Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.     

高通量技术在萜类合成酶筛选中的应用

萜类化合物种类繁多,生物活性多样,在食品、药品与化妆品等行业中具有广泛的应用。萜类化合物多来源于植物,然而随着合成生物学的快速发展,相较于传统的天然植物提取与化学合成方法,利用工程微生物进行萜类化合物异源合成的方法显得更为经济与环保。萜类合成酶的催化活性及合成产物的结构特性是萜类化合物异源合成的关键。通过蛋白定向进化与理性设计可以有针对性地优化萜类合成酶的催化性能及产物专一性,但该方案需要一个特异的筛选方法来实现蛋白突变体库的高通量筛选。近年来,一系列高通量筛选方法的建立使得萜类合成酶的筛选变得更加灵敏与高效。本文对近期建立的萜类合成酶高通量筛选方法进行了综述,简要概述了各种筛选方法的基本原理与优缺点,并对高通量筛选技术在萜类合成酶改造中的应用做出了展望。     

Direct formation of the sesquiterpeonid ether liguloxide by a terpene synthase in Senecio scandens

Key message

SsLOS directly catalyzed formation of the sesquiterpenoid ether liguloxide in the medicinal plant Senecio scandens.

Abstract

Terpene synthases determine the diversity of terpene skeletons and corresponding terpenoid natural products. Oxygenated groups introduced in catalysis of terpene synthases are important for solubility, potential bioactivity and further elaboration of terpenoids. Here we identified one terpene synthase, SsLOS, in the Chinese medicinal plant Senecio scandens. SsLOS acted as the sesquiterpene synthase and utilized (E,E)-farnesyl diphosphate as the substrate to produce a blend of sesquiterpenoids. GC–MS analysis and NMR structure identification demonstrated that SsLOS directly produced the sesquiterpenoid ether, liguloxide, as well as its alcoholic isomer, 6-epi-guaia-2(3)-en-11-ol. Homology modeling and site-directed mutagenesis were combined to explore the catalytic mechanism of SsLOS. A few key residues were identified in the active site and hedycaryol was identified as the neutral intermediate of SsLOS catalysis. The plausible catalytic mechanism was proposed as well. Altogether, SsLOS was identified and characterized as the sesquiterpenoid ether synthase, which is the second terpenoid ether synthase after 1,8-cineol synthase, suggesting some insights for the universal mechanism of terpene synthases using the water molecule in the catalytic cavity.

     

植物萜类生物合成中的后修饰酶

萜类化合物由于其结构类型丰富多样而被称为"terpenome".除了参与植物生长发育、环境应答等生理过程,萜类化合物还应用于医药、有机化工等领域.萜类的生物合成大致可分为前体形成、骨架构建以及后修饰三部分,基本骨架通常由萜类合酶催化形成,进一步在后修饰酶的作用下产生数以万计的萜类化合物.结合我们对香茶菜二萜生物合成的初步研究结果,本文主要针对近年来植物萜类生物合成中的一些有代表性的后修饰酶包括P450单氧酶、双键还原酶、酰基转移酶和糖基转移酶,进行研究现状分析与展望.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

The use of Aspergillus niger cultures for biotransformation of terpenoids

Aspergillus niger is a well-known fungus that has been used for many different biotransformations of organic compounds. The terpenoids include a large variety of natural hydrocarbons and their derivatives, mostly obtained from plant essential oils, but some obtained from animals or fungi. They may be acyclic or have one or more rings of various sizes, and they show a variety of biological activities that include antibacterial, antifungal, antiparasitic, antiviral, and anticancer activities. Terpenoids are classified as monoterpenoids (C₁₀), sesquiterpenoids (C₁₅), diterpenoids (C₂₀), triterpenoids (C₃₀), and others. This review summarizes experimental processes that use cultures of various A. niger strains to carry out stereoselective biochemical reactions in terpenoids, including related epoxides, lactones, N-phenylcarbamates, and saponins, to produce metabolites that may be useful as flavors and fragrances or as new experimental drug candidates. Cultures of A. niger that add hydroxyl, carbonyl, and other groups at specific positions or reduce double bonds have resulted in the production of valuable new compounds.     

Positioning Bacillus subtilis as terpenoid cell factory

Increasing demands for bioactive compounds have motivated researchers to employ micro-organisms to produce complex natural products. Currently, Bacillus subtilis has been attracting lots of attention to be developed into terpenoids cell factories due to its generally recognized safe status and high isoprene precursor biosynthesis capacity by endogenous methylerythritol phosphate (MEP) pathway. In this review, we describe the up-to-date knowledge of each enzyme in MEP pathway and the subsequent steps of isomerization and condensation of C5 isoprene precursors. In addition, several representative terpene synthases expressed in B. subtilis and the engineering steps to improve corresponding terpenoids production are systematically discussed. Furthermore, the current available genetic tools are mentioned as along with promising strategies to improve terpenoids in B. subtilis, hoping to inspire future directions in metabolic engineering of B. subtilis for further terpenoid cell factory development.     

Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.     

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants

The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.     

Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus

Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secretes an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defence in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α‐pinene synthase (PmTPS4) and longifolene synthase (PmTPS21), identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defence strategy in resistant masson pines. Up‐regulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defence against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α‐pinene, while PmTPS21 produced α‐pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene‐defence mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene.     

NO inhibitory phytochemicals as potential anti-inflammatory agents from the twigs of Trigonostemon heterophyllus

Studies on the relationship of nitric oxide (NO) and inflammation have revealed that compounds with NO inhibitory effects are potentially useful for inflammation and related inflammatory disorders. A phytochemical investigation to obtain new NO inhibitors resulted in the isolation of two new cleistanthane diterpenoids (1 and 2) and 11 known terpenoids (3–13) from Trigonostemon heterophyllus. The structures of these terpenoids were established by analysis of their NMR, MS, and electronic circular dichroism (ECD) data. Compounds 1 and 2 possess rare 3,4-seco-cleistanthane diterpenoid skeletons. All of the isolates were evaluated biologically for their NO inhibitory effects in lipopolysaccharide (LPS)-induced murine microglial BV-2 cells and compounds 1, 6, and 8–10 showed strong NO inhibitory effects with IC₅₀ values less than 40 μM. Using Western blotting experiments and molecular docking, the possible mechanism of NO inhibition was investigated.     

The Activity and Isoform Complement of Glutamine Synthetase in Panicum Species Differing in Photosynthetic Pathway

Anion exchange chromatography and immunoprecipitation have been used to demonstrate the presence of two forms (GS₁, and GS₂) of glutamine synthetase in the leaves of nine species of Panicum representative of C₃, C₄ and C₃-C₄ intermediate-type photosynthesis. GS₂ from the Panicum species, P. miliaceum and P. maximum was more thermostable than GS₁, GS₁, and GS₂ from P. laxum were equally thermostable but GS₂ from all the Panicum species examined was more sensitive to inhibition by N-ethylmaleimide than GS₁. GS₁, and GS₂ were characterised as being cytoplasmic and chloroplastic isoforms respectively by their reaction with N-ethylmaleimide and by immunoprecipitation with antibodies raised against the cytosolic isoform in barley and the chloroplastic form in tobacco. C₃ species were found to have higher activity of the chloroplastic isoform of glutamine synthetase than C₄ species. C₃-C₄ intermediate species had total leaf glutamine synthetase activities similar to those in C₃ species but were found to have a lower chloroplastic isoform content. The results are consistent with the reassimilation of photorespiratory ammonia by chloroplastic glutamine synthetase.     

萜类合成酶定向进化的新思路

萜类化合物是天然产物中种类最多且主要存在于植物和微生物体内的一类化合物。随着越来越多具有应用价值的萜类化合物被挖掘,其应用前景引起了人们的关注,但由于含量低、提取成本高等缺点,因此制约了萜类化合物的广泛应用。合成生物学的兴起,为异源合成具有应用价值的萜类化合物提供了新思路,使构建定向、高效的微生物细胞工厂成为现实。萜类合成酶常作为萜类化合物异源合成代谢调控的靶酶,但天然的萜类合成酶存在催化效率低、底物专一性差、立体/区域选择性差、稳定性差等问题,严重影响萜类化合物的产量。萜类合成酶的定向进化可以有效地解决上述问题,为实现微生物细胞工厂异源、高效合成萜类化合物奠定基础。本文综述了近年来酶的定向进化技术的最新进展及应用,并提出了萜类合成酶定向进化的策略。     

The biochemistry of homoterpenes--common constituents of floral and herbivore-induced plant volatile bouquets

Volatile organic compounds emitted by plants mediate a variety of interactions between plants and other organisms. The irregular acyclic homoterpenes, 4,8-dimethylnona-1,3,7-triene (DMNT) and 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), are among the most widespread volatiles produced by angiosperms with emissions from flowers and from vegetative tissues upon herbivore feeding. Special attention has been placed on the role of homoterpenes in attracting parasitoids and predators of herbivores and has sparked interest in engineering homoterpene formation to improve biological pest control. The biosynthesis of DMNT and TMTT proceeds in two enzymatic steps: the formation of the tertiary C₁₅-, and C₂₀-alcohols, (E)-nerolidol and (E,E)-geranyl linalool, respectively, catalyzed by terpene synthases, and the subsequent oxidative degradation of both alcohols by a single cytochrome P450 monooxygenase (P450). In Arabidopsis thaliana, the herbivore-induced biosynthesis of TMTT is catalyzed by the concerted activities of the (E,E)-geranyllinalool synthase, AtGES, and CYP82G1, a P450 of the so far uncharacterized plant CYP82 family. TMTT formation is in part controlled at the level of AtGES expression. Co-expression of AtGES with CYP82G1 at wound sites allows for an efficient conversion of the alcohol intermediate. The identified homoterpene biosynthesis genes in Arabidopsis and related genes from other plant species provide tools to engineer homoterpene formation and to address questions of the regulation and specific activities of homoterpenes in plant-herbivore interactions.     

Genome-wide analysis of terpene synthases in soybean: functional characterization of GmTPS3

Terpenes (terpenoids or isoprenoids) constitute a large class of plant natural products and play numerous functional roles in primary and secondary metabolism as well as inecological interactions. This study presents a genomic analysis of 23 putative soybean (Glycine max) terpene synthase genes (GmTPSs) distributed over 10 of 20 chromosomes. The GmTPSs are grouped into six types based on gene architecture and sequence identity. Sequence alignment indicates that most GmTPSs contain the conserved aspartate-rich DDX₂D motif, and two clades encoded by TPS-a and TPS-b contain variations of an arginine-rich RRX₈W motif. Quantitative real-time PCR analysis demonstrated that GmTPSs were predominantly expressed in reproductive organs. Heterologous expression followed by enzymatic assay suggested that GmTPS3 functions as a geraniol synthase. We also generated transgenic tobacco plants ectopically expressing GmTPS3. In dual-choice feeding-preference and force-feeding assays, the transgenic tobacco lines expressing GmTPS3 exhibited enhanced resistance to cotton leafworms and an increased level of geraniol. Taken together, these data provide a comprehensive understanding of the TPS family in soybeans and suggest a promising approach to engineering transgenic plants with enhanced insect resistance.     

Plant Terpenoids： Biosynthesis and Ecological Functions

Among plant secondary metabolites terpenolds are a structurally most diverse group; they function as phytoalexins In plant direct defense, or as signals In Indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the Investigation of the ecological role of plant terpenolds. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes Include the synthesis of C5 precursor isopentenyl diphosphate （IPP） and Its allylic isomer dlmethylallyl dlphosphate （DMAPP）, the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases （TPSs） play a key role In volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to Increase terpenoid production. This review mainly summarizes the recent research progress In elucidating the ecological role of terpenoids and characterization of the enzymes Involved in the terpenold biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.