Targeted Deletions Uncover Previously Uncharacterised C1-fixing Cluster Genes as Essential for Autotrophy in Clostridium autoethanogenum
Recycling carbon oxides from industrial off-gases and gasified organic waste using gas fermentation offers a sustainable route for biomanufacturing of fuels and chemicals. Acetogens are attractive biocatalysts here and fix CO and CO2 via the Wood-Ljungdahl pathway (WLP). However, the incomplete functional annotation of the associated C1-fixing gene cluster limits rational engineering of acetogen…
Research reveals crucial genes enabling autotrophy in Clostridium autoethanogenum. Utilizing targeted CRISPR/Cas9 gene deletions, scientists analyzed five previously unidentified genes within the C1-fixing cluster, essential for converting carbon oxides into fuels and chemicals via the Wood-Ljungdahl pathway. Deletions of acsV, lpdA, cooC, and cooC2 genes demonstrated their necessity for autotrophic growth, while gcvH deletion affected autotrophy by altering carbon flux.
Amino acid supplementation and computational studies indicated lpdA and gcvH's roles in C1 and redox metabolism, not glycine or serine biosynthesis. Genetic and physiological analyses showed CooC and CooC2, ATPases in the CooC-family, had distinct growth roles, with cooC2 deletion impairing heterotrophy. Even with disabled autotrophy, cells retaining acsV showed metabolic activity, suggesting acsV's role in ATP-dependent corrinoid activation.
CRISPRi-mediated repression of acsV didn't impair autotrophy, only causing altered carbon partitioning. These findings elucidate the physiological significance of C1-fixing cluster genes in acetogens, supporting their potential as gas-fermenting cell factories.
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