Microfluidic-Assisted Evolution of a pyridoxal 5'-phosphate (PLP) synthase to optimize Vitamin B6 production

• Grant number: 25/21832-1
• Support Opportunities: Scholarships abroad - Research Internship - Master's degree
• Start date: January 31, 2026
• End date: July 30, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Leticia Maria Zanphorlin
• Grantee: Vitor Akio Oya
• Supervisor: Volker Sieber
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Campinas , SP, Brazil
• Institution abroad: Technical University of Munich, Straubing (TUM), Germany
• Associated to the scholarship: 24/08161-8 - Biocatalytic systems for the production of vitamin B6 from industrially relevant lignocellulosic waste, BP.MS

Abstract

Vitamin B6 is an essential coenzyme involved in more physiological processes than any other vitamin. In a previous project, we developed a cell-free enzymatic cascade capable of synthesizing pyridoxal-5-phosphate (PLP), the active form of vitamin B6, from xylose. To enhance PLP production, a Sequence Similarity Network (SSN) was applied to identify enzyme homologs with higher thermal stability and activity than the original candidates. After successive rounds of optimization, the cascade achieved a production of 0.91 mM PLP from lignocellulosic hydrolysate within 24 hours. Despite these advances, the final enzyme of the cascade, PLP synthase (BcPdxS), remains a bottleneck due to its relative low activity and thermal stability-limitations commonly observed among PLP synthases. Furthermore, the cascade efficiency is hindered by the thermodynamic landscape of the system, leading to the accumulation of glyceraldehyde 3-phosphate. This problem could be partially solved if the last enzyme is fast enough to prevent the accumulation of the intermediates. Therefore, this project proposes the Microfluidic-Assisted Directed Evolution of the BcPdxS, aiming at the enhancement of the catalytic activity and improve even more the biocatalytic system to produce vitamin B6 out of xylose. The specific objectives of this project are: (I) synthesis and experimental standardization of BcPdxS; (II) generation of mutant libraries and high-throughput screening; and (III) biochemical characterization of improved variants. This research exchange will not only provide a new approach for directed evolution in Brazil but also strengthen the national expertise in cell-free biocatalysis and enzymology, fostering innovations that contribute to a circular economy and sustainable development.