Engineering the Distal Loci of SAM Synthase for High-Yield Synthesis of SAM Using Whole-Cell Catalysis

S-adenosyl methionine (SAM) is a vital metabolic intermediate with wide applications ranging from medicine to agriculture. The high-yield synthesis of SAM in Escherichia coli using whole-cell catalysis offers many advantages, including environmental friendliness. However, the heterologous expression of SAM synthase (SAM2) from Saccharomyces cerevisiae in E. coli mainly suffers from low enzymatic activity. In this study, we propose a novel molecular design strategy targeting distal sites to address these limitations. When combined with expression optimization, this strategy enabled the development of a highly efficient E. coli whole-cell catalytic system. Through distal site engineering, an I189 V/V266H double mutant was designed and obtained, which resulted in a 1353.08% increase in enzymatic activity, a substantial improvement in thermal stability, and a 524.62% enhancement in whole-cell catalytic yield. Molecular dynamics simulations and structural analysis revealed that the distal site mutations synergistically enhanced the enzyme structural stability and optimized substrate binding. Using a green feeding strategy (45 mM ATP), the system achieved a conversion rate of 91.3% within 12 h at an E. coli OD600 of 60, yielding 16.39 g/L of SAM─the highest production reported to date. Ion-exchange resin-based separation and purification yielded a SAM recovery rate of up to 82.5% and a product purity exceeding 95%. This work not only pioneers a distal site-based molecular design for SAM synthetase modification and establishes an integrated whole-cell catalytic synthesis system, but also provides a promising green and sustainable strategy for the high-yield synthesis of SAM and SAM-dependent chemicals.