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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 ?-sesquiphellandrene synthase as it was demonstrated to be functional in producing ?-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 ?-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 ?-sesquiphellandrene by 1.25-1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

SUBMITTER: Song AA

PROVIDER: S-EPMC3530464 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

Song Adelene Ai-Lian AA Abdullah Janna Ong JO Abdullah Mohd Puad MP Shafee Norazizah N Othman Roohaida R Tan Ee-Fun EF Noor Normah Mohd NM Raha Abdul Rahim AR

PloS one 20121226 12

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 ...[more]

PMID: 23300671

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Project description:The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters.IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide access to this chemodiversity for the discovery and synthesis of molecules with new bioactivities. The identification and successful cloning of the previously elusive hirsutene synthase from the S. hirsutum provide important insights and strategies for biosynthetic gene discovery in Basidiomycota. The finding of a terpene synthase-HMGS fusion, the discovery of other sesquiterpenoid biosynthetic gene clusters with dedicated HMGS genes, and HMGS gene duplications in fungal genomes give new importance to the role of HMGS as a key regulatory enzyme in isoprenoid and sterol biosynthesis that should be exploited for metabolic engineering.

Project description:ObjectiveIn addition to inducing a self-limited myopathy, statin use is associated with an immune-mediated necrotizing myopathy (IMNM), with autoantibodies that recognize ∼200-kd and ∼100-kd autoantigens. The purpose of this study was to identify these molecules to help clarify the disease mechanism and facilitate diagnosis.MethodsThe effect of statin treatment on autoantigen expression was addressed by immunoprecipitation using sera from patients. The identity of the ∼100-kd autoantigen was confirmed by immunoprecipitation of in vitro-translated 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) protein. HMGCR expression in muscle was analyzed by immunofluorescence. A cohort of myopathy patients was screened for anti-HMGCR autoantibodies by enzyme-linked immunosorbent assay and genotyped for the rs4149056 C allele, a predictor of self-limited statin myopathy.ResultsStatin exposure induced expression of the ∼200-kd/∼100-kd autoantigens in cultured cells. HMGCR was identified as the ∼100-kd autoantigen. Competition experiments demonstrated no distinct autoantibodies recognizing the ∼200-kd protein. In muscle biopsy tissues from anti-HMGCR-positive patients, HMGCR expression was up-regulated in cells expressing neural cell adhesion molecule, a marker of muscle regeneration. Anti-HMGCR autoantibodies were found in 45 of 750 patients presenting to the Johns Hopkins Myositis Center (6%). Among patients ages 50 years and older, 92.3% had taken statins. The prevalence of the rs4149056 C allele was not increased in patients with anti-HMGCR.ConclusionStatins up-regulate the expression of HMGCR, the major target of autoantibodies in statin-associated IMNM. Regenerating muscle cells express high levels of HMGCR, which may sustain the immune response even after statins are discontinued. These studies demonstrate a mechanistic link between an environmental trigger and the development of sustained autoimmunity. Detection of anti-HMGCR autoantibodies may facilitate diagnosis and direct therapy.

Dataset Information

Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

Publications

Overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in the lactococcal mevalonate pathway for heterologous plant sesquiterpene production.

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