Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast.

While sanguinarine has gained recognition for antimicrobial and antineoplastic activities, its complex conjugated structure and low abundance in plants impede broad applications. Here, we demonstrate the complete biosynthesis of sanguinarine and halogenated derivatives using highly engineered yeast strains. To overcome sanguinarine cytotoxicity, we establish a splicing intein-mediated temperature-responsive gene expression system (SIMTeGES), a simple strategy that decouples cell growth from product synthesis without sacrificing protein activity. To debottleneck sanguinarine biosynthesis, we identify two reticuline oxidases and facilitated functional expression of flavoproteins and cytochrome P450 enzymes via protein molecular engineering. After comprehensive metabolic engineering, we report the production of sanguinarine at a titer of 448.64 mg L-1. Additionally, our engineered strain enables the biosynthesis of fluorinated sanguinarine, showcasing the biotransformation of halogenated derivatives through more than 15 biocatalytic steps. This work serves as a blueprint for utilizing yeast as a scalable platform for biomanufacturing diverse benzylisoquinoline alkaloids and derivatives.

Development of a Pichia pastoris cell factory for efficient production of germacrene A: a precursor of ß-elemene.

ß-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to ß-elemene by a one-step reaction in vitro. In this study, we constructed an engineered Pichia pastoris cell factory for producing germacrene A. We rerouted the fluxes towards germacrene A biosynthesis through the optimization of the linker sequences between germacrene A synthase (GAS) and farnesyl pyrophosphate synthase (ERG20), overexpression of important pathway genes (i.e., IDI1, tHMG1, and ACS), and multi-copy integration of related expression cassettes. In combination with medium optimization and bioprocess engineering, the final titer of germacrene A in a 1 L fermenter reached 1.9 g/L through fed-batch fermentation. This represents the first report on the production of germacrene A in P. pastoris and demonstrates its advantage in producing terpenoids and other value-added natural products.

De Novo Biosynthesis of Vindoline and Catharanthine in Saccharomyces cerevisiae.

Vinblastine has been used clinically as one of the most potent therapeutics for the treatment of several types of cancer. However, the traditional plant extraction method suffers from unreliable supply, low abundance, and extremely high cost. Here, we use synthetic biology approach to engineer Saccharomyces cerevisiae for de novo biosynthesis of vindoline and catharanthine, which can be coupled chemically or biologically to vinblastine. On the basis of a platform strain with sufficient supply of precursors and cofactors for biosynthesis, we reconstituted, debottlenecked, and optimized the biosynthetic pathways for the production of vindoline and catharanthine. The vindoline biosynthetic pathway represents one of the most complicated pathways ever reconstituted in microbial cell factories. Using shake flask fermentation, our engineered yeast strains were able to produce catharanthine and vindoline at a titer of 527.1 and 305.1 µg·liter-1, respectively, without accumulating detectable amount of pathway intermediates. This study establishes a representative example for the production of valuable plant natural products in yeast.