Engineering of a Nepetalactol-Producing Platform Strain of <i>Saccharomyces cerevisiae</i> for the Production of Plant Seco-Iridoids

Campbell, Alex; Bauchart, Philippe; Gold, Nicholas D.; Zhu, Yun; Luca, Vincenzo De; Martin, Vincent J. J.

doi:10.1021/acssynbio.5b00289

Cited by 45 publications

(48 citation statements)

References 82 publications

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“…Thus, it is likely that different cyclases, unrelated to NEPS, are operating in other iridoid producing species. The absence of such cyclases from iridoid pathways reconstituted in microbial organisms may have negatively impacted yields [36][37][38] . The NEPS cyclases described here, especially NEPS2, a dedicated cistrans cyclase, can be incorporated into such systems to improve yield.…”

Section: Discussionmentioning

confidence: 99%

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Lichman

Kamileen

Tichman

et al. 2018

Preprint

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Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyses the activation of its substrate 8-oxogeranial into a reactive enol intermediate but does not catalyse the subsequent cyclisation into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) which catalyse the stereoselective cyclisation of the enol intermediate into nepetalactol isomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterisation of the NEPS3 cyclase reveals it binds to NAD + yet does not utilise it chemically for a nonoxidoreductive formal [4+2] cyclisation. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis. Main paperNepetalactones 1 are volatile natural products produced by plants of the genus Nepeta, notably catmint (Nepeta mussinii syn racemosa) and catnip (N. cataria) (Fig. 1a) 1,2 . These compounds are responsible for the stimulatory effects these plants have on cats [3][4][5] . Moreover, certain insects use nepetalactones as sex pheromones, so production of these compounds by the plant also impacts interactions with insects 6 . Notably, the bridgehead stereocentres (carbons 4a and 7a) vary between 2 and within 2,4,7 Nepeta species. N. mussinii individuals, for example, produce different ratios of cis-trans 1a, cis-cis 1b and trans-cis-nepetalactone 1c 7 . Variation in stereoisomer ratio may influence the repellence of insect herbivores 8,9 . While the ratio of stereoisomers may be responsible for important biological effects, the mechanism of stereocontrol in nepetalactone biosynthesis is not known.Nepetalactones are iridoids, non-canonical monoterpenoids containing a cyclopentanopyran ring. Canonical cyclic terpenoids (e.g. (-)-limonene) are biosynthesised from linear precursors by terpene synthases (Fig. 1b) 10 . These enzymes activate linear precursors either by loss of pyrophosphate or protonation [10][11][12] . The resulting carbocations generated cyclise rapidly to form an array of cyclic products 13 . Therefore, in canonical terpenoid biosynthesis, activation and cyclisation of precursors are coupled and occur in the same enzyme active site.In plant iridoid biosynthesis, geranyl pyrophosphate is hydrolysed and oxidised into 8-oxogeranial 2 14 . This precursor then undergoes a two-step activation-cyclisation process, analogous to canonical terpene synthesis (Fig. 1c) 15 . Unlike canonical terpene synthesis, however, activation is achieved by reduction, and the intermediate is not a carbocation, but the enol or enolate species 3. Cyclisation of this intermediate yields cis-trans-nepetalactol 4a along with iridodial side products 5 (Fig. 1c).

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Section: Discussionmentioning

confidence: 99%

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Lichman

Kamileen

Tichman

et al. 2018

Preprint

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“…As substrates of GOR and ISY, the α,β-unsaturated alcohol 1 and aldehyde 2, respectively , are subject to significant crosstalk and interference with the endogenous yeast redox metabolism. For example, efforts in de novo iridoid production were derailed when it was discovered that saturated shunt products accumulate when the iridoid pathway is expressed in yeast (Campbell et al, 2016). The α,β-unsaturated aldehydes present in the linear iridoid precursor 2 are required in the cyclization mechanism of ISY towards the formation of nepetalactol 3 (Lindner et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

“…While several reports have described the reduction of the double bonds in monoterpene pathway intermediates by yeast (Gramatica et al, 1982; King and Richard Dickinson, 2000; Steyer et al, 2013), the exact mechanism has remained unresolved particularly with regard to crosstalk between exogenous biosynthetic enzymes (GOR and ISY) and endogenous yeast enzymes. However the low benchmark titers of 8-hydroxygeraniol 2 achieved to date (5.3 mg/L) (Campbell et al, 2016), in combination with the obscured mechanistic details of shunt product formation have significantly limited the reconstitution of nepetalactol 3 production and overall MIA pathway engineering in yeast.…”

Section: Introductionmentioning

confidence: 99%

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Billingsley

DeNicola

Barber

et al. 2017

Metabolic Engineering

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Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous ‘ene’-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in α,β-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.

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“…[256a] Such low titers of MIAs can be due to the leakage of metabolic fluxes to nonproductive intermediates along the long target pathways, such as in the step from geraniol to citronellol-instead of going toward the desired product of 10-hydroxygeraniol. [259] The complete MIA biosynthetic pathways leading to the end products, such as vinblastine and camptothecin, still have not been fully understood. Fortunately, breakthroughs are being made, such as elucidation of the major steps leading to vindoline from tabersonin, [260] increasing the possibility of the complete biosynthesis of vinblastine or vincristine by microbial platforms.…”

Section: Identification Of Unknown Pathway Enzymesmentioning

confidence: 99%

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

Park

Yang

et al. 2017

Adv. Biosys.

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Natural products have been attracting much interest around the world for their diverse applications, especially in drug and food industries. Plants have been a major source of many different natural products. However, plants are affected by weather and environmental conditions and their successful extraction is rather limited. Chemical synthesis is inefficient due to the complexity of their chemical structures involving enantioselectivity and regioselectivity. For these reasons, an alternative means of overproducing valuable natural products using microorganisms has emerged. In recent years, various metabolic engineering strategies have been developed for the production of natural products by microorganisms. Here, the strategies taken to produce natural products are reviewed. For convenience, natural products are classified into four main categories: terpenoids, phenylpropanoids, polyketides, and alkaloids. For each product category, the strategies for establishing and rewiring the metabolic network for heterologous natural product biosynthesis, systems approaches undertaken to optimize production hosts, and the strategies for fermentation optimization are reviewed. Taken together, metabolic engineering has enabled microorganisms to serve as a prominent platform for natural compounds production. This article examines both the conventional and novel strategies of metabolic engineering, providing general strategies for complex natural compound production through the development of robust microbial‐cell factories.

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Engineering of a Nepetalactol-Producing Platform Strain of Saccharomyces cerevisiae for the Production of Plant Seco-Iridoids

Cited by 45 publications

References 82 publications

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

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