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Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism.

ABSTRACT: BACKGROUND:Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H2 production, including the hydrogenase maturase hydG and NiFe hydrogenase ech, were deleted from the chromosome of C. thermocellum. While ethanol yield increased, the growth rate of ?hydG decreased substantially compared to wild type. RESULTS:Addition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ?hydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ?hydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated in C. thermocellum ?hydG in the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ?hydG. Clostridium thermocellum ?hydG?ech had a higher growth rate than ?hydG in the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate. CONCLUSIONS:Sulfur metabolism is perturbed in C. thermocellum ?hydG strains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.

SUBMITTER: Biswas R

PROVIDER: S-EPMC5209896 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Improved growth rate in <i>Clostridium thermocellum</i> hydrogenase mutant via perturbed sulfur metabolism.

Biswas Ranjita R Wilson Charlotte M CM Giannone Richard J RJ Klingeman Dawn M DM Rydzak Thomas T Shah Manesh B MB Hettich Robert L RL Brown Steven D SD Guss Adam M AM

Biotechnology for biofuels 20170103

<h4>Background</h4>Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium <i>Clostridium thermocellum</i> is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H<sub>2</sub> production, including the hydrogenase maturase <i>hydG</i> and NiFe hydrogenase < ...[more]

PMID: 28053665

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Project description:Clostridium thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly consume cellulose and its existing ethanol production pathway. C. thermocellum converts cellulose and cellobiose to lactate, formate, acetate, H2, ethanol, amino acids, and other products. Elimination of the pathways leading to products such as H2 could redirect carbon flux towards ethanol production. Rather than delete each hydrogenase individually, we targeted a hydrogenase maturase gene (hydG), which is involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes by assembling the active site. This functionally inactivated all three Fe-Fe hydrogenases simultaneously, as they were unable to make active enzymes. In the ∆hydG mutant, the [NiFe] hydrogenase-encoding ech was also deleted to obtain a mutant that functionally lacks all hydrogenase. The ethanol yield increased nearly 2-fold in ∆hydG∆ech compared to wild type, and H2 production was below the detection limit. Interestingly, ∆hydG and ∆hydG∆ech exhibited improved growth in the presence of acetate in the medium. Transcriptomic and proteomic analysis reveal that genes related to sulfate transport and metabolism were up-regulated in the presence of added acetate in ∆hydG, resulting in altered sulfur metabolism. Further genomic analysis of this strain revealed a mutation in the bi-functional alcohol/aldehyde dehydrogenase adhE gene, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities, whereas the wild type strain can only utilize NADH. This is the exact same adhE mutation found in ethanol-tolerant C. thermocellum strain E50C, but ∆hydG∆ech is not more ethanol tolerant than the wild type. Targeting protein post-translational modification is a promising new approach to target multiple enzymes simultaneously for metabolic engineering. A seventeen array study using total RNA recovered from fermentation cultures of three strains (parental, ∆hydG and ∆hydG∆ech) of Clostridium thermocellum DSM1313. Cells were harvested at an OD 0.4-0.5 from cultures grown in the presence of additional 5mM acetate and compared to untreated controls. At least two biological replicates were performed for each treatment and control condition.

Dataset Information

Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism.

Publications

Improved growth rate in <i>Clostridium thermocellum</i> hydrogenase mutant via perturbed sulfur metabolism.

Similar Datasets

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