Metabolic engineering of Methylorubrum extorquens PA1 for cinnamic acid production from ethanol

Developing a microbial platform capable of converting ethanol into value-added products is vital for next-generation carbon recycling biorefineries. Methylorubrum extorquens PA1 is a facultative methylotroph that assimilates ethanol; however, its metabolic potential for aromatic synthesis from ethanol has not been explored. Here, we report the first demonstration of de novo cinnamic acid (CA) production from ethanol in a methylotrophic bacterium. Metabolite profiling revealed that ethanol assimilation was limited by inefficient conversion of acetate to acetyl-CoA. Overexpression of endogenous acetyl-CoA synthase (MeACS1) suppressed extracellular acetate accumulation and improved growth on ethanol. To establish the CA biosynthetic module, we introduced Streptomyces maritimus phenylalanine ammonia-lyase (SmEncP), enabling conversion of L-phenylalanine into CA. To enhance precursor supply, we strengthened the shikimate pathway and phenylalanine biosynthetic pathway by co-expressing feedback-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EcAroG fbr ) and chorismate mutase/prephenate dehydrogenase (EcPheA fbr ) from Escherichia coli . Further, endogenous transketolase (MeTkt) was overexpressed to enhance erythrose-4-phosphate supply. The resulting strain PAc1/pTE102-CA3 produced 1.75 mg L⁻ 1 of CA from ethanol in test tube culture, 2.6-fold that obtained from methanol. Fed-batch cultivation with ethanol feeding achieved 52.34 mg L⁻ 1 of CA, demonstrating stable conversion of ethanol to value-added chemicals without notable growth inhibition or acetate accumulation. This study establishes M. extorquens PA1 as a promising chassis for ethanol-based aromatic biosynthesis and suggests that relieving acetyl-CoA bottlenecks is likely critical for efficient ethanol assimilation. This work provides ethanol-driven aromatic biosynthesis in methylotrophs and offers a foundation for further engineering of C₂-assimilating bacteria toward renewable chemical production.