Rebalancing Redox to Improve Biobutanol Production by Clostridium tyrobutyricum

Ou, Jianfa; Xu, Ningning; Fierst, Janna L.; Yang, Shang‐Tian; Liu, Margaret

doi:10.3390/bioengineering3010002

Cited by 11 publications

(13 citation statements)

References 34 publications

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“…To test this, we altered the cellular redox balance by adding formate to cultures or exposing cells to O 2 and studied their effects on transcription of adhA1 and adhA2 genes in C. beijerinckii. Supplementation of cultures with formate has been shown to provide a surplus of electrons for cells (23,24), whereas limited exposure to O 2 causes oxidative stress to clostridial cells (10,25). We observed that the transcript levels of adhA1 and adhA2 in the wild-type C. beijerinckii were significantly upregulated by formate supplementation and downregulated by limited exposure to O 2 ( Fig.…”

Section: Resultsmentioning

confidence: 66%

Ferrous-Iron-Activated Transcriptional Factor AdhR Regulates Redox Homeostasis in Clostridium beijerinckii

Yang

Nie

Xiao

et al. 2020

Appl Environ Microbiol

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The AdhR regulatory protein is an activator of σ54-dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii. Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA+ domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ54 showed large shifts in intracellular redox state indicated by the NADH/NAD+ ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe2+-activated transcriptional regulator AdhR and σ54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckii. IMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a σ54-dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii. AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of σ54 in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia.

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Section: Resultsmentioning

confidence: 66%

Ferrous-Iron-Activated Transcriptional Factor AdhR Regulates Redox Homeostasis in Clostridium beijerinckii

Yang

Nie

Xiao

et al. 2020

Appl Environ Microbiol

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“…As ethanol and n-butanol biosynthesis pathways are NAD(P)Hdependent, redox engineering has been applied to alter the electron flow to favor alcohols, instead of hydrogen, production in solventogenic clostridia (Cui et al, 2019;Lo et al, 2017;Ma et al, 2015b). Especially, ferredoxin NAD(P) + reductase has been overexpressed in C. acetobutylicum for increased butanol production (Bennett & Thakker, 2017;Qi et al, 2018).…”

Section: Effects Of Overexpressing Fnr On Fermentationmentioning

confidence: 99%

Engineering Clostridium cellulovorans for highly selective n‐butanol production from cellulose in consolidated bioprocessing

Teng

Hou

et al. 2021

Biotech & Bioengineering

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Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA CA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P) + oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol.The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr CA -thlA CAhbd CT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

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“…Therefore, various cofactor engineering strategies have been developed to regulate the redox balance and improve the yield and efficiency of microbial cell factories [108]. To maintain redox balance in the 1-butanol synthetic pathway, four strategies were employed, i.e., regulation of endogenous cofactor systems [37], heterologous cofactor regeneration systems [48], modifying cofactor preferences [60,109], and synthetic cofactor [32,49,110]. For example, endogenous cofactor regeneration strategy and acetyl-CoA were coupled as driving forces to direct the carbon flux in E. coli, through which 1-butanol production was improved to a level comparable to that produced by the Clostridium species, with a 1-butanol titer of 30 g/L and increasing the yield to 70% of the theoretical level [37].…”

Section: Cofactor Imbalancementioning

confidence: 99%

Genetic engineering of non-native hosts for 1-butanol production and its challenges: a review

Nawab

Wang

et al. 2020

Microb Cell Fact

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Background: Owing to the increase in energy consumption, fossil fuel resources are gradually depleting which has led to the growing environmental concerns; therefore, scientists are being urged to produce sustainable and ecofriendly fuels. Thus, there is a growing interest in the generation of biofuels from renewable energy resources using microbial fermentation. Main text: Butanol is a promising biofuel that can substitute for gasoline; unfortunately, natural microorganisms pose challenges for the economical production of 1-butanol at an industrial scale. The availability of genetic and molecular tools to engineer existing native pathways or create synthetic pathways have made non-native hosts a good choice for the production of 1-butanol from renewable resources. Non-native hosts have several distinct advantages, including using of cost-efficient feedstock, solvent tolerant and reduction of contamination risk. Therefore, engineering non-native hosts to produce biofuels is a promising approach towards achieving sustainability. This paper reviews the currently employed strategies and synthetic biology approaches used to produce 1-butanol in non-native hosts over the past few years. In addition, current challenges faced in using non-native hosts and the possible solutions that can help improve 1-butanol production are also discussed. Conclusion: Non-native organisms have the potential to realize commercial production of 1-butanol from renewable resources. Future research should focus on substrate utilization, cofactor imbalance, and promoter selection to boost 1-butanol production in non-native hosts. Moreover, the application of robust genetic engineering approaches is required for metabolic engineering of microorganisms to make them industrially feasible for 1-butanol production.

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Rebalancing Redox to Improve Biobutanol Production by Clostridium tyrobutyricum

Cited by 11 publications

References 34 publications

Ferrous-Iron-Activated Transcriptional Factor AdhR Regulates Redox Homeostasis in Clostridium beijerinckii

Ferrous-Iron-Activated Transcriptional Factor AdhR Regulates Redox Homeostasis in Clostridium beijerinckii

Engineering Clostridium cellulovorans for highly selective n‐butanol production from cellulose in consolidated bioprocessing

Genetic engineering of non-native hosts for 1-butanol production and its challenges: a review

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