by C. beijerinckii when butyrate was added to cultures. Our results reveal SigL as a novel engineering target for improving solvent production by C. beijerinckii and other solvent‐producing clostridia. Moreover, this study gains an insight into regulation of alcohol metabolism in diverse clostridia.

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“…Lowing yeast extract concentrations (0.05 g/L) in the medium resulted in higher ABE production of 134 mM, low sugar uptake and acid product rates [8]. Overexpressing aldehyde/alcohol dehydrogenase and CoA-transferase in Clostridium beijerinckii was able to prevent "acid crash" and increase butanol production [40]. Syngas fermentation with Clostridium carboxidivorans at a low temperature has been reported to enhance butanol production by lowering metabolic rates at 25 °C [11].…”

Section: Effect Of Ethanol Washing On Abe Production In Shf Processesmentioning

confidence: 99%

Effect of Residual Extractable Lignin on Acetone-Butanol-Ethanol Production in SHF and SSF Processes

Shi

2020

Preprint

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Background Lignin played an important role in biochemical conversion of biomass to biofuels. A significant amount of lignin is precipitated on the surface of pretreated substrates after organosolv pretreatment. The effect of this residual lignin on enzymatic hydrolysis has been well understood, however, their effect on subsequent ABE fermentation is still unknown. Results To determine the effect of residual extractable lignin on Acetone-Butanol-Ethanol (ABE) fermentation in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes, we compared ABE production from ethanol washed and unwashed substrates. The ethanol organosolv pretreated loblolly pine (OPLP) was used as the substrate. It was observed that butanol production from OPLP-UW (unwashed) and OPLP-W(washed) reached 8.16 and 1.69 g/L, respectively in SHF. The results showed that ABE production in SHF from OPLP-UW prevents an “acid crash” as comparing the OPLP-W. In SSF process, the “acid crash” occurred for both OPLP-W and OPLP-UW. The inhibitory extractable lignin intensified the “acid crash” for OPLP-UW and resulted in less ABE production than OPLP-W. The addition of detoxified prehydrolysates in SSF processes shortened the fermentation time and could potentially prevent the “acid crash”. Conclusions The results suggested that the residual extractable lignin in high sugar concentration could help ABE production by lowering the metabolic rate and preventing “acid crash” in SHF processes. However, it became unfavorable in SSF due to its inhibition of both enzymatic hydrolysis and ABE fermentation with low initial sugar concentration. It is essential to remove extractable lignin of substrates for ABE production in SSF processes. Also, a higher initial sugar concentration is needed to prevent the “acid crash” in SSF processes.

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Enhanced robustness in acetone-butanol-ethanol fermentation with engineered Clostridium beijerinckii overexpressing adhE2 and ctfAB

Cited by 48 publications

References 45 publications

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

Control of solvent production by sigma‐54 factor and the transcriptional activator AdhR in Clostridium beijerinckii

Effect of Residual Extractable Lignin on Acetone-Butanol-Ethanol Production in SHF and SSF Processes

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