Dataset Information

Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting Clostridium ljungdahlii.

ABSTRACT: Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO₂/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, we identified a RpiR family transcription factor (TF) that can regulate the autotrophic growth and carbon fixation of C. ljungdahlii. After deletion of the corresponding gene, the resulting mutant strain exhibited significantly impaired growth in gas fermentation, thus reducing the production of acetic acid and ethanol. In contrast, the overexpression of this TF gene could promote cell growth, indicating a positive regulatory effect of this TF in C. ljungdahlii. Thus, we named the TF as GssR (growth and solvent synthesis regulator). Through the following comparative transcriptomic analysis and biochemical verification, we discovered three important genes (encoding pyruvate carboxylase, carbon hunger protein CstA, and a BlaI family transcription factor) that were directly regulated by GssR. Furthermore, an upstream regulator, BirA, that could directly bind to gssR was found; thus, these two regulators may form a cascade regulation and jointly affect the physiology and metabolism of C. ljungdahlii. These findings substantively expand our understanding on the metabolic regulation of carbon fixation in gas-fermenting Clostridium species.

SUBMITTER: Zhang H

PROVIDER: S-EPMC10458427 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting Clostridium ljungdahlii.

Zhang Huan H Zhang Can C Nie Xiaoqun X Wu Yuwei Y Yang Chen C Jiang Weihong W Gu Yang Y

Microorganisms 20230731 8

Clostridium ljungdahlii is a representative autotrophic acetogen capable of producing multiple chemicals from one-carbon gases (CO2/CO). The metabolic and regulatory networks of this carbon-fixing bacterium are interesting, but still remain minimally explored. Here, based on bioinformatics analysis followed by functional screening, we identified a RpiR family transcription factor (TF) that can regulate the autotrophic growth and carbon fixation of C. ljungdahlii. After d ...[more]

PMID: 37630531

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Project description:UnlabelledIt has been predicted that the Rnf complex of Clostridium ljungdahlii is a proton-translocating ferredoxin:NAD(+) oxidoreductase which contributes to ATP synthesis by an H(+)-translocating ATPase under both autotrophic and heterotrophic growth conditions. The recent development of methods for genetic manipulation of C. ljungdahlii made it possible to evaluate the possible role of the Rnf complex in energy conservation. Disruption of the C. ljungdahlii rnf operon inhibited autotrophic growth. ATP synthesis, proton gradient, membrane potential, and proton motive force collapsed in the Rnf-deficient mutant with H(2) as the electron source and CO(2) as the electron acceptor. Heterotrophic growth was hindered in the absence of a functional Rnf complex, as ATP synthesis, proton gradient, and proton motive force were significantly reduced with fructose as the electron donor. Growth of the Rnf-deficient mutant was also inhibited when no source of fixed nitrogen was provided. These results demonstrate that the Rnf complex of C. ljungdahlii is responsible for translocation of protons across the membrane to elicit energy conservation during acetogenesis and is a multifunctional device also implicated in nitrogen fixation.ImportanceMechanisms for energy conservation in the acetogen Clostridium ljungdahlii are of interest because of its potential value as a chassis for the production of biocommodities with novel electron donors such as carbon monoxide, syngas, and electrons derived from electrodes. Characterizing the components implicated in the chemiosmotic ATP synthesis during acetogenesis by C. ljungdahlii is a prerequisite for the development of highly productive strains. The Rnf complex has been considered the prime candidate to be the pump responsible for the formation of an ion gradient coupled with ATP synthesis in multiple acetogens. However, experimental evidence for a proton-pumping Rnf complex has been lacking. This study establishes the C. ljungdahlii Rnf complex as a proton-translocating ferredoxin:NAD(+) oxidoreductase and demonstrates that C. ljungdahlii has the potential of becoming a model organism to study proton translocation, electron transport, and other functions of the Rnf complex in energy conservation or other processes.

Project description:BackgroundClostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.ResultsWe have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.ConclusionsThe developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Project description:Background: The global demand for affordable carbon has never been stronger, and there is an imperative in many industrial processes to use waste streams to make products. Gas-fermenting acetogens offer a potential solution and several commercial gas fermentation plants are currently under construction. As energy limits acetogen metabolism, supply of H2 should diminish substrate loss to CO2 and facilitate production of reduced and energy-intensive products. However, the effects of H2 supply on CO-grown acetogens have yet to be experimentally quantified under controlled growth conditions. Results: Here, we quantify the effects of H2 supplementation by comparing growth on CO, syngas, and a high-H2 CO gas mix using chemostat cultures of Clostridium autoethanogenum. Cultures were characterised at the molecular level using metabolomics, proteomics, gas analysis, and a genome-scale metabolic model (GEM). CO-limited chemostats operated at two steady-state biomass concentrations facilitated co-utilisation of CO and H2. We show that H2 supply strongly impacts carbon distribution with a four-fold reduction in substrate loss as CO2 (61% vs. 17%) and a proportional increase of flux to ethanol (15% vs. 61%). Notably, H2 supplementation lowers the molar acetate/ethanol ratio by five-fold. At the molecular level, quantitative proteome analysis showed no obvious changes leading to these metabolic rearrangements suggesting the involvement of post-translational regulation. Metabolic modelling showed that H2 availability provided reducing power via H2 oxidation and saved redox as cells reduced all the CO2 to formate directly using H2 in the Wood-Ljungdahl pathway. Modelling further indicated that the methylene-THF reductase reaction was ferredoxin-reducing under all conditions. In combination with proteomics, modelling also showed that ethanol was synthesised through the acetaldehyde:ferredoxin oxidoreductase (AOR) activity. Conclusions: Our quantitative molecular analysis revealed that H2 drives rearrangements at several layers of metabolism and provides novel links between carbon, energy, and redox metabolism advancing our understanding of energy conservation in acetogens. We conclude that H2 supply can substantially increase the efficiency of gas fermentation and thus the feed gas composition can be considered an important factor in developing gas fermentation-based bioprocesses.

Dataset Information

Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting Clostridium ljungdahlii.

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Pleiotropic Regulator GssR Positively Regulates Autotrophic Growth of Gas-Fermenting <i>Clostridium ljungdahlii</i>.

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