# Elucidating the roles of essential genes in autotrophic metabolism and cell morphology of Clostridium ljungdahlii by CRISPRi

## TL;DR

This study uses CRISPRi to explore essential genes in Clostridium ljungdahlii, revealing their roles in autotrophic metabolism and cell shape.

## Contribution

The study introduces a CRISPRi system to investigate essential genes in C. ljungdahlii, revealing their roles in autotrophic growth and cell division.

## Key findings

- Repression of PFOR1, PFOR2, AOR1, AOR2, and GAP-I genes impairs autotrophic growth and ethanol production.
- Downregulation of ftsZ leads to elongated cell morphology, indicating its role in cell shape regulation.

## Abstract

Understanding the function of essential genes in Clostridium ljungdahlii is critical for unraveling its autotrophic metabolism and optimizing its potential as a platform for syngas fermentation. However, study on essential genes of this species remains insufficient. Here, we employed an inducible CRISPR interference (CRISPRi) system to investigate the roles of key metabolic and cell division genes in C. ljungdahlii. Targeted repression of genes encoding pyruvate:ferredoxin oxidoreductase (PFOR1, PFOR2), acetaldehyde:ferredoxin oxidoreductase (AOR1, AOR2), and glyceraldehyde phosphate hydrogenase type I (GAP-I) revealed their essential contributions to autotrophic growth, as knockdown strains exhibited impaired growth and reduced ethanol production. Furthermore, downregulation of the cell division gene ftsZ resulted in elongated cell morphology, highlighting its critical role in cell shape regulation. These findings provide new insights into the functional importance of essential genes in C. ljungdahlii and demonstrate how targeted gene repression can advance our understanding of autotrophic metabolism and cellular processes.

The online version contains supplementary material available at 10.1007/s00253-026-13714-3.