Engineering Clostridium ljungdahlii for efficient 3-hydroxybutyrate production from one-carbon gases

3-Hydroxybutyrate (3-HB) has attracted great attention due to its importance as a building block in the production of high-value chemicals and polymers. Autotrophic Clostridium species have the potential to convert one-carbon (C1) gases (CO and CO 2 ) into a range of bulk chemicals and fuels, and thus have been modified to synthesize 3-HB using C1 gases. However, the metabolic engineering of Clostridium strains capable of efficiently producing 3-HB from CO/CO 2 still has huge space for further improvements. Here, we achieved rapid generation of a 3-HB-producing Clostridium ljungdahlii strain library based on the transposon-mediated chromosomal integration of pathway genes. Through the following phenotypic screening, multiple 3-HB high-producing C. ljungdahlii strains were found. The best-performing strain 3HB-02 was further optimized by reinforcing the expression of an endogenous hbd2 gene that encodes a crucial 3-hydroxybutyryl-CoA dehydrogenase, yielding an engineered strain capable of producing 3.0 g/L and 10.3 g/L of 3-HB in batch and continuous gas fermentation, respectively. Therefore, this work has facilitated the ability of gas-fermenting Clostridium strains to produce 3-HB from C1 gases, laying the foundation for the industrial production of this bulk chemical in gas fermentation. Based on metabolic engineering combined with phenotypic screening, we have developed a 3-hydroxybutyrate (3-HB)-producing autotrophic bacterium and realized the efficient synthesis of the bulk chemical 3-HB by using CO and CO 2 as the carbon sources. • Rapid generation of 3-HB-producing Clostridium ljungdahlii chassis via genome engineering • The crucial 3-HB pathway genes were integrated onto the chromosome for stable expression • The 3-HB-producing C. ljungdahlii strain exhibited good genetic stability • The titer of 3-HB reached 10.3 g/L in continuous gas fermentation