Enhancing vitamin B5 biosynthesis by multimodule optimization and protein engineering

Abstract

Vitamin B₅ is a crucial water-soluble vitamin widely used in pharmaceuticals, food and animal feed. Traditionally, chemical enzymatic methods, which are not environmentally benign, predominate in the industrial production of VB₅. Herein, we present a metabolically engineered Escherichia coli platform for sustainable VB₅ production. The key strategies included (1) reinforcing the R-pantoate biosynthetic pathway, (2) redirecting carbon flux from the TCA cycle, and (3) constructing a one-carbon module to enhance precursor supply. Rational design of the acetohydroxyacid isomeroreductase (AHAIR), a critical metabolic node, improved its specificity for acetyl-lactate, thereby increasing the R-pantoate flux. The optimized strain DPAC4 achieved 148.31 g L⁻¹ VB₅ in a 5 L bioreactor over 96 hours with β-alanine supplementation, yielding 0.43 g g⁻¹ glucose and a productivity of 1.54 g L⁻¹ h⁻¹. To eliminate dependence on the β-alanine supplement, a fully biosynthetic route (FBRV) was developed in strain DPAS3, enabling 65.12 g L⁻¹ VB₅ production in 60 hours (0.93 g L⁻¹ h⁻¹) without supplementation. This work demonstrates the synergy of the modular pathway and enzyme engineering for precise metabolic control, advancing the industrial feasibility of VB₅ biomanufacturing. Our approach provides a blueprint for sustainable chemical synthesis through tailored microbial chassis design.