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

Bo-Yong Zhang,Tian-Jiao Chen,Ping Zhu,Jing-Jing Chen,Ting Gong,Hao Wang,Jin-Ling Yang

doi:10.3390/catal11101240

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.

Highlights

Acetyl-CoA is a ubiquitous substance found in organisms and participates in various metabolisms of life
In our previous work [12], we found that 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) could acetylate 10-deacetylbaccatin III (10-DAB) into baccatin III with acetyl-CoA as the acetyl donor, and de-acetylate baccatin III into 10-DAB under alkaline conditions
We systematically investigated the enzymatic approach to producing acetyl-CoA using DBAT as the catalyst, which included the screening of proper acetyl donors and the optimization of reaction conditions

Summary

Introduction

Acetyl-CoA is a ubiquitous substance found in organisms and participates in various metabolisms of life It converges three nutrients (sugar, fat, and protein) into one common metabolic route—the tricarboxylic acid cycle and oxidative phosphorylation, through which these nutrients are oxidized into CO2 and water, with the concomitant production of ATP, a high-energy phosphate compound [1]. Acetyl-CoA ( written as acetyl-S-CoA) is an acetylated form of coenzyme A (CoASH), and the acetyl group is docked on the sulfhydryl residue of CoA by a high-energy thioester bond This product is not a bulk commodity and is costly in the market. Irving et al utilized carnitine acetyltransferase to prepare acetyl-CoA from acetylcarnitine and CoASH [8] In another approach, acetyl-CoA was synthesized by acetyl-CoA synthetase from acetic acid and CoASH supplemented with ATP and Mg2+ as the cofactors [9]. Using pyruvate and CoASH as the substrates, acetyl-CoA can be synthesized by the combination of pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase in the presence of NAD [11]

Methods

Results

Discussion

Conclusion