Scopoletin, a plant-derived coumarin, exhibits diverse pharmacological activities. Nowadays, scopoletin is mainly produced by plant extraction. In this study, a Saccharomyces cerevisiae strain capable of de novo scopoletin biosynthesis was constructed by successively integrating ten heterologous genes into a p -coumaric acid producing strain. The following strategies were employed to improve the yield of scopoletin: (1) A high-yield caffeic acid platform strain (301.98 mg/L) was established by increasing the HpaBC copy number, integration of EcaroL and RgTAL , feedback-resistant mutant expression cassette ( ARO3 D154N / ARO4 G141S / ARO7 K229L ), and knockout competing pathway genes ( ARO10 / PDC5 ); (2) Two distinct pathways from caffeic acid to scopoletin were introduced and selected; (3) Integration of heterologous genes AtHCT and AtC3’H , to construct the pathway from p -coumaroyl-CoA to caffeoyl-CoA; (4) Attenuating PYK1 by introducing the mutant gene of PYK1 D146N to increase PEP supply; (5) Overexpression of cofactor regeneration genes ( ADO1 , SAH1 , and ZWF1 ) to strengthen the supply of SAM and NADPH; (6) Adjustment of the fermentation medium. The engineered strain produced scopoletin of 55.32 mg/L in shake flasks. To our knowledge, this is the first report of de novo scopoletin biosynthesis in S. cerevisiae . These results establish new insights for microbial production of scopoletin and coumarin derivatives.
Ye Zhang,Zihua Li,Yu Liu,Xuecong Cen,Dehua Liu,Zhen Chen
The economic viability of current bio-production systems is often limited by its low productivity due to slow cell growth and low substrate uptake rate. The fastest-growing bacterium Vibrio natriegens is a highly promising next-generation workhorse of the biotechnology industry which can utilize various industrially relevant carbon sources with high substrate uptake rates. Here, we demonstrate the first systematic engineering example of V. natriegens for the heterologous production of 1,3-propanediol (1,3-PDO) from glycerol. Systems metabolic engineering strategies have been applied in this study to develop a superior 1,3-PDO producer, including: (1) heterologous pathway construction and optimization; (2) engineering cellular transcriptional regulators and global transcriptomic analysis; (3) enhancing intracellular reducing power by cofactor engineering; (4) reducing the accumulation of toxic intermediate by pathway engineering; (5) systematic engineering of glycerol oxidation pathway to eliminate byproduct formation. A final engineered strain can efficiently produce 1,3-PDO with a titer of 56.2 g/L, a yield of 0.61 mol/mol, and an average productivity of 2.36 g/L/h. The strategies described in this study would be useful for engineering V. natriegens as a potential chassis for the production of other useful chemicals and biofuels.
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