Toward the Heterologous Biosynthesis of Plant Natural Products: Gene Discovery and Characterization

Wang, Huiyan; Guo, Hao; Wang, Ning; Huo, Yi-Xin

doi:10.1021/acssynbio.1c00315

Cited by 15 publications

(14 citation statements)

References 105 publications

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“…The metabolic engineering of high value natural products in the native producer plants is often challenging, due to demanding propagation conditions, low gene transformation efficiencies, complex genetic regulation, and cross-talk between the engineered and native biosynthetic pathways. , As an alternative strategy, biosynthetic pathways of high-value plant natural products, such as benzylisoquinoline and tropane alkaloids or diterpenes such as taxol precursors, have been reconstituted in microbial heterologous hosts . Recently, the biosynthesis of the vinblastine precursors vindoline and catharanthine has been reconstituted in Saccharomyces cerevisiae to produce microgram per liter titers after extensive optimization .…”

Section: Introductionmentioning

confidence: 99%

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

et al. 2022

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Vinblastine is a chemotherapy agent produced by the plant Catharanthus roseus in small quantities. Currently, vinblastine is sourced by isolation or semisynthesis. Nicotiana benthamiana is a plant heterologous host that can be used for reconstitution of biosynthetic pathways as an alternative natural product sourcing strategy. Recently, the biosynthesis of the latestage vinblastine precursors precondylocarpine acetate, catharanthine, and tabersonine have been fully elucidated. However, the large number of enzymes involved in the pathway and the unstable nature of intermediates make the reconstitution of late-stage vinblastine precursor biosynthesis challenging. We used the N. benthamiana chassis and a state-of-art modular vector assembly to optimize the six biosynthetic steps leading to production of precondylocarpine acetate from the central intermediate strictosidine (∼2.7 mg per 1 g frozen tissue). After selecting the optimal regulatory element combination, we constructed four transcriptional unit assemblies and tested their efficiency. Finally, we successfully reconstituted the biosynthetic steps leading to production of catharanthine and tabersonine.

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Section: Introductionmentioning

confidence: 99%

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

et al. 2022

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“…Recently, the biotransformation of raw organic materials into bioactive compounds has emerged as a powerful method for solving problem of the rapidly increasing demand for natural products. 31 The production of high-value-added natural products, such as ginsenosides, requires multistep enzyme reactions to perform specific conversions or modifications, which are necessary to enhance biological activities. The CK enzymatic conversion pathway is composed of three enzymes, Abf22-3, 17 BglPm, 18 and BglBX10, 19,20 that selectively remove sugar moieties from the ginsenoside Rc, Rd, and F2, respectively (Figure 3A).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Next, we devised another in vivo assembly application that was large and complex to further demonstrate the applicability of our split intein system. Recently, the biotransformation of raw organic materials into bioactive compounds has emerged as a powerful method for solving the problem of the rapidly increasing demand for natural products . The production of high-value-added natural products, such as ginsenosides, requires multistep enzyme reactions to perform specific conversions or modifications, which are necessary to enhance biological activities.…”

Section: Resultsmentioning

confidence: 99%

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside

Lee

et al. 2022

ACS Synth. Biol.

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Cascade reaction systems, such as protein fusion and synthetic protein scaffold systems, can both spatially control the metabolic flux and boost the productivity of multistep enzymatic reactions. Despite many efforts to generate fusion proteins, this task remains challenging due to the limited expression of complex enzymes. Therefore, we developed a novel fusion system that bypasses the limited expression of complex enzymes via a post-translational linkage. Here, we report a split intein-mediated cascade system wherein orthogonal split inteins serve as adapters for protein ligation. A genetically programmable, self-assembled, and traceless split intein was utilized to generate a biocatalytic cascade to produce the ginsenoside compound K (CK) with various pharmacological activities, including anticarcinogenic, anti-inflammatory, and antidiabetic effects. We used two types of split inteins, consensus atypical (Cat) and Rma DnaB, to form a covalent scaffold with the three enzymes involved in the CK conversion pathway. The multienzymatic complex with a size greater than 240 kDa was successfully assembled in a soluble form and exhibited specific activity toward ginsenoside conversion. Furthermore, our split intein cascade system significantly increased the CK conversion rate and reduced the production time by more than 2-fold. Our multienzymatic cascade system that uses split inteins can be utilized as a platform for regulating multimeric bioconversion pathways and boosting the production of various high-value substances.

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“…Microbial synthesis is an attractive alternative for the production of triterpenoids, traditionally manufactured by extraction from plant materials. − Most triterpenoids are biosynthesized from 2,3-oxidosqualene, a linear 30-carbon compound, and a few triterpenoids are synthesized via squalene and lanosterol. In the past decade, Saccharomyces cerevisiae has become a successful chassis for the production of various triterpenoids as well as other terpenoids because it not only provides the precursors through its native mevalonate (MVA) pathway but also the ER membrane for expression of the enzymes involved in triterpenoid biosynthesis, such as cytochrome P450 monooxygenases (P450s) and cytochrome P450 reductases (CPRs). − …”

Section: Introductionmentioning

confidence: 99%

Engineering Critical Amino Acid Residues of Lanosterol Synthase to Improve the Production of Triterpenoids in Saccharomyces cerevisiae

et al. 2022

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Triterpenoids are a subgroup of terpenoids and have wide applications in the food, cosmetics, and pharmaceutical industries. The heterologous production of various triterpenoids in Saccharomyces cerevisiae, as well as other microbes, has been successfully implemented as these production hosts not only produce the precursor of triterpenoids 2,3-oxidosqualene by the mevalonate pathway but also allow simple expression of plant membrane-anchored enzymes. Nevertheless, 2,3oxidosqualene is natively converted to lanosterol catalyzed by the endogenous lanosterol synthase (Erg7p), causing low production of recombinant triterpenoids. While simple deletion of ERG7 was not effective, in this study, the critical amino acid residues of Erg7p were engineered to lower this critical enzyme activity. The engineered S. cerevisiae indeed accumulated 2,3-oxidosqualene up to 180 mg/L. Engineering triterpenoid synthesis into the ERG7-modified strain resulted in 7.3-and 3-fold increases in the titers of dammarane-type and lupane-type triterpenoids, respectively. This study presents an efficient inducer-free strategy for lowering Erg7p activity, thereby providing 2,3-oxidosqualene for the enhanced production of various triterpenoids.

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Toward the Heterologous Biosynthesis of Plant Natural Products: Gene Discovery and Characterization

Cited by 15 publications

References 105 publications

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

Novel Split Intein-Mediated Enzymatic Channeling Accelerates the Multimeric Bioconversion Pathway of Ginsenoside

Engineering Critical Amino Acid Residues of Lanosterol Synthase to Improve the Production of Triterpenoids in Saccharomyces cerevisiae

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