Targeted Deletions Uncover Previously Uncharacterised C1-fixing Cluster Genes as Essential for Autotrophy in Clostridium autoethanogenum

Targeted single-gene deletions in Clostridium autoethanogenum show acsV, lpdA, cooC, and cooC2 are essential for autotrophic growth but not heterotrophy.

• Why it matters: In acetogens, incomplete annotation of the C1-fixing gene cluster limits rational engineering for gas fermentation using the Wood-Ljungdahl pathway.
• What they did: Using targeted CRISPR/Cas9 deletions, CRISPRi, physiological assays, and comparative computational structural analyses, the study quantified growth and carbon flux after deleting five conserved, previously uncharacterised C1-fixing cluster genes.
• The result: Deleting gcvH reduced autotrophic growth and shifted carbon flux from acetate toward 2,3-butanediol, while acetate tests showed {Delta}acsV retained metabolic activity despite abolished autotrophy and CRISPRi repression of acsV altered carbon partitioning without impairing autotrophy.

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