Deletion of genes linked to the C₁-fixing gene cluster affects growth, by-products, and proteome ofClostridium autoethanogenum

Abstract

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in theC1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions - hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) - directly adjacent and expressed at similar levels to theC1-fixing gene cluster in the gas-fermenting model-acetogenClostridium autoethanogenum. Targeted deletion of either thehpornbpgene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both∆hpand∆nbpstrains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of theC1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

Autotrophic adaptive laboratory evolution of the acetogenClostridium autoethanogenumdelivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

“…Glycerol stock of DSM 10061 was revived in PETC-MES medium (28) supplemented with 5 g/L of fructose, 1.5 g/L of YE, and 0.4 g/L of cysteine-HCl·H 2 O as the reducing agent. Cells were sub-cultured from exponential growth phase into the same medium but with fructose replaced by CO as the carbon source: this culture was Round 1 of UT ALE (Fig.…”

“…The detailed steps of our CRISPR/Cas9-aided homologous recombination workflow for genetic engineering include designing and selecting sgRNAs, plasmid construction and electroporation into C. autoethanogenum JA1-1, screening and plasmid curing, and confirmation of transformants were described before (28). Briefly, the homology arms (HAs) for the CLAU_3129 ( spo0A ) deletion plasmid were obtained by PCR amplifying the 1 kb upstream and downstream regions of the spo0A gene.…”

“…Sampling, sample preparation, LC-MS/MS analysis using data-independent acquisition (DIA), and DIA MS data analysis were performed as described before (28). Briefly, cell pellets were treated with a chaotrope-based lysis buffer and bead beating, lysates alkylated and processed, and proteins digested.…”

“…Autotrophic cultures for sporulation tests were the same as described for proteome analysis while heterotrophic cultures were grown in 10 mL of YTF (28) in 50ml serum bottles and incubated horizontally at 37°C with orbital shaking at 120 RPM under strictly anaerobic conditions. Samples from the stationary phase during ~40 and ~20 days of autotrophic and heterotrophic incubation, respectively, were observed on glass slips covered with an agarose pad using phase-contrast microscopy (Zeiss Observer Z1) at ×1000 magnification.…”

“…We used CRISPR/Cas9-aided homologous recombination (28) for reverse genetic engineering of JA1-1. Mutations #4 and #5 occurred across LT and UQ ALE (Fig.…”