A two-step evolutionary process establishes a non-native vitamin B6 pathway in Bacillus subtilis.

Pyridoxal 5'-phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy-xylulose-5-phosphate (DXP)-independent pathway, which is present in the Gram-positive model bacterium Bacillus subtilis and the longer DXP-dependent pathway, which has been intensively studied in the Gram-negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so-called 'underground metabolism', which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non-native DXP-dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non-native enzymes catalysing the last steps of the DXP-dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non-native vitamin B6 pathway. The introduction of non-native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances.