Enzymatic cascade transformation of renewable C1 and C2 alcohols into 3-hydroxypropionaldehyde with a high conversion rate

Abstract

C1 and C2 chemicals play a significant role in various industrial, environmental, and scientific research applications due to their versatility, renewable potential, and importance as building blocks in organic synthesis. Consequently, advancements in the discovery of novel biocatalysts and the development of new bioconversion pathways for C1 and C2 chemicals have garnered significant attention in the manufacturing industry. Methanol and ethanol, inexpensive alcohols derived from greenhouse gases (GHGs) and renewable feedstocks, were enzymatically condensed into 3-hydroxypropionaldehyde (3-HPA) using alcohol oxidase and carboligase. We discovered a new soluble alcohol oxidase from Hypoxylon sp. (HpAOx) that was successfully expressed in Escherichia coli, alongside a novel 2-deoxyribose-5-phosphate aldolase from Paenibacillus sp. (PaDERA) serving as the carboligase. Purified HpAOx catalyzed the conversion of methanol and ethanol to formaldehyde and acetaldehyde, respectively, which were subsequently condensed by PaDERA into 3-HPA. To enhance the biocatalytic performance, HpAOx was modified via structure-based enzyme engineering. The variant of HpAOx (HpAOxR339A) showed a higher activity than that of HpAOx wild-type (HpAOxWT) for the bioconversion of alcohols. A one-pot enzymatic cascade reaction, involving HpAOxR339A and PaDERA, facilitated the efficient synthesis of 3-HPA with high bioconversion from methanol and ethanol. Furthermore, a hydrogen peroxide (H2O2) elimination system based on a catalase (CAT) was integrated into the system, leading to a 2-fold higher production of 3-HPA. Ultimately, 3-HPA was produced at a concentration of 18.3 mM from alcohols (25 mM) using HpAOxR339A and PaDERA with CAT. Our findings present a novel soluble AOx and a carboligase for 3-HPA production from low-cost alcohols, achieving the highest conversion rate reported to date. These biocatalysts provide a promising strategy for producing value-added products from C1/C2 chemicals, contributing to efforts aimed at reducing the impact of GHG emissions and advancing a circular carbon economy.