Yarrowia lipolytica is a promising industrial host, yet its metabolic engineering potential remains limited by insufficient genetic tools. Here, we engineered a synthetic Transcription Activation Toolkit (TAT) based on LacI–VPRH, a chimeric protein fusing the prokaryotic DNA-binding domain LacI with the eukaryotic activation domain VPRH. Systematic optimization of LacO copy numbers and core promoter composition achieved up to 205-fold gene activation. The TAT platform was further expanded to construct bidirectional expression systems and enable multiplexed gene control. Applied to resveratrol biosynthesis via a “push-pull” strategy, CRISPR/Cas9-mediated integration of TAT-controlled synthetic promoters upregulated the shikimate pathway genes aroM10 and aroC alongside the rate-limiting enzyme ST1, achieving a shake-flask titer of 2.715 g/L─the highest reported to date. Additionally, an IPTG-inducible “turn-on” system (TAT-2.0) incorporating the antiLacI9 mutant was developed for small-molecule-responsive transcriptional control. Collectively, the modular TAT system provides a versatile strategy for precise metabolic pathway optimization in Y. lipolytica.
Yunhe Li, Wen-Ping Wei, Ping Zhang et al.· Journal of Agricultural and...· 0 citations
Taxol (paclitaxel) is a frontline anticancer drug widely applied for the treatment of breast, ovarian and lung cancers. Currently, its supply mainly relies on the semi-synthesis using baccatin III from Taxus plants. Heterologous biosynthesis of baccatin III in microorganisms offers a promising solution to alleviate global Taxol supply shortage, but remains challenging due to pathway complexity. Here, we report a novel taxusin-mediated biosynthetic pathway for baccatin III production via the identification of C13 deacetylase, elucidation of the exact sequence underlying C1 hydroxylation, and stepwise enzymatic functional validation. Through protein engineering of the promiscuous C1 and C5 hydroxylases, coupled with the distribution of pathway modules in Saccharomyces cerevisiae and Escherichia coli, we achieved the de novo biosynthesis of baccatin III. Collectively, our findings remodel the current biosynthetic framework governing the formation of Taxol precursors and highlight the great potential of microbial cell factories for the production of complex plant-derived therapeutic compounds. Highlights • Discovery of C13 deacetylase reveals a novel biosynthetic route to baccatin III via taxusin • Stepwise verification of the complete biosynthetic route to baccatin III through taxusin and baccatin VI • Single-site mutation reversed the product selectivity of T1OH and converted T5OH into a specific taxoid C5 hydroxylase • Complete biosynthesis of baccatin III in engineered microbes
