AUTHOR=Huang Jia-jun , Wei Tao , Ye Zhi-wei , Zheng Qian-wang , Jiang Bing-hua , Han Wen-feng , Ye An-qi , Han Pei-yun , Guo Li-qiong , Lin Jun-fang TITLE=Microbial Cell Factory of Baccatin III Preparation in Escherichia coli by Increasing DBAT Thermostability and in vivo Acetyl-CoA Supply JOURNAL=Frontiers in Microbiology VOLUME=12 YEAR=2022 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.803490 DOI=10.3389/fmicb.2021.803490 ISSN=1664-302X ABSTRACT=

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbatS189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbatS189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.