Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Abstract

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

A review on Lactococcus lactis: from food to factory

“…Our research group has also successfully expressed two plant terpene synthases from orchid [65] and kesum ( Persicaria minor ) [66] in L. lactis leading to the production of germacrene D and β-sesquiphellandrene, respectively. Interestingly, L. lactis uses the mevalonate pathway (MVA) for terpenoid biosynthesis, a pathway more commonly found in eukaryotes.…”

Engineering the lactococcal mevalonate pathway for increased sesquiterpene production

“…Sequences in brackets encode his‐tag, which was used for identification and purification. PCR conditions were as previously described (Song et al ., , b). For pNZ: PMSTS : mvk and pNZ: PMSTS : mvk : mvaA (abbreviated p PMM from this point), the PMSTS (for Persicaria minor sesquiterpene synthase) and mvk genes were first amplified separately.…”

“…Conditions used for PCR amplification were as described in Song et al . () except that the annealing temperature for the mvk gene was 42 °C. The PMSTS and mvk were then fused together by overlap extension PCR.…”

“…To verify that PMSTS, mevalonate kinase ( mvk ) and HMGR ( mvaA ) were expressed properly, clones harbouring pNZ: PMSTS:mvk and p PMM were induced in conditions as per Song et al . () with the following modifications. Cells were harvested in PBS, and no desalting step was conducted as it was not necessary to repeat the in vitro enzyme assays in the current study.…”

Engineered biosynthesis of natural products in heterologous hosts

“…Different sesquiterpene synthases were used to catalyze the synthesis of different sesquiterpenes from FPP (Figure 3). β-sesquiphellandrene, with efficient anti-microbial and anti-oxidant activity, was synthesized by PMSTS ( Persicaria minor sesquiterpene synthase) 28, 29 . Valencene, an odorant molecule, was synthesized by CsPTS ( Citrus sinensis valencene synthase), and could be further oxidized to (+)-nootkatol, (−)-nootkatol or (−)-nootkatone by different kinds of P450 cytochrome oxidase (CYP71D51v2 from Nicotiana tabacum , CYP71AV8 from Cichorium intybus and CnVO from C. nootkatensis ) 31, 32 .…”