Microbial Synthesis of Plant Alkaloids

Pyne, Michael E.; Narcross, Lauren; Hansen, Christian; Campbell, Alex; Davies, Meghan; Bourgeois, Leanne; Fossati, Elena; Martin, Vincent J. J.

doi:10.1007/978-3-319-67903-7_5

Cited by 3 publications

(4 citation statements)

References 138 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nepetalactone has higher in planta yields than MIAs, yet the mass market demand would preclude plant-sourced nepetalactone for commercial scale production due to prohibitive costs. Elucidation of the strictosidine biosynthetic pathway has enabled de novo production of nepetalactol and strictosidine in the budding yeast S. cerevisiae . Nepetalactone production was reported in yeast; however, this was achieved using an incomplete pathway and supplying pathway intermediates .…”

mentioning

confidence: 99%

Engineering Yeast for De Novo Synthesis of the Insect Repellent Nepetalactone

2021

Self Cite

View full text Add to dashboard Cite

While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe for the first time de novo nepetalactone synthesis in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introducing eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, and copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity and increasing geraniol-8-hydroxylase gene copy number improved 8-hydroxygeraniol titers. The accumulation of an unwanted metabolite implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologues were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. This optimized pathway will benefit the development of high yielding strains for the scale up production of nepetalactone.

show abstract

mentioning

confidence: 99%

Engineering Yeast for De Novo Synthesis of the Insect Repellent Nepetalactone

2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…13 Protein engineering stands as an effective approach for enhancing the substrate specificity of enzymes participating in the metabolic pathways. 9 Leonard et al 14 successfully reprogrammed promiscuous enzymes acting as regulatory nodes through protein engineering, ultimately achieving overproduction and selectivity control of the diterpenoid levopimaradiene in Escherichia coli. Bian et al 15 demonstrated that terpene synthases with substrate promiscuity are widespread and enhanced the production of certain terpenoids in Escherichia coli exploiting engineered mutants.…”

Section: ■ Introductionmentioning

confidence: 99%

“…In the iridoid metabolic pathway (Figure ), geraniol ( 1 ) undergoes successive catalysis by geraniol 8-oxidase (G8O) and 8-hydroxygeraniol oxidoreductase (8-HGO) to yield 8-oxogeranial ( 5 ). , Subsequently, compound 5 is transformed into the unstable intermediate 8-oxocitronellyl enol under the catalysis of iridoid synthase (ISY). This unstable intermediate then spontaneously undergoes cyclization, resulting in the formation of structure 6 . ,,, ISY, an enzyme belonging to the superfamily of short-chain dehydrogenase/reductase (SDR), , has been demonstrated to exhibit promiscuity with low substrate preference, resulting in the diversion of pathway flux to off-target routes. ,− Among the off-target routes, the main byproduct is citronellol ( 4 ). In the metabolic pathway of 4 , ISY can reduce geranial ( 2 ), the oxidation products of 1 , into citronellal ( 3 ); then, 3 will be converted into 4 by endogenous alcohol dehydrogenase (ADH) or 8-HGO.…”

Section: Introductionmentioning

confidence: 99%

“…Protein engineering stands as an effective approach for enhancing the substrate specificity of enzymes participating in the metabolic pathways . Leonard et al successfully reprogrammed promiscuous enzymes acting as regulatory nodes through protein engineering, ultimately achieving overproduction and selectivity control of the diterpenoid levopimaradiene in Escherichia coli .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Enhancing Substrate Preference of Iridoid Synthase via Focused Polarity-Steric Mutagenesis Scanning

Yu,

Ye,

Wang

et al. 2024

Chem Bio Eng.

View full text Add to dashboard Cite

Nepetalactol serves as the scaffold for most iridoids, which exhibit a wide range of biological and pharmacological activities. Iridoid synthase (ISY) plays a crucial role in the in vivo synthesis of nepetalactol from 8-oxogeranial. However, the substrate promiscuity of ISY could result in a deviation of flux toward off-target routes. In this work, the substrate preference (SP, the ratio of activity for 8-oxogeranial to geranial) of ISY for nepetalactol was improved by directed evolution. First, the strategy of focused polarity-steric mutagenesis scanning (FPSMS) was performed to construct a small mutant library with NmISY2 from Nepeta mussinii as an object. Four amino acid residues with varying polarity and steric hindrance, including alanine, aspartic acid, serine, and arginine, were incorporated to scan hot spots. Consequently, four sites of W109, M217, K343, and W345 with a significant impact on the substrate preference of NmISY2 were found. Then, the four sites were combined by a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy. As a result, the mutant W345D/K343M/W109Y (3M+) was obtained with a significantly increased SP value for 6 from 8.5 to 293.1. Molecular dynamics simulations revealed that the steric hindrance and polarity of the substrate tunnel played pivotal roles in the SP value of NmISY2. Notably, upon integration of 3M+ into Pichia pastoris, the de novo titer of 6 increased by 24.9 times, reaching 15.8 mg/L. This study offers a strategic approach to improving the substrate preference of enzymes.

show abstract