Exploring auxotrophy and engineering vitamin B₆ prototrophy in the acetogen Clostridium sp. AWRP

Gas fermentation using acetogenic bacteria requires a chemically defined minimal medium to be established. This approach not only helps in creating a cost-effective medium but also allows for a thorough exploration of their metabolic potential. In this study, the auxotrophy of the acetogen Clostridium sp. AWRP was investigated through genomic analysis and growth performance in formulated media. It was found that the strain needs pantothenate and biotin and that substituting vitamin B₆ from pyridoxine to pyridoxamine or pyridoxal-5'-phosphate is crucial for growth. The determined chemically defined minimal medium supported both heterotrophic (using fructose as a substrate) and autotrophic (using syngas as a substrate) growth of the AWRP strain. To overcome the vitamin B₆ auxotrophy, the pdxST genes responsible for vitamin B₆ biosynthesis were introduced into the AWRP strain using plasmid-based gene expression system and CRISPR/Cas12a genome-editing technology. As a result, the genetically engineered strains were able to grow successfully without vitamin B₆. This chemically defined minimal medium will enhance the fermentation performance of AWRP.

Importance: The identification of auxotrophy in Clostridium sp. AWRP underpins subsequent investigations into its physiology and metabolism. Additionally, the development of a chemically defined minimal medium specific to this acetogenic bacterium will enable reproducible industrial processes. This innovation is particularly significant for the bioconversion of carbon monoxide and/or dioxide into commercially valuable chemicals through the process of gas fermentation.