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

Area of Science:

• Microbiology
• Biochemistry
• Synthetic Biology

Background:

• Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, is essential for numerous protein functions.
• Two de novo PLP biosynthesis pathways exist: the DXP-independent pathway in Bacillus subtilis and the DXP-dependent pathway in Escherichia coli.
• Bacteria possess promiscuous enzymes ('underground metabolism') that can contribute to the evolution of new metabolic pathways.

Purpose of the Study:

• To investigate the feasibility of using a truncated, non-native DXP-dependent PLP pathway from E. coli in B. subtilis.
• To determine if B. subtilis can adapt to synthesize PLP using components of the E. coli pathway.

Main Methods:

• Employing adaptive laboratory evolution (ALE) to drive adaptation in B. subtilis.
• Introducing two non-native enzymes from the E. coli DXP-dependent PLP pathway into B. subtilis.
• Analyzing genomic alterations that occurred during ALE.

Main Results:

• ALE enabled vitamin B6 auxotrophic B. subtilis to grow as rapidly as the wild type.
• The successful growth was attributed to two introduced non-native enzymes and two genomic modifications.
• This demonstrates the functional integration of a partial non-native pathway into the host's metabolism.

Conclusions:

• The 'underground metabolism' in B. subtilis supports the creation of a novel PLP synthesis pathway using parts of a non-native system.
• Introducing heterologous enzymes and metabolic rewiring can engineer new pathways for producing valuable compounds.
• This approach holds potential for optimizing the production of high-value substances in microbial hosts.