Directed evolution of an integral membrane monooxygenase unlocks its full potential

Area of Science:

• Biocatalysis and Enzyme Engineering
• Sustainable Chemistry
• Metabolic Engineering

Background:

• Traditional chemical synthesis of 2,6-(bis)hydroxymethylpyridine from lutidine is inefficient and uses toxic reagents.
• Existing biocatalytic routes using xylene monooxygenase (XylMA) require a multi-step process prone to side reactions and intermediate accumulation.

Purpose of the Study:

• To engineer XylMA for direct dihydroxylation of lutidine, enhancing atom efficiency and sustainability.
• To overcome limitations of existing biocatalytic methods for API intermediate production.

Main Methods:

• Screened 50,000 XylMA mutants using high-throughput mass spectrometry to identify key residues for substrate specificity.
• Employed directed evolution to substitute the M116 residue, creating the M116G XylMA mutant.
• Scaled up the biocatalytic reaction in recombinant E. coli.

Main Results:

• Identified M116 as crucial for XylM substrate specificity; M116G substitution enabled direct dihydroxylation of lutidine.
• The engineered M116G XylMA mutant facilitated a two-step reaction, reducing metabolic burden and side products.
• Achieved product concentrations of 17 g L⁻¹ at a space-time yield of 1.45 g L⁻¹h⁻¹ in a 450 L scale-up.

Conclusions:

• Enzyme engineering of XylMA significantly enhances process efficiency for API intermediate production.
• The M116G XylMA mutant demonstrates the potential of recombinant transmembrane hydroxylases for sustainable industrial biocatalysis.
• This approach offers a highly atom-efficient and environmentally friendly alternative to chemical synthesis.