Mining and engineering of ene-reductases from marine sediment metagenome for prochiral ACE inhibitor synthesis

The development of sustainable biocatalytic processes for pharmaceutical synthesis represents a major goal in green chemistry. Ene-reductases (ERs) are attractive biocatalysts for asymmetric hydrogenation of activated alkenes, yet their industrial application is often constrained by limited substrate scope and stability. In this study, we explored the deep-sea sediment metagenome of the South China Sea and identified 41 putative ER genes, with 22 successfully solubly expressed in <i>Escherichia coli</i>. Biochemical characterization revealed broad substrate specificity, achieving up to 90% conversion for diverse α,β-unsaturated compounds. Notably, three enzymes (S2gene2614772, S2gene1139, and S2gene22028) exhibited exceptional adaptability, maintaining high activity over a wide pH range (5.5-8.5) and at low temperatures (15°C). However, none of the wild-type ERs showed significant activity toward the prochiral substrate 2-oxo-4-phenyl-3-butenoic acid, a key intermediate for angiotensin-converting enzyme inhibitors (ACEIs). Through directed evolution, we obtained a mutant (S2gene22028-G102S) with 30-fold enhanced activity, reaching 90% conversion at 10 mM substrate. Scale-up synthesis (5 mmol substrate) afforded 2-oxo-4-phenylbutyric acid (OPBA) at 11 mg/mL, demonstrating industrial potential. This study highlights marine metagenomes as valuable sources of novel ERs and provides an efficient biocatalytic route to ACEI precursors.IMPORTANCEThe development of sustainable biocatalysts for pharmaceutical synthesis is a pivotal goal in green chemistry. This study leverages the untapped enzymatic diversity of the South China Sea deep-sea sediment metagenome to discover novel ene-reductases (ERs). We not only identified robust ERs with broad substrate promiscuity and exceptional adaptability to low temperature and pH fluctuations but also successfully engineered a variant to overcome the key biocatalytic challenge in the synthesis of 2-oxo-4-phenylbutyric acid (OPBA), a critical precursor to angiotensin-converting enzyme inhibitors. Our work underscores marine metagenomes as a valuable reservoir for discovering industrially relevant biocatalysts and demonstrates the power of combining metagenomic mining with protein engineering to enable greener manufacturing routes for high-value pharmaceuticals.