Aims Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677 center dot 4 USD mol(-1)), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin & x2162;, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. Methods and Results In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin & x2162;-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30 center dot 95 USD mol(-1), about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2 center dot 13-fold of that when using acetyl-CoA. In thein vivosystem, the yield increased 24 center dot 17%, which may indicate its cooperation with acetyl-CoA. Conclusion The success of open-whole-cell synthesis andin vivosynthesis of baccatin & x2162; by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin & x2162;. Significance and Impact of the Study All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin & x2162; semi-synthesis.