Screening of Acetyl Donors and the Robust Enzymatic Synthesis of Acetyl-CoA by 10-Deacetylbaccatin III-10-β-O-acetyltransferase

Abstract

Acetyl-CoA is the precursor of many bio-manufacturing products and is also the hub of the cellular metabolism of energy and substances. However, acetyl-CoA is not a bulk commodity and its application is hindered due to its high cost and low yield. In this study, we screened acetyl donor candidates and utilized 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) in the synthesis of acetyl-CoA with CoASH as the acetyl acceptor. Among the tested candidates, acetylsalicylic acid methyl ester was identified to be the best acetyl donor, followed by acetyl-trans-resveratrol, acetylsalicylic acid ethyl ester, acetylsalicylsalicylic acid, and 4-acetoxyacetanilide. The enzymatic reaction conditions were optimized and the maximum yield of acetyl-CoA reached 14.82 mg/mL, which is the highest yield among all reported approaches to date. Meanwhile, 4.22 mg/mL of the by-product salicylic acid methyl ester, which is another industrial material, was produced. Additionally, a preliminary purification process for acetyl-CoA was established, in which 40 mg acetyl-CoA (HPLC purity > 98%) was acquired from the finished 20 mL reaction system (feeding 46 mg CoASH and 34 mg ASME) with a recovery rate of 86%. Our study lays the foundation for the large-scale production of acetyl-CoA by an enzymatic approach and will promote its application in different fields.