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Plant natural products (PNPs) exhibit a wide range of biological activities and have essential applications in various fields such as medicine, agriculture, and flavors. Given their natural limitations, the production of high-value PNPs using microbial cell factories has become an effective alternative in recent years. However, host metabolic burden caused by its massive accumulation has become one of the main challenges for efficient PNP production. Therefore, it is necessary to strengthen the transmembrane transport process of PNPs. This review introduces the discovery and mining of PNP transporters to directly mediate PNP transmembrane transportation both intracellularly and extracellularly. In addition to transporter engineering, this review also summarizes several auxiliary strategies (such as small molecules, environmental changes, and vesicles assisted transport) for strengthening PNP transportation. Finally, this review is concluded with the applications and future perspectives of transportation engineering in the construction and optimization of PNP microbial cell factories.

Abstract 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.

Paclitaxel is a renowned broad-spectrum anticancer drug. With the establishment of a chromosome-level high-quality reference genome map of Taxus, recent research on paclitaxel biosynthesis has flourished. The oxetane ring is a distinctive chemical moiety of paclitaxel, and three recent studies have proposed different enzymes involved in its formation, reflecting divergent opinions on whether the pathway proceeds via acetylation followed by epoxidation or vice versa. Subsequently, researchers have elucidated gene clusters responsible for the biosynthesis of the key intermediate baccatin III. Despite varying reports, two studies successfully achieved heterologous biosynthesis of baccatin III by transient expression in tobacco. Taxadiene 5α-hydroxylase (T5αH), the first cytochrome P450 in the pathway, exhibited varied product profiles upon heterologous expression systems, contrasting with observations in native Taxus species, probably due to differences in partner proteins or cellular microenvironments. Further elucidation of biosynthesis mechanisms, including the reaction order and the promiscuity of key enzymes, is anticipated through collaborative efforts among botanists, chemists, and synthetic biologists.

Liu et al. optimize vindoline biosynthesis in yeast from tabersonine by altering the copy numbers of pathway genes, pairing CYPs with CPRs, engineering the microenvironment for functional expression of CYPs, enhancing cofactor supplies, and optimizing fermentation conditions. By doing so, the authors increased the production of vindoline to a final titer as high as ∼16.5 mg/L.

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.