Identification of a primary-secondary alcohol dehydrogenase loss-of-function mutant induced by random mutagenesis in Clostridium ljungdahlii for syngas-based acetone production

Acetogenic bacteria represent a promising platform for the production of valuable chemical compounds from a variety of alternative, low value feedstock. Clostridium ljungdahlii utilizes syngas for the formation of native fermentation products such as ethanol, acetate and 2,3-butandiol as well as non-native biomolecules including acetone via expression of heterologous acetone biosynthesis gene clusters. A primary-secondary alcohol dehydrogenase in C. ljungdahlii reduces acetone to isopropanol thereby decreasing the acetone yield and raising separation issues in downstream processes.

Here we present the N-methyl-N’-nitro-nitrosoguanidine-mediated generation of C. ljungdahlii strain 8.1.23 with almost completely eliminated acetone reducing activity. A protocol for the mutagenesis on C. ljungdahlii was established. Whole genome sequencing revealed a mutation frequency of approximately 37 point mutations per 1Mbp. A glycine to aspartate exchange was identified as molecular basis for the desired primary-secondary alcohol dehydrogenase inactivation. Recombinant expression of wildtype and mutant enzyme in E. coli confirmed its reducing activity towards acetone as well as its functional inactivation, respectively. Transformation of C. ljungdahlii wildtype with an acetone biosynthesis gene cluster resulted in predominant formation of isopropanol. Instead, the main product in acetone gene cluster expressing C. ljungdahlii strain 8.1.23 was almost exclusively acetone.

A combined approach of random mutagenesis and high throughput screening resulted in C. ljungdahlii strain 8.1.23. This novel C. ljungdahlii strain represents a potential platform for syngas-based acetone production. Adaption of the efficient screening protocol may also apply to the rapid development of additional C. ljungdahlii strains with superior product tolerance, growth characteristics or product formation rates.