Reshaping Phosphatase Substrate Preference for Controlled Biosynthesis Using a "Design-Build-Test-Learn" Framework.

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time-consuming trial-and-error process. This study presents a quantum mechanics (QM)-incorporated design-build-test-learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N-acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field-based methods, resulting in a 1.4-fold increase in substrate preference (k/K) toward GlcNAc-6-phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol reduction in activation energy. Furthermore, an iterative computer-aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5-fold increase in k/K and a 59% decrease in k/K is highly desirable compared to the wild type in the GlcNAc-producing chassis. The GlcNAc titer increases to 217.3 g L with a yield of 0.597 g (g glucose) in a 50-L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts.