Expanding catalytic promiscuity of a bifunctional terpene synthase through a single mutation-induced change in hydrogen-bond network within the catalytic pocket

Fungal bifunctional terpene synthases(BFTSs)catalyze the formation of numerous di-/sester-/tri-terpenes skeletons.However,the mechanism in controlling the cyclization pattern of terpene scaffolds is rarely deciphered for further application of tuning the catalytic promiscuity of terpene synthases for expanding the chemical space.In this study,we expanded the catalytic promiscuity of Fusarium oxysporum fusoxypene synthase(FoFS)by a single mutation at L89,leading to the production of three new sesterterpenes.Further computational analysis revealed that the reconstitution of the hydrogen-bond(H-bond)network of second-shell residues around the active site of FoFS influences the orientation of the aromatic residue W69 within the first-shell catalytic pocket.Thus,the dynamic orientation of W69 alters the carbocation transport,leading to the production of diverse ring system skeletons.These findings enhance our knowledge on understanding the molecular mechanisms,which could be applied on protein engineering terpene synthases on regulating the terpene skeletons.

Functional characterization of(E)-β-caryophyllene synthase from lima bean and its up-regulation by spider mites and alamethicin

（E）-β-Caryophyllene is a sesquiterpene compound widely distributed in plants and functions in plant defence. However, little is known about the sequence and function of （E）-β-caryophyllene synthase in lima bean （Phaseolus lunatus）. Here, we report a new full-length cDNA （PICAHS） encoding （E）-β-caryophyllene synthase, a possible key enzyme of plant defence. The cDNA of PICAHS contains an open reading frame of 1 761 bp, encoding a protein of 586 amino acids with a predicted mass of 67.95 kDa. The deduced amino acid sequence shows 52% identity with sesquiterpene synthase MtCAHS of Med- icago truncatula. Based on phylogenetic analysis, PICAHS is classified as the terpene synthases （TPS）-a subfamily. The recombinant enzyme, expressed in Escherichia coil, catalysed the formation of a major product （E）-β-caryophyllene （82%） and a minor product a-humulene （18%） from farnesyl dJphosphate. Real-time quantitative PCR （qRT-PCR） analysis found that the PICAHS transcript was significantly up-regulated in leaves after treatment with spider mites and alamethicin （ALA）, suggesting its ecological function in plant defence.

华山松alpha-pinene synthase基因和(-)-limonene synthase基因的克隆及表达

【目的】研究华山松alpha-pinene synthase基因和(-)-limonene synthase基因及其表达特性,为揭示其在华山松抵御华山松大小蠹和蓝变真菌秦岭细粘束孢的入侵机制提供理论依据。【方法】运用PCR和RACE技术克隆华山松alpha-pinene synthase基因和(-)-limonene synthase基因cDNA序列;利用生物信息学方法分析基因的核苷酸序列和编码蛋白的氨基酸序列,并构建系统发育树;通过实时荧光定量PCR技术分析华山松alpha-pinene synthase基因和(-)-limonene synthase基因在MeJA处理和秦岭细粘束孢侵染后的表达差异。【结果】华山松alpha-pinene synthase基因编码区全长为1887 bp,编码的蛋白由628个氨基酸组成,分子质量为71.59 kDa,理论等电点为5.86。华山松(-)-limonene synthase基因编码区全长为1905 bp,编码的蛋白由634个氨基酸组成,分子质量为73.01 kDa,理论等电点为5.97。经实时荧光定量PCR分析,MeJA处理后alpha-pinene synthase基因和(-)-limonene synthase基因在不同时间均被诱导表达,其中alpha-pinene synthase基因表达量在第2天达到最高峰,(-)-limonene synthase基因表达量在第4天达到最高峰;秦岭细粘束孢侵染后alpha-pinene synthase基因和(-)-limonene synthase基因均被诱导表达,但表达量在处理后不同时间有明显差异。结果表明华山松alpha-pinene synthase基因和(-)-limonene synthase基因是响应MeJA处理(模拟华山松大小蠹入侵危害)和秦岭细粘束孢侵染重要的基因。【结论】华山松alpha-pinene synthase基因和(-)-limonene synthase基因能够响应MeJA处理和秦岭细粘束孢侵染,这对进一步揭示寄主华山松抵御华山松大小蠹和蓝变真菌入侵机制提供了理论依据。

石斛属花香物质的合成及相关基因调控研究进展

大部分兰科石斛属植物都具有极高的药用和观赏价值。全球大约有1200~1500种石斛属植物,在我国分布的有76种,主要分布在西南、华南和台湾等地,其中云南最多,有62个种。花香是观赏植物重要的品质性状,随着分子生物学发展,在花香物质的生物合成途径、相关基因调节机制和基因工程方面等取得了较大进展,对近年来报道的石斛属26种植物中17种主要赋香成分和植物花香的生物合成途径、相关酶与基因调节机制的研究成果进行了综述,并对今后的研究和发展方向等进行了展望,旨在为今后植物花香性状的改良提供参考。

基于全基因组的栀子花香成分合成TPS基因系统分析

栀子(Gardenia jasminoides J.Ellis)花色洁白且香气怡人,不仅具有极高的观赏价值,还是重要天然香料来源。萜类是栀子花独特香气的主要组成成分,但合成该类挥发性产物的关键萜类合酶(TPS)尚未鉴定。本研究基于栀子高质量基因组,通过转录组数据、系统发育树和保守结构域分析等,全面挖掘栀子萜类花香成分合成相关的TPS基因。结果显示,栀子基因组共注释到44个GjTPS基因,不均匀分布于11条染色体,其中9个GjTPS参与串联复制事件。所有GjTPS被划分为5个亚家族,其中27个属于被子植物特有的TPS-a、TPS-b和TPS-g分支。结合栀子5个器官的转录组数据及实时荧光定量PCR分析,筛选出5个候选GjTPS基因,其在盛开期的花器官中特异高表达,且含有DDXXD和NSE/DTE活性基序。其中,GjTPS1、GjTPS2、GjTPS3和GjTPS27为TPS-b分支酶,推测为芳樟醇或罗勒烯等栀子主要花香成分合成酶,而TPS-a分支的GjTPS18可能是金合欢烯合酶。

紫丁香花香成分鉴定及关键TPS基因的功能分析

萜烯合成酶(TPS,terpene synthase)基因是萜类化合物生物合成途径中的关键基因,其在植物萜烯代谢中起到重要的作用。本研究以紫丁香(Syringa oblata Lindl.ex Carr.)花瓣为材料,在优化固相微萃取花香化合物条件的基础上,对4个不同花发育时期(花蕾期、初开期、盛开期和衰败期)的花香化合物进行鉴定分析;结合基因组和转录组数据筛选到了SoTPS2和SoTPS3关键候选基因,并对其进行克隆和功能研究。结果表明:(1)最适的萃取条件为30℃萃取40 min;4个花发育时期的花香总释放量呈先升高后下降的趋势,盛花期达到最高;4个时期中萜烯类化合物的释放量占花香总释放量的比例均最高,其中以单萜罗勒烯的释放量最高。(2)SoTPS2和SoTPS3基因编码区长度分别为1731 bp和1779 bp,编码氨基酸分别为576个和592个,编码的蛋白具有Terpene_cyclase_plant_C1保守结构域,属于Isoprenoid_Biosyn_C1超家族;实时荧光定量表明2个基因均在花瓣中表达量最高,在不同花发育时期表达量均呈先升高后下降趋势,在盛开期表达量最高,与单萜罗勒烯的释放量呈正相关。(3)在金鱼草(Antirrhinum majus L.)花瓣中异源瞬时过表达SoTPS2和SoTPS3基因,发现罗勒烯释放量与对照组相比分别显著增加10.91倍和23.67倍。综上表明,紫丁香花香主要成分为单萜类化合物,SoTPS2和SoTPS3可以影响紫丁香单萜化合物,尤其是罗勒烯的合成。

The chromosome-level genome of double-petal phenotype jasmine provides insights into the biosynthesis of floral scent

Jasmine(Jasminum sambac Aiton)is a well-known cultivated plant species for its fragrant flowers used in the perfume industry and cosmetics.However,the genetic basis of its floral scent is largely unknown.In this study,using PacBio,Illumina,10×Genomics and highthroughput chromosome conformation capture(Hi-C)sequencing technologies,a high-quality chromosome-level reference genome for J.sambac was obtained,exploiting a double-petal phenotype cultivar‘Shuangbanmoli’(JSSB).The results showed that the final assembled genome of JSSB is 580.33 Mb in size(contig N50=1.05 Mb;scaffold N50=45.07 Mb)with a total of 39618 predicted protein-coding genes.Our analyses revealed that the JSSB genome has undergone an ancient whole-genome duplication(WGD)event at 91.68 million years ago(Mya).It was estimated that J.sambac diverged from the lineage leading to Olea europaea and Osmanthus fragrans about 28.8 Mya.On the basis of a combination of genomic,transcriptomic and metabolomic analyses,a range of floral scent volatiles and genes were identified involved in the benzenoid/phenylpropanoid and terpenoid biosynthesis pathways.The results provide new insights into the molecular mechanism of its fragrance biosynthesis in jasmine.

澳洲香水石斛花香成分及DeTPS6的克隆与表达

花香是评价石斛商业价值的重要性状之一。为探究石斛花香物质的形成机理,本研究采用顶空固相微萃取-气相色谱-质谱联用法(HS-SPME-GC-MS)、转录组测序和PCR技术分析了不同花期澳洲香水石斛花香物质变化及其与萜类合成关键酶基因表达间的关系。结果表明,澳洲香水石斛中共检测到17种花香物质,其中1,4-二甲氧基苯、α-蒎烯和苯乙醇的含量相对较高,盛花期的花香释放量呈现明显的昼夜节律变化和组织特异性。基于转录组数据,筛选到77个与萜类合成相关的差异表达基因(DEGs),并成功克隆DeTPS6的全长,为1749 bp。DeTPS6蛋白具有萜类合成酶家族的保守结构域,属于TPS-b亚家族。亚细胞定位分析结果显示,DeTPS6定位于胞质。实时荧光定量PCR(qRT-PCR)分析结果显示,DeTPS6在澳洲香水石斛的盛花期中高表达,且呈现显著的昼升夜降的变化趋势,与花香物质的含量呈正相关。本研究为DeTPS6生物学功能的探讨提供了理论基础。

栀子TPS基因家族鉴定及与萜类物质代谢的相关性分析

TPS基因家族是萜类化合物合成的末端关键酶,在栀子花香的形成中起重要作用。为了明确栀子TPS基因家族成员的基本特征,利用生物信息学方法对栀子TPS基因进行家族成员鉴定;通过外源激素喷施试验进行转录组测序,结合顶空固相微萃取和气相色谱-质谱联用技术,分析不同外源激素浓度下栀子花朵TPS基因家族成员表达水平和代谢物含量变化关系。结果表明:从栀子基因组中共鉴定获得GjTPS家族成员41个,编码氨基酸380~849个,含有外显子5~15个不等,GjTPS家族成员定位在叶绿体中,并且不均匀地分布在10条染色体上;共线性分析表明栀子与其同科植物中粒咖啡的TPS基因有更近的亲缘关系。系统发育分析结果显示GjTPS基因分为5个亚家族,TPS-a、TPS-b亚家族包含了大多数GjTPS家族成员。GjTPS家族成员的大多数启动子顺式作用元件在植物激素响应类别中,并且包含MYC基序的茉莉酸甲酯响应元件是其中大类;通过转录组数据得到GjTPS02、GjTPS12、GjTPS14、GjTPS20、GjTPS21、GjTPS22这6个基因在各处理的表达量较高。对基因表达量与萜类物质含量变化进行相关性分析,发现1.25 mmol·L^(-1)的茉莉酸甲酯有助于栀子TPS基因家族成员的表达及萜类物质的释放;GjTPS12、GjTPS14、GjTPS20、GjTPS21、GjTPS22这5个基因可能参与栀子花香调控。本研究结果可为后续栀子TPS基因家族功能特性研究提供借鉴。

油楠SgTPS7的克隆及其在萜类生物合成和非生物胁迫中的功能

【目的】萜类化合物在植物生理和防御中发挥着重要作用。萜类合成酶基因(TPS)是萜类生物合成途径中的关键酶,热带泌油树种油楠(Sindora glabra)茎部含有大量萜类树脂油,探究油楠SgTPS在萜类物质合成和非生物胁迫响应中的功能,为通过合成生物学生产萜类提供重要基因资源,有助于拓展植物萜类合成途径与调控机制的认识。【方法】利用油楠基因组和转录组数据从油楠茎部组织克隆SgTPS7,并对其进行生物信息学分析;表达纯化SgTPS7蛋白利用GC-MS鉴定其催化功能;采用RT-qPCR技术分析SgTPS7的表达模式。【结果】油楠SgTPS7编码区序列全长1650 bp。经同源比对分析发现,SgTPS7与苏木亚科古巴香胶树(Copaifera officinalis)CoTPS2的相似性最高,达92.3%,与其他科属植物的亲缘关系较远,属于苏木亚科特异的TPS基因。体外酶活试验表明,SgTPS7主要催化法尼基焦磷酸产生大根香叶烯D,同时催化香叶基焦磷酸产生芳樟醇。在非生物胁迫处理条件下,SgTPS7的表达差异显著。高温胁迫处理后,SgTPS7在叶片和茎中的表达均在24 h达到峰值,而在根部的表达则呈下降趋势;长期干旱处理显著影响SgTPS7的表达,在处理5 d后SgTPS7在叶和茎中的表达均达到最高,而根部SgTPS7的表达在10 d后达到最高。【结论】SgTPS7为多功能萜类合成酶基因,其在响应高温和干旱两种非生物胁迫中可能具有重要的作用。

青钱柳萜烯合成酶TPS家族鉴定分析

萜烯是植物中一类重要的化合物,不仅在细胞膜上扮演着电子传递的角色,还参与植物光合作用,并作为植物激素的主要成分。萜烯合成酶TPS是合成萜烯的关键酶,对于植物生长和抵御疾病具有重要意义。研究对青钱柳的TPS基因家族进行了全面的鉴定和分析,发现青钱柳共有63个TPS基因家族成员,这些基因的蛋白质长度在260至854个氨基酸之间,并且分布在青钱柳的11条染色体上。通过启动子顺式作用元件分析,预测青钱柳的TPS基因家族可能参与了多种生物学反应。该研究为进一步探究青钱柳TPS的生理功能和应对环境胁迫的机制提供了理论支持。

萜类化合物的非常规生物合成研究进展

萜类化合物是自然界中广泛存在的一类具有重要生理功能和显著生物活性的天然产物,在食品、医疗及日化行业有着广泛的应用。在萜类化合物的生物合成途径中,萜类合酶往往决定了萜类碳骨架的种类和结构新颖性,细胞色素P450酶等后修饰酶则可对碳骨架进行多种修饰,最终形成结构和功能都具有丰富多样性的萜类化合物。近年来,随着基因测序技术与合成生物学的发展,大量植物和微生物来源的萜类生物合成酶被表征,令人兴奋的是,其中包含一些与经典萜类合酶不同的非常规萜类合酶,它们亦可催化生成独特的环化萜类骨架。与此同时,利用组合生物合成等策略,人们创造了许多新颖的非天然萜类化合物,进一步丰富了萜类资源库。本文综述了近5年在非常规萜类环化酶与组合生物合成途径等方面取得的最新研究进展,以期为未来新型萜类化合物的发现和生物合成提供启示。本文首先综述了新发现的具有萜类环化功能的新酶,包含Ⅰ型萜类合酶新亚族、非角鲨烯来源三萜合酶、UbiA型萜类环化酶、细胞色素P450氧化酶、甲基转移酶、钒依赖卤素过氧化物酶、卤代酸脱卤酶等,同时还对其序列、功能和可能的环化机制进行了介绍,有助于理解自然界中萜类生物合成酶的进化起源和发现新颖萜类化合物。然后,本文介绍了非常规萜类衍生物的组合生物合成,通过将萜类合酶与甲基转移酶、天然或人工细胞色素P450氧化酶进行组合,产生了一系列包含非常规C_(11)、C_(16)骨架以及具有不同氧化形式的非天然萜类化合物,可为往后萜类化合物的结构创新研究带来启发。这些新颖酶元件的挖掘与新型组合生物合成途径的构建,将进一步拓宽萜类化合物的结构多样性和化学空间,有望为临床萜类药物研发提供更多的潜在小分子。

基因组深度挖掘驱动微生物萜类化合物高效发现

萜类天然产物广泛分布于动物(包括海洋无脊椎动物)、植物、微生物中,具有复杂的化学结构和丰富的生物活性。人们通过从植物和微生物中直接分离提取的方式获得了大量萜类天然产物,然而随着越来越多化合物被发现,使用基于自然筛选的传统挖掘方式很难获得新的萜类天然产物。随着基因组测序技术和合成生物学使能技术的不断发展,我们进入了基因组挖掘驱动天然产物发现的时代,萜类天然产物的挖掘也进入了“井喷式”发现新阶段。针对基因组挖掘在微生物萜类天然产物发现方面的应用,本文综述了近年来使用的主要研究策略和最新研究进展,介绍了多种高效微生物底盘、基因组深度挖掘策略、人工智能与自动化平台等驱动的萜类化合物挖掘的最新研究进展,讨论了基因组挖掘萜类天然产物面临的挑战,展望了未来萜类化合物创新发现的发展趋势。通过在多种微生物中强化前体供应途径,人们打造了多个萜类化合物合成底盘,突破了异源合成萜类天然产物时“产量低”和“产物难获取”的瓶颈;针对萜类天然产物生物合成基因簇或萜类合酶进行深度挖掘,可以有效地解决“重复发现”和“集中度低”的难题;随着人工智能和自动化技术在合成生物学领域的发展和应用,萜类化合物的发现也进入了高通量智能发现时期,显著地改善了“研究通量低”的现状,高效获得了大量新结构萜类天然产物。在未来,更多萜类化合物将开发成药物、进入工业化生产应用,更多萜类“暗物质”会走进我们视野。

银杏GbTPS3基因克隆和功能表征

目的:基于银杏转录组深度测序,通过构建原核表达系统工程菌株,系统研究银杏中萜类合成酶的功能。方法:根据蛋白功能的预测,利用不同的原核表达体系进行功能表征,GC-MS检测分析。结果:银杏GbTPS3基因可以同时表达两个单萜化合物(龙脑和2-蒎烷醇)。结论:银杏GbTPS3为Class I型萜类合成酶,主要负责参与萜类化合物起始的合成反应。其特点是碳骨架重排的随机性可以产生不同的碳正离子中间体,在底物唯一的情况下,单一萜类合成酶往往会产生多种产物。

The chromosome-scale genome of Phoebe bournei reveals contrasting fates of terpene synthase (TPS)-a and TPS-b subfamilies

Terpenoids,including aromatic volatile monoterpenoids and sesquiterpenoids,function in defense against pathogens and herbivores.Phoebe trees are remarkable for their scented wood and decay resistance.Unlike other Lauraceae species investigated to date,Phoebe species predominantly accumulate sesquiterpenoids instead of monoterpenoids.Limited genomic data restrict the elucidation of terpenoid variation and functions.Here,we present a chromosome-scale genome assembly of a Lauraceae tree,Phoebe bournei,and identify 72 full-length terpene synthase(TPS)genes.Genome-level comparison shows pervasive lineage-specific duplication and contraction of TPS subfamilies,which have contributed to the extreme terpenoid variation within Lauraceae species.Although the TPS-a and TPS-b subfamilies were both expanded via tandem duplication in P.bournei,more TPS-a copies were retained and constitutively expressed,whereas more TPS-b copies were lost.The TPS-a genes on chromosome 8 functionally diverged to synthesize eight highly accumulated sesquiterpenes in P.bournei.The essential oil of P.bournei and its main component,b-caryophyllene,exhibited antifungal activities against the three most widespread canker pathogens of trees.The TPS-a and TPS-b subfamilies have experienced contrasting fates over the evolution of P.bournei.The abundant sesquiterpenoids produced by TPS-a proteins contribute to the excellent pathogen resistance of P.bournei trees.Overall,this study sheds light on the evolution and adaptation of terpenoids in Lauraceae and provides valuable resources for boosting plant immunity against pathogens in various trees and crops.

SNARE-RNAi Results in Higher Terpene Emission from Ectopically Expressed Caryophyllene Synthase in Nicotiana benthamiana

Plants produce numerous terpenes and much effort has been dedicated to the identification and charac- terization of the terpene biosynthetic genes. However, little is known about how terpenes are transported within the cell and from the cell into the apoplast. To investigate a putative role of vesicle fusion in this pro- cess, we used Agrobacterium tumefaciens-mediated transient coexpression in Nicotiana benthamiana of an MtVAMP721e-RNAi construct （Vi） with either a caryophyllene synthase or a linalool synthase, respec- tively. Headspace analysis of the leaves showed that caryophyllene or linalool emission increased about five-fold when N. benthamiana VAMP72 function was blocked. RNA sequencing and protein ubiquitination analysis of the agroinflltrated N. benthamiana leaf extracts suggested that increased terpene emissions may be attributed to proteasome malfunction based on three observations： leaves with TPS＋Vi showed （1） a higher level of a DsRed marker protein, （2） a higher level of ubiquitinated proteins, and （3） coordinated induced expression of multiple proteasome genes, presumably caused by the lack of proteasome- mediated feedback regulation. However, caryophyllene or linalool did not inhibit proteasome-related pro- tease activity in the in vitro assays. While the results are not conclusive for a role of vesicle fusion in terpene transport, they do show a strong interaction between inhibition of vesicle fusion and ectopic expression of certain terpenes. The results have potential applications in metabolic engineering.

Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases

Class I terpene synthase(TPS)generates bioactive terpenoids with diverse backbones.Sesterterpene synthase(sester-TPS,C25),a branch of class I TPSs,was recently identified in Brassicaceae.However,the catalytic mechanisms of sester-TPSs are not fully understood.Here,we first identified three nonclustered functional sester-TPSs(AtTPS06,AtTPS22,and AtTPS29)in Arabidopsis thaliana.AtTPS06 utilizes a type-B cyclization mechanism,whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism.We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs.We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism:(1)mainly due to the outward shift of helix G,plant sester-TPSs have a larger catalytic pocket than do mono-,sesqui-,and di-TPSs to accommodate GFPP;(2)type-A sester-TPSs have more aromatic residues(five or six)in their catalytic pocket than classic TPSs(two or three),which also determines whether the type-A or type-B cyclization mechanism is active;and(3)the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs.Altogether,this study improves our understanding of the catalytic mechanism of plant sester-TPS,which ultimately enables the rational engineering of sesterterpenoids for future applications.

A likely autotetraploidization event shaped the Chinese mahogany(Toona sinensis)genome

Chinese mahogany(Toona sinensis) is of considerable medical and economic importance, and its genome has been deciphered. However, the process underlying its polyploidy is unclear, and the chromosomal evolutionary trajectory is poorly understood. Here, by reanalysing the T.sinensis genome, we found evidence of a tetraploidization event(T. sinensis special tetraploidization, TST) that occurred approximately 15-17 million years ago(MYA) after the core eudicot-common hexaploidization(ECH or gamma) event. We characterized the synonymous nucleotide substitution rates(Ks values) of collinear genes and found that T. sinensis genes affected by the TST evolve at a slower rate than Acer yangbiense genes. Furthermore, we identified homologous genes related to polyploidization and speciation and constructed multiple alignments with different reference genomes. Notably, the significant balance of gene retention and loss characterized in the two TST-derived subgenomes suggests an autopolyploid nature of the TST. Moreover, we deduced the chromosomal karyotypes of the two subgenomes and identified 7chromosomal fusions that have shaped the T. sinensis genome;more information is available on a newly constructed karyotype platform(http://www.cgrpoee.top/Toona_sinensis/index.html). The T. sinensis genome preserves the ancestral chromosome structure of dicotyledons well and could serve as a good reference for understanding genomic changes in other Meliaceae and Sapindales plants. In addition, we verified that tandem duplication and the ECH have promoted the expansion of terpene synthase(TPS) genes;conversely, the TST seems to have inhibited expansion of these genes. This present effort has clarified the polyploidy events of the T. sinensis genome, filled gaps in the history of karyotype evolution, and laid a solid foundation for further genomic studies in the Meliaceae research community and beyond.

香叶醇及其糖苷在茶树中的积累和对红茶香气的贡献

采用液相质谱联用仪LC-MS、气相质谱联用仪GC-MS分析不同茶树品种、不同组织器官、不同时间香叶醇及其糖苷含量的变化,结果显示:香叶基樱草糖苷在中国变种(Camellia sinensis var. sinensis)中的含量显著高于阿萨姆变种(C.sinensis var. assamica)。香叶基樱草糖苷在茶树幼嫩叶片中含量最高,在根中的含量最低,总体表现出嫩叶>茎>花>根。3月份茶树嫩梢中的香叶醇积累量最高,随着月份的增加而逐渐降低。在祁门红茶加工过程中,香叶基樱草糖苷含量在揉捻阶段显著降低。黄山地区6个品种红茶中,皖茶4号红茶中的香叶醇含量最高;6个品种红茶茶汤中香叶醇的气味活性值(OAV)均>1,分布范围3.93~28.38。Pearson相关性分析显示,有7个萜类合成酶候选基因表达量与香叶基樱草糖苷含量变化呈显著相关。本研究结果为进一步探究香叶醇积累差异成因及形成机制提供理论参考。

Enzyme engineering in microbial biosynthesis of terpenoids:progress and perspectives

Terpenoids,known for their structural and functional diversity,are highly valued,especially in food,cosmetics,and cleaning products.Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids.However,the natural enzymes involved in the synthesis of terpenoids have problems such as low activity,poor specificity,and insufficient stability,which limit the biosynthesis efficiency.Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids.By modifying the structures and functions of key enzymes,researchers have significantly improved the catalytic activity,specificity,and stability of enzymes related to terpenoid synthesis,providing strong support for the sustainable production of terpenoids.This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids,including improving enzyme activity and stability,changing specificity,and promoting mass transfer through multi-enzyme collaboration.Additionally,this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.