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.

Study on Pore Structure and Fractal Characterization during Thermal Evolution of Oil Shale Experiments.

In order to better study the characteristics of the pore structure and to explore the influence factors of its fractal dimensions during the thermal evolution of oil shale, the immature oil shale (T max = 433 °C, TOC = 28.00%) of the Ordos Basin Extension Group was selected to simulate the whole thermal evolution process from immature to over mature in a semiopen system. Organic geochemical data show that the thermal simulation hydrocarbon generation threshold is between 300 and 400 °C. According to AIP-SEM observation, the pore types of the samples are different in different thermal simulation stages. The fractal dimensions are calculated by low-temperature N2 adsorption data using the fractal Frenkel-Halsey-Hill fractal model. The average surface fractal dimension (D 1) is 2.26, indicating that the pore (<4 nm) surface is relatively smooth. The average pore structure fractal dimension (D 2) is 2.49, indicating that the pore (>4 nm) structure is complex. Through the exploration of the relationship between fractal dimensions and organic geochemistry, whole rock X-ray diffraction, and N2 adsorption data, it is found that fractal dimensions have different degrees of correlation with thermal maturity, mineral composition, TOC content, and pore parameters. Through comprehensive research, it shows that hydrocarbon generation and expulsion, oil and gas cracking, and organic matter carbonization have important effects on the pore structure and fractal characteristics of oil shale.