Biocatalytic Cascade of Sebacic Acid Production with In Situ Co-Factor Regeneration Enabled by Engineering of an Alcohol Dehydrogenase

Abstract

Sebacic acid (1,10-decanedioic acid) is an important chemical intermediate. Traditional chemical oxidation methods for sebacic acid production do not conform with “green” manufacturing. With the rapid development of enzymatic technologies, a biocatalytic cascade method based on the Baeyer–Villiger monooxygenase was developed. The most attractive point of the method is the oleic acid that can be utilized as raw material, which is abundant in nature. However, this bio-catalysis process needs co-factor electron carriers, and the high cost of the co-factor limits its progress. In this piece of work, a co-factor in situ regeneration system between ADH from Micrococcus luteus WIUJH20 (MlADH) and BVMO is proposed. Since the co-factors of both enzymes are different, switching the co-factor preference of native MlADH from NAD+ to NADP+ is necessary. Switching research was carried out based on in silico simulation, and the sites of Tyr36, Asp 37, Ala38, and Val39 were selected for mutation investigation. The experimental results demonstrated that mutants of MlADH_D37G and MlADH_D37G/A38T/V39K would utilize NADP+ efficiently, and the mutant of MlADH_D37G/A38T/V39K demonstrated the highest sebacic acid yield with the combination of BVMO. The results indicated that the in situ co-factor generation system is successfully developed, which would improve the efficiency of the biocatalytic cascade for sebacic acid production and is helpful for simplifying product isolation, thus, reducing the cost of the enzymatic transformations process.

Keywords

• co-factor regeneration
• alcohol dehydrogenase
• sebacic acid
• enzymatic cascade reaction
• in silico simulation