Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

Bat-Erdene, Undramaa; Billingsley, John M; Turner, W. C.; Lichman, Benjamin R.; Ippoliti, Francesca M.; Garg, Neil K.; O’Connor, Sarah E.; Tang, Tang; Yi, Mingdong

doi:10.1021/acscatal.1c02267

Cited by 23 publications

(26 citation statements)

References 47 publications

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“…We therefore hypothesized that addition of the MLPL from Nepeta mussinii (a.k.a Nepeta racemosa ), which is specific for the stereochemistry found in strictosidine, would enhance flux through the pathway in N. benthamiana . Recent efforts at reconstitution in yeast 31 and cell-free 42 systems have also indicated that this enzyme substantially enhances yield.…”

Section: Resultsmentioning

confidence: 99%

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Dudley

Guerrero

et al. 2022

Commun Biol

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Monoterpene indole alkaloids (MIAs) are a diverse class of plant natural products that include a number of medicinally important compounds. We set out to reconstitute the pathway for strictosidine, a key intermediate of all MIAs, from central metabolism in Nicotiana benthamiana. A disadvantage of this host is that its rich background metabolism results in the derivatization of some heterologously produced molecules. Here we use transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates and produce plant lines with targeted mutations in the genes encoding them. Expression of the early MIA pathway in these lines produces a more favorable product profile. Strictosidine biosynthesis was successfully reconstituted, with the best yields obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) is critical for improving flux through the iridoid pathway. The removal of endogenous glycosyltransferases does not impact the yields of strictosidine, highlighting that the metabolic flux of the pathway enzymes to a stable biosynthetic intermediate minimizes the need to engineer the endogenous metabolism of the host. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

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Section: Resultsmentioning

confidence: 99%

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Dudley

Guerrero

et al. 2022

Commun Biol

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“…We therefore hypothesized that addition of the MLPL from Nepeta mussinii (a.k.a Nepeta racemosa ), which is specific for the stereochemistry found in strictosidine, would enhance flux through the pathway in N. benthamiana. Recent efforts at reconstitution in yeast 31 and cell-free 66 systems have also indicated that this enzyme substantially enhances yield.…”

Section: Resultsmentioning

confidence: 99%

“…We hypothesise that MLPL might similarly enhance secoiridoid metabolic engineering in microorganisms such as yeast (Brown et al 2015). Indeed, a cell-free in vitro one-pot enzyme cascade included MLPL with nine other pathway enzymes, accessory proteins, and cofactor regeneration enzymes to produce ~1 g/L nepetalactone (Bat-Erdene et al 2021).…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Dudley

Guerrero

et al. 2021

Preprint

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Monoterpene indole alkaloids (MIAs) are a diverse and important class of plant natural products that include a number of medicinally significant compounds, often present at low concentrations within their native plant species. The complex biosynthesis of MIAs requires the assembly of tryptamine with a secoiridoid to produce the central intermediate, strictosidine, from which all known MIAs derive. Structural complexity makes chemical synthesis challenging, but recent efforts to identify the biosynthetic enzymes provide options for pathway reconstruction in a heterologous host. Previous attempts have had limited success, with yield in microorganisms limited by the poor expression of some enzymes. Here, we reconstitute the pathway for strictosidine biosynthesis from central metabolism without the need for supplementation of any metabolite precursors or intermediates in Nicotiana benthamiana. The best yields were obtained by the co-expression of 14 enzymes, of which a major latex protein-like enzyme (MLPL) from Nepeta (catmint) was critical for improving flux through the secoiridoid pathway. The production of strictosidine in planta expands the range of MIA products amenable to biological synthesis.

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“…The cell-free biocatalytic synthesis of the corrin metabolite hydrogenobyrinic acid from aminolevulinic acid, which uses 12 overexpressed enzymes for catalyzing 17 reaction steps in one pot, has been achieved with an overall yield of 20% (Roessner et al, 1994;1995). A cell-free system containing nine enzymes, including auxiliary enzymes and cofactor regenerating enzymes, enabled the one-pot synthesis of nepetalactol or nepetalactone starting from geraniol at a much higher product concentration of about 1 g L −1 than the highest titer described for microbial systems (Bat-Erdene et al, 2021). The use of cell-free systems is highly attractive due to its modularity and continues to attract much interest in cell-free metabolic engineering (Dudley et al, 2015;Swartz, 2018) and synthetic biochemistry (Bowie et al, 2020).…”

Section: Cell-free Versus Whole-cell Biocatalytic Systemsmentioning

confidence: 99%

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

Wohlgemuth

Littlechild²

2022

Front. Bioeng. Biotechnol.

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The biosynthesis of metabolites from available starting materials is becoming an ever important area due to the increasing demands within the life science research area. Access to metabolites is making essential contributions to analytical, diagnostic, therapeutic and different industrial applications. These molecules can be synthesized by the enzymes of biological systems under sustainable process conditions. The facile synthetic access to the metabolite and metabolite-like molecular space is of fundamental importance. The increasing knowledge within molecular biology, enzyme discovery and production together with their biochemical and structural properties offers excellent opportunities for using modular cell-free biocatalytic systems. This reduces the complexity of synthesizing metabolites using biological whole-cell approaches or by classical chemical synthesis. A systems biocatalysis approach can provide a wealth of optimized enzymes for the biosynthesis of already identified and new metabolite molecules.

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Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

Cited by 23 publications

References 47 publications

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

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