2023, Vol. 50
表1(Table 1)
微生物异戊烯基转移酶研究进展
扩展功能
文章信息
- 王云娇, 张宁, 李炳学
- WANG Yunjiao, ZHANG Ning, LI Bingxue
- 微生物异戊烯基转移酶研究进展
- Advances in the prenyltransferases in microorganisms
- 微生物学通报, 2023, 50(10): 4639-4654
- Microbiology China, 2023, 50(10): 4639-4654
- DOI: 10.13344/j.microbiol.china.230078
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文章历史
- 收稿日期: 2023-02-07
- 接受日期: 2023-04-04
- 网络首发日期: 2023-05-06
王云娇, 张宁, 李炳学. 微生物异戊烯基转移酶研究进展[J]. 微生物学通报, 2023, 50(10): 4639-4654.
WANG Yunjiao, ZHANG Ning, LI Bingxue. Advances in the prenyltransferases in microorganisms[J]. Microbiology China, 2023, 50(10): 4639-4654.
微生物异戊烯基转移酶研究进展
1. 沈阳农业大学生物科学技术学院, 辽宁 沈阳 110866;
2. 沈阳农业大学土地与环境学院, 辽宁 沈阳 110866
2. 沈阳农业大学土地与环境学院, 辽宁 沈阳 110866
摘要: 类异戊二烯(isoprenoids)是最具化学多样性的一种天然分子家族，参与微生物中类胡萝卜素、甾醇等次生代谢物的合成，这类物质在工业大规模生产中具有广阔的商业前景。异戊烯基转移酶是类异戊二烯合成途径中的关键酶，其活性及编码基因的转录水平参与调节次生代谢物产量，在类异戊二烯化合物生物合成途径中发挥重要作用。本文重点归纳了微生物中异戊烯基转移酶的发现与鉴定，分析其结构特点与链长决定机制，讨论异戊烯基转移酶家族之间的复杂进化，概述酶基因表达调控的应用以及生物合成研究现状，为深入研究异戊烯基转移酶作用机理及各领域中的应用提供思路。
关键词:
类异戊二烯 异戊烯基转移酶 进化分析 转录调控
Advances in the prenyltransferases in microorganisms
1. College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, Liaoning, China;
2. College of Land and Environment, Shenyang Agricultural University, Shenyang 110866, Liaoning, China
2. College of Land and Environment, Shenyang Agricultural University, Shenyang 110866, Liaoning, China
Abstract: Isoprenoids, a family of natural molecules with high chemical diversity are involved in the synthesis of secondary metabolites such as carotenoids and sterols in microorganisms, demonstrating a promising commercial prospect for industrial production. Prenyltransferase (PT), a key enzyme in the synthesis of isoprenoids, regulates the production of secondary metabolites. In this paper, we review the recent studies about the isolation and identification of prenyltransferases in microorganisms and introduce their structural features, chain length determination mechanisms, and complex evolution. Furthermore, we overview the applications of the regulation of enzyme gene expression and the current status of research on biosynthesis, aiming to provide ideas for in-depth study of the mechanism of action and applications of prenyltransferases.
Keywords:
isoprenoids prenyltransferase phylogenetic analysis transcriptional regulation
类异戊二烯化合物又称萜类化合物，是由2个或2个以上的异戊二烯单元构成。类异戊二烯家族是最古老、存在种类最多的一类天然化合物，截至目前，已知的类异戊二烯化合物有80 000多种[1]。其结构多样化使它们具有了许多重要生化功能，例如：作为电子传递链中醌类化合物前体；构成古细菌中细胞膜；参与蛋白的修饰及靶向定位；参与类胡萝卜素的合成；参与单萜、倍半萜、二萜等植物防御化合物的合成，而且其商业用途也在不断扩大，包括其他潜在应用如杀虫剂、可再生聚合物和生物燃料等[2]。这类天然化合物的合成通常被分为4个阶段(图 1)，第1阶段，合成基本的异戊二烯单元；第2阶段，2个及2个以上的异戊二烯单元通过头尾缩合生成不同萜类前体物质；第3阶段，不同碳原子数的萜类化合物的合成，如单萜、二萜和倍半萜等，即萜类核心骨架的合成过程；第4阶段，在不同修饰酶催化下合成具有结构多样性和多种生物学功能的萜类化合物[3]。
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| 图 1 萜类化合物合成途径 Figure 1 Biosynthetic pathway of terpenoids. |
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类异戊二烯化合物在自然界中普遍存在，从细胞成分到次生代谢产物，它们在各个领域的应用意味着与工业有着紧密的相关性，如红豆杉中具有抗癌药用价值的紫杉醇的合成，青蒿中有抗疟功能的青蒿素以及合成橡胶等[4]，这类化合物的利用价值很高，在合成过程中都需要异戊烯基转移酶的参与，植物的生长周期、合成工艺成本以及市场上产量的需求通常会成为一大难题。目前，由于微生物的生物合成工艺成本低、操作便捷、周期短等特点，逐渐替代了传统合成工艺。例如，白杨中异戊烯基转移酶基因IspS引入大肠杆菌对其进行优化，重组菌中异戊二烯产物干重可达60 μg/L[5]，改变甲羟戊酸途径中基因序列使mRNA的稳定性和加工过程发生变化，获得甲羟戊酸产量提高7倍的菌株等[6]。因此，对微生物异戊烯基转移酶的研究十分有必要。
1 类异戊二烯合成途径概述Ruzicka等[7]在1953年提出的“异戊二烯定则”中提到所有天然萜类都源自简单的5C结构单元：异戊烯基焦磷酸(isoprenyl diphosphate, IPP)及其同分异构体二甲基烯丙基焦磷酸(dimethylallyl diphosphate, DMAPP)。IPP和DMAPP有两种独立的非同源合成途径，一种称为甲羟戊酸途径(mevalonate pathway, MVA途径)，另一种为2C-甲基-d-赤藓糖醇-4-磷酸途径(1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d- erythritol 4-phosphate pathway, DOXP/MEP途径，也称为非甲羟戊酸途径)[8-9]，古细菌、真菌和动物通过MVA途径合成类异戊二烯化合物，DOXP/MEP途径存在于大多数细菌和原生生物中，高等植物则可以同时利用这两种途径[10-11]。MVA途径始于乙酰辅酶A在硫代酶的2次缩合下生成乙酰乙酰辅酶A (acetoacetyl-CoA)，然后由3-羟基-3-甲基戊二酰辅酶A合酶(3-hydroxy- 3-methylglutary CoA synthase, HMGS)催化与乙酰辅酶A的第三分子缩合，生成3-羟基-3-甲基戊二酰辅酶A (HMG-CoA)，随后被HMG-CoA还原酶(3-hydroxy-3-methyl glutaryl coenzyme A reductase, HMGR)还原为甲羟戊酸，甲羟戊酸通过磷酸化、脱羧最终得到IPP[12]。DOXP/MEP途径中，IPP和DMAPP的合成始于二磷酸硫胺在DOXP合酶作用下缩合形成DOXP。DOXP被DOXP还原异构酶(1-deoxy-d-xylulose-5- phosphate reductoisomerase, DXR)还原为MEP；随后被修饰、磷酸化，通过2-c-甲基-d-赤藓糖醇2, 4-环二磷酸合酶合成2-c-甲基-d-赤藓糖醇2, 4-环二磷酸(2-C-methyl-d-erythritol 2, 4-cyclodiphosphate, ME-cPP)，被羟甲基丁烯基4-二磷酸合酶(4-hydroxy-3-methylbut-2-enyl diphosphate synthase, HDS)催化生成羟甲基丁烯基4-二磷酸[1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate, HMBPP]；最后，HMBPP还原酶[4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase, HDR]同时合成IPP和DMAPP[13]，该途径最初被命名为非甲羟戊酸途径或Rohmer途径。鉴定出该途径第一步后，该途径名称以第一个中间体[Deoxyxylulose(DX)5-phosphate, DOXP]命名，然而按照与MVA途径相同的命名规则，以该途径中目前被认为是第一个前体的甲基赤藓糖醇4-磷酸(methylerythritol 4-phosphate, MEP)来命名更被大众所接受[14]。
一分子5C异戊二烯单元DMAPP与多个IPP缩合，生成不同碳链长度的萜类化合物前体物质牻牛儿基焦磷酸(geranyl diphosphate, GPP，也称为香叶基焦磷酸)、法尼基焦磷酸(farnesyl diphosphate, FPP)、牻牛儿基牻牛儿基焦磷酸(geranylgeranyl diphosphate, GGPP，也称香叶基香叶基二磷酸)、香叶基法尼基焦磷酸(geranylfarnesyl diphosphate, GFPP)[15]。在不同萜类环化酶的作用下将萜类化合物的前体物质进行环化，生成单萜(monoternope, 10C)、倍半萜(sesquiterpene, 15C)、二萜(diterpene, 20C)、三萜(triterpene, 30C)、四萜(tetraterpene, 40C)等萜类核心骨架[16]。随后这些不同碳原子数萜类化合物核心骨架经过细胞色素P450、羰基化酶、酰基转移酶等修饰酶的催化，合成不同结构具有多种生物学功能的萜类化合物[17]。
2 异戊烯基转移酶所有异戊二烯化合物的骨架都是由类异戊二烯合酶(isoprenyl diphosphate synthases, IPPS或IDS)又称异戊烯基转移酶(prenyltransferase, PT)催化合成或修饰的[18]。该酶家族各个成员都有不同的特点。根据催化产物链延伸过程中产生的不同立体化学结构cis(Z)和trans(E)，分为顺式异戊烯基转移酶(cis-prenyltransferase)和反式异戊烯基转移酶(trans-prenyltransferase)[19]，E家族(反式异戊烯基转移酶家族)成员通常负责合成途径早期中较短碳链异戊二烯焦磷酸，Z家族(顺式异戊烯基转移酶家族)成员合成更长链异戊二烯焦磷酸[20]。根据合成产物的碳链长度，反式异戊烯基转移酶又可以进一步分为短链(short-chain prenyl diphosphate synthases, SCIPPS; C10−C25)、中链(medium-chain prenyl diphosphate synthases, MCIPP; C30−C35)和长链(long-chain prenyl diphosphate synthases, LCIPPS; C40−C50)异戊烯基转移酶[21]。这些异戊烯基转移酶合成的类异戊二烯化合物可以作为生命活动中必需的初级代谢物和次生代谢物前体，具有许多商业、农业和药理价值。目前对异戊二烯类物质的研究主要集中在高等植物中，其合成途径已基本清晰，但真菌、昆虫等生物中异戊二烯类物质合成的相关研究还处于摸索阶段，因此，对合成途径中酶基因的克隆表达与功能验证则是该研究必不可少的基础。在微生物中，主要负责催化合成类异戊二烯化合物合成的异戊烯基转移酶包括法尼基焦磷酸合酶(farnesyl diphosphate synthase, FPPS)、牻牛儿基牻牛儿基焦磷酸合酶(geranylgeranyl diphosphate synthase, GGPPS)和香叶基法尼基焦磷酸合酶(geranylfarnesyl diphosphate synthase, GFPPS)。
2.1 法尼基焦磷酸合酶(FPPS)法尼基焦磷酸合酶(farnesyl diphosphate synthase, FPPS, EC 2.5.1.10)是MVA途径中重要的分支酶，负责催化DMAPP与IPP连续缩合合成FPP，FPP是一些代谢物质如甾醇、辅酶Q、类胡萝卜素等的前体[22]。1975年，Eberhardt等在酿酒酵母中纯化出同源转移酶[23]，1994年首次获得了X晶体射线结构的异戊二烯转移酶[24]，为陆续在不同物种中发现的异戊烯基转移酶提供参考进行同源建模。这些先驱者的探索为FPPS的后续研究奠定了基础。目前已发现的FPPS为亚基大小为32−44 kDa的同源二聚体，在微生物细胞质中以单拷贝的形式表达[25]。随后对该酶的催化反应过程进行探索发现，FPPS的活性需要一定浓度二价阳离子的参与，如细菌Methanobacterium thermoautotrophicum中FPPS在Mg2+浓度为10 mmol/L时表现出的活性最大，当Mn2+在浓度低于2.5 mmol/L时比Mg2+更有效，但较高浓度的Mn2+抑制活性[26]。
2.2 牻牛儿基牻牛儿基焦磷酸合酶(GGPPS)牻牛儿基牻牛儿基焦磷酸合酶(geranylgeranyl diphosphate synthases, GGPPS, EC 2.5.1.29)，也称香叶基香叶基焦磷酸合酶，催化IPP连续加成DMAPP、GPP和FPP合成GGPP，为类胡萝卜素、二萜类化合物、叶绿素、泛醌和牻牛儿基牻牛儿基化蛋白等提供必需的前体物质，但酵母与哺乳动物中的GGPPS接受FPP作为烯丙基底物[27]。目前GGPPS已在古菌、细菌及真核生物中分离获得。许多类胡萝卜素合成酶基因通过在大肠杆菌中异源表达而产生色素来验证基因功能，牻牛儿基牻牛儿基焦磷酸合酶参与合成类胡萝卜素且作为色素合成通路中关键限速酶之一，欧文氏菌(Erwinia herbicola)中参与类胡萝卜素合成关键基因crtE、crtI和crtB与载体pACYC184构建了重组质粒pAC-LYC[28]。大肠杆菌中缺乏合成20C的GGPPS，且GGPPS必须与其他类胡萝卜素合成酶基因异源表达才可以在大肠杆菌中合成并积累40C的类胡萝卜素[29]，Sandmann等[30]克隆了粗糙脉孢霉(Neurospora crassa)中牻牛儿基牻牛儿基焦磷酸合酶基因al-3取代重组质粒PAC-LYC中crtE，转化到大肠杆菌中后观察到了色素的积累，异源互补验证了Al-3具有CrtE酶功能。大部分掷孢酵母可以积累类胡萝卜素，魏娜等[31]在草莓果实上发现并分离了一株高产类胡萝卜素的掷孢酵母Sporobolomyces pararoseus (GenBank登录号为HM749332)，命名为NGR。Yan等[32]对NGR中crtE基因进行了分离克隆；Li等通过构建异源互补检测体系验证NGR中crtE是参与类胡萝卜素合成的关键基因之一，阻碍其表达将不能合成GGPP，导致下游八氢番茄红素合成酶/番茄红素环化酶基因ctrtYB和八氢番茄红素去饱和酶基因crtI编码的蛋白无法利用GGPP最终合成番茄红素[33]。此外，NGR中CrtE主要利用FPP为底物合成GGPP[32]，但NGR中FPPS基因功能尚未被鉴定，GGPPS与FPPS蛋白间相互作用的关系也有待进一步探讨。
2.3 香叶基法尼基焦磷酸合酶(GFPPS)香叶基法尼基焦磷酸合酶(geranylfarnesyl diphosphate synthases, GFPPS)，最初从嗜盐杆菌Natronomonas pharaonis DSM 2160中克隆出[34]，并在嗜盐杆菌膜上分离出含有C20−C25的类异戊二烯化合物，通常以GGPP为底物时活性最高[35]。随后陆续从细菌中被分离出来(表 1)，GFPPS主要参与二萜化合物的合成，产物GFPP是二倍半萜的前体物质。
表 1 部分已分离与鉴定的异戊烯基转移酶基因
Table 1 Isolation and identification of partial isopentenyl transferase genes
| Gene | Organism | GenBank accession No.a | References |
| AtFPPS | Arabidopsis thaliana | AAB07248 | [36] |
| MrFPPS | Matricaria recutita (L.) | AHN96159 | [37] |
| CrFPPS | Catharanthus roseus | ADO95193 | [38] |
| ispA | Escherichia coli | BAA00599 | [39] |
| GsFPPS | Geobacillus stearothermophilus | BAA02551 | [40] |
| MtFPPS | Mycobacterium tuberculosis H37Rv | WP_157154909 | [41] |
| erg20 | Saccharomyces cerevisiae | AAA34606 | [42] |
| PrFPPS | Phaffia rhodozyma | KJ140284 | [43] |
| erg20 | Trichoderma reesei | AFX82678 | [44] |
| HsFPPS | Homo sapiens | AAA52423 | [45] |
| GgFPPS | Gallus gallus | P08836 | [24] |
| LtuGGPPS | Liriodendron tulipifera | UOO00991 | [46] |
| AtGGPPS | Arabidopsis thaliana | AAA32797 | [47] |
| idsA | Corynebacterium glutamicum | WP_220312883 | [48] |
| GGPPS | Deinococcus radiodurans | WP_010888034 | [49] |
| NdGGPPS | Nonlabens dokdonensis DSW-6 | 6KD7_A | [50] |
| DbGGPS | Dunaliella salina | APW83741 | [51] |
| GaGGPS | Geoglobus acetivorans | A0A0A7GEY4 | [52] |
| BTS1 | Saccharomyces cerevisiae | Q12051 | [53] |
| NpGFPS | Natronomonas pharaonis DSM 2160 | Q3IPL1 | [34] |
| ApGFPPS | Aeropyrum pernix | BAA88983 | [35] |
| MmGFPPS | Methanosarcina mazei Go1 | Q8PYS1 | [54] |
| a：不同物种的异戊烯基转移酶基因登录号从NCBI获得 a: Isopentenyl transferase genes accessions of different species were from NCBI (https://www.ncbi.nlm.nih.gov/). |
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在异戊烯基转移酶家族中，法尼基焦磷酸合酶基因与牻牛儿基牻牛儿基焦磷酸合酶基因在微生物中的研究较为广泛，通常利用构建原核表达体系、实时荧光定量、体外酶活反应以及LC-MS或GC-MS等方法，结合生物信息学分析验证基因功能[55-57]。
2.4 蛋白异戊烯基转移酶从广义上而言，异戊烯基转移酶分为两大类，除上述提到的类异戊二烯合酶，还包括蛋白异戊烯基转移酶(protein prenyltransferase)[58]，这类酶将类异戊二烯基团共价添加到羧基末端或附近的半胱氨酸残基，这种蛋白质修饰方式称为蛋白质异戊二烯化(prenylation of proteins)[59]，广泛存在于真核细胞中。异戊二烯化修饰主要包括2种：(1) 法尼基蛋白转移酶(farnesyl- transferase, FTase, EC 2.5.1.58)催化的法尼基化修饰；(2) 牻牛儿基牻牛儿基蛋白转移酶Ⅰ (geranylgeranyl-transferase Ⅰ, GGTase Ⅰ, EC 2.5.1.59)和牻牛儿基牻牛儿基蛋白转移酶Ⅱ (geranylgeranyl-transferase Ⅱ, GGTase Ⅱ, EC 2.5.1.60)催化的牻牛儿基牻牛儿基化修饰[60]。FTase和GGTase Ⅰ是由相同分子量的α亚基和相似结构的β亚基构成的异源二聚体[61]，可以识别含有CAAX基序的蛋白(C：半胱氨酸，A：脂肪族氨基酸，X：任意氨基酸)，其中X残基是决定蛋白异戊二烯化修饰方式的关键，当X为丝氨酸、甲硫氨酸、丙氨酸和谷氨酰胺时，蛋白被法尼基化修饰；当X为亮氨酸或异亮氨酸时，该蛋白被牻牛儿基牻牛儿基化；当X为苯丙氨酸时，蛋白既可被法尼基化修饰又可被牻牛儿基牻牛儿基化修饰[62]，而被GGTase Ⅱ识别修饰的蛋白质羧基端结构是CXC或者GGGCCC (C：半胱氨酸，G：甘氨酸，X：任意氨基酸)。其中被异戊二烯化修饰的最大的蛋白家族是小G蛋白超家族(也被称为GTPases)，GTPase中的Ras亚家族主要负责调节基因表达、Rho/Rac调节细胞骨架重组和基因表达，已有研究揭示，Ras蛋白必须经过法尼基化修饰才可以定位到细胞膜去促进肿瘤的发生[63]；Rab调节细胞内囊泡运输等[64] (更多的被修饰蛋白见表 2)。但FTase与GGTase Ⅰ的底物特异性不是绝对的[71]。例如，当FTase被抑制时，K-RasB蛋白可能会成为GGTase Ⅰ的底物进行牻牛儿基牻牛儿基化修饰[72]。
表 2 部分异戊二烯化修饰的蛋白及其功能
Table 2 A family of prenylated proteins and their functions
| Prenylation | Protein | GTPase | Function | References |
| Farnesylation | Ras | + | Regulation of gene expression, cardiac hypertrophy and fibrosis | [65] |
| Lamin A/Lamin B | ‒ | Nuclear membrane components | [66] | |
| Geranylgeranylation | Rho | + | Regulation of cytoskeletal reorganization and gene expression Assembly of actin stress fibers |
[67] |
| Rab | + | Vesicular transportation | [68] | |
| Rac | + | Plasma membrane secretion | [69] | |
| Cdc42/G25K | ‒ | Membrane localization | [70] | |
| +：属于；‒：不属于 +: Member of GTPases; ‒: Not member of GTPases. |
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FPPS是研究最广泛的反式异戊烯基转移酶。由α螺旋组成并形成大中心腔，空腔由10个包含保守残基的反平行α螺旋组成[24]。将已被鉴定不同物种中的反式异戊二烯基转移酶蛋白序列进行比对，得到的比对结果显示这些蛋白序列中都包含几个保守区域，其中2个保守区中存在高度保守的天冬氨酸基序，靠近N端的被称为第一个富含天冬氨酸的基序(first aspartate rich motif, FARM)，靠近C端的被命名为第二个富含天冬氨酸的基序(second aspartate rich motif, SARM)，产物链长通常由FARM及其上游的五位氨基酸残基所构成的区域决定，这个区域被称为是反式异戊烯基转移酶的链长决定区(chain-length determination domain, CLD)[73-75]。根据FARM不同特点可将FPPS与GGPPS分为以下3种类型(图 2)。
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| 图 2 调节终产物链长的异戊烯基转移酶基序的结构 Figure 2 Structure of prenyl diphosphate synthase motif regulating the chain-length of final products. |
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Ⅰ型FPPS的FARM基序为DDXXD (D：天冬氨酸，X：任意氨基酸)，在FARM上游的第4个和第5个位置均为芳香族氨基酸残基；Ⅱ型FPPS的FARM中有4个任意氨基酸插入(DDXXXXD)，在FARM上游的第5位存在一个芳香族氨基酸；Ⅲ型FPPS的FARM基序为DDXXD，芳香族氨基酸位于FARM上游第5位[41, 76]。
Ⅰ型GGPPS的FARM基序为DDXXD，在FARM上游的第5个位置为芳香族氨基酸残基；Ⅱ型GGPPS的FARM中有4个任意氨基酸插入(DDXXXXD)，在FARM上游的第4和第5位不存在芳香族氨基酸；Ⅲ型GGPPS特点与I型GGPPS相似，但FARM中插入的2个残基之一是谷氨酸，Ⅲ型GGPPS上游第4位和第5位也不存在芳香族氨基酸。此外，Ⅲ型GGPPS主要利用FPP为底物[77]。
3.2 链长决定机制当在FARM上游的第4或第5位引入突变时，催化活性改变使产物类异戊二烯焦磷酸链长出现变化。几十年来在高度保守区域中氨基酸的定点突变研究，揭示了它们在酶的底物结合和催化活性方面起到了关键作用。嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)的FPPS突变体S113F (表示氨基酸序列中第113位丝氨酸替换为苯丙氨酸，下文其他突变表达形式相同)产物FPP/GPP摩尔比为1:27，主要产物为GPP[78]，Ohnuma等[74]在嗜热芽孢杆菌中构建了20个FPPS突变体，将FARM上游第5位即序列中第81位氨基酸Y进行取代，产生链长为C20‒C30的产物，野生型酶活产物为15C FPP，将第81位氨基酸替换为较小侧链氨基酸A时，酶活反应产物变为GGPP，甚至HexPP。
在前人的研究中，这些氨基酸残基在酶催化活性以及酶底物结合过程中都具有重要功能，为进一步了解蛋白序列中参与结合和催化的残基以及酶的链长选择性提供信息。
Kloer等[79]将DMAPP与IPP异戊烯基转移酶的反应空腔中结合位点分别命名为“烯丙基”或“A位”和“异戊烯基”或“I位”，催化反应如图 3所示，在反应过程中，一分子的IPP与蛋白反应腔中I位点结合，随后IPP中的C4与DMAPP的C1相连，使得IPP的链被延长，延长后的焦磷酸产物必须从I位移动到A位，以便新的IPP去占据I位开启新一轮链的延伸，此外，他们还指出A位点是由3个Mg2+和2个天冬氨酸基序形成的，I位点中不含离子，只含有带正电荷的氨基酸侧链，并不是每个天冬氨酸基序都含有金属离子。
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| 图 3 IPP在I位点与DMAPP在A位点的缩合反应示意图 Figure 3 Sketch of the condensation reaction of IPP at the I-site and DMAPP at the A-site. |
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为探究异戊烯基转移酶家族的进化关系，在早期研究中，许多研究课题组从不同的角度构建了反式异戊烯基转移酶的系统发育树[35, 80-82]，一些研究认为所有反式异戊烯基转移酶家族来自于同一祖先，并认为古细菌GGPPSs是与这一祖先亲缘关系最近的一类酶[81-82]。1994年，Chen等[82]提出13个异戊烯基转移酶(FPPS, GGPPS, HexPPS)是从一个共同祖先进化成3个不同的簇，这些异戊烯基转移酶可能从同样参与代谢途径的另一个酶进化而来。Vandermoten等[21]构建进化树表明反式异戊烯基转移酶可能来自同一原核祖先，并提出FPPS与GGPPS两种酶之间可能不存在从彼此进化而来的关系，而是通过共同祖先水平基因转移而产生这一观点。这些观点仍存在争议[35]，一直作为异戊烯基转移酶进化讨论的热点问题。
本文根据从NCBI (https://www.ncbi.nlm.nih.gov/)中检索并下载的38个反式异戊烯基转移酶蛋白序列，使用Clustal W算法对氨基酸序列进行比对，采用遗传分析软件MEGA 11 Neighbor-Joining法构建系统发育树，通过iTOL (https://itol.embl.de/)美化(图 4)。我们的系统发育树包含了不同物种中的短链异戊烯基转移酶，其中GPPS看起来几乎只存在于动物和植物中，与Vandermoten等[21]的研究结果一致。进化树的第二大分支中，GGPPS与FPPS明显地被分成了两簇，其中一簇为真核中的异戊烯基转移酶；另一簇为细菌与古菌中的异戊烯基转移酶以及植物GGPPSs，这两簇中酶的分布可能表明了其进化关系与酶链长无关，且分类间的FPPS-FPPS亲缘关系比种内FPPS-GGPPS亲缘关系更近(如图 4中真菌与细菌FPPS，红酵母FPPS与红酵母GGPPS的亲缘关系)，这些可能说明这2种酶并非由彼此进化而来，更可能是通过独立进化获得。可见，反式异戊烯基转移酶可能起源于共同祖先，但它们的进化似乎十分复杂，并不能仅仅通过简单的构建系统发育树分析得出结论。
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| 图 4 基于反式异戊烯基转移酶蛋白序列及neighbor-joining法构建的系统发育树 Figure 4 Phylogenetic tree analysis of trans-IPPS based on the amino acid sequences by the neighbor-joining method. 分支上的数据表示Bootstrap检验的支持百分率；自展支持值(Bootstrap)≥30%的显示在各个进化分支节点上；括号内为GenBank登录号 Protein sequences of trans-IPPS were used for alignment by Clustal W, and the phylogenetic tree was developed by the method of neighbor-joining tested with bootstrap 1 000 using MEGA 11. AtGPPS: Arabidopsis thaliana; PcGPPS: Pogostemon cablin; IcGPPS: Ips confusus; PpGPPS: Pardosa pseudoannulata. MtFPPS: Mycobacterium tuberculosis; CkFPPS: Clostridium kluyveri; LpFPPS: Lactococcus piscium; GsFPPS: Geobacillus stearothermophilus; GgFPPS: Gallus gallus; MmFPPS: Mus musculus; HsFPPS: Homo sapiens; BtFPPS: Bos taurus; SbFPPS: Sanghuangporus baumii; RtFPPS: Rhodotorula toruloides; PrFPPS: Phaffia rhodozyma; AtFPPS: Arabidopsis thaliana; CaFPPS: Capsicum annuum; SmFPPS: Salvia miltiorrhiza; PgFPPS: Panax ginseng; HaFPPS: Helianthus annuus; AaFPPS: Artemisia annua; PsGGPPS: Pontimonas salivibrio; PmGGPPS: Petrimonas mucosa; SaGGPPS: Sulfolobus acidocaldarius; GaGGPPS: Geoglobus acetivorans; SmGGPPS: Salvia miltiorrhiza; EbGGPPS: Eschenbachia blinii; JsGGPPS: Jasminum sambac; AtGGPPS: Arabidopsis thaliana; PsGGPPS: Pissodes strobi; AgGGPPS: Aphis gossypii; CcGGPPS: Ceratitis capitata; MmGGPPS: Mus musculus; HsGGPPS: Homo sapiens; RtGGPPS: Rhodotorula toruloides; NcGGPPS: Neurospora crassa; PeGGPPS: Penicillium expansum; FfGGPPS: Fusarium fujikuroi. The confidence values over 30% from 1 000 replicate bootstrap samplings are shown at each node; GenBank accession numbers are shown in the parentheses. |
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法尼基焦磷酸合酶与牻牛儿基牻牛儿基焦磷酸合酶位于类异戊二烯合成途径分支点，参与不同代谢合成途径，其编码基因的表达情况以及酶活性的变化可能影响其下游基因的转录水平，调控下游各个分支中的碳流量。Piłsyk等[44]过表达木霉(Trichoderma reesei)中编码法尼基焦磷酸合酶的基因erg20，结果表明过表达的erg20显著增强了FPP合酶的活性，并激发了FPPS下游的某种异戊烯基转移酶活性。麦角甾醇是酵母的重要膜成分，与哺乳动物系统中的胆固醇类似，影响膜流动性、渗透性和膜结合酶的活性，是MVA途径主要的终产物之一[83]。对酿酒酵母(Saccharomyces cerevisiae)中FPPS蛋白进行定点诱变，通过调节FPPS活性来控制FPP的可用性，结果表明当FPPS活性降低至20%时麦角甾醇的合成量降低至50%[84]。类胡萝卜素是一种脂溶性类异戊二烯化合物，普遍存在于高等植物、细菌和真菌中[85]。接合菌毛霉(Mucor circinelloides)主要积累β-胡萝卜素，Csernetics等[86]克隆了异戊烯焦磷酸异构酶基因(ipi)并与法尼基焦磷酸合酶基因(isoA)和牻牛儿基牻牛儿基焦磷酸合酶(carG)基因一起过表达，试验结果显示，影响类胡萝卜素的关键基因首先是出于carG的调控，其次ipi和isoA基因也有助于积累合成40C的类胡萝卜素前体GGPP。此外，红法夫酵母(Xanthophyllomyces dendrorhous)是为数不多可以合成虾青素的生物之一，过表达红法夫酵母中crtE以及结合培养条件的优化使菌体中虾青素的含量相比野生型提高8倍[87]。
6 类异戊二烯化合物生物合成应用由于大多数类异戊二烯化合物在天然植物资源中含量较低且结构复杂，难以通过化学合成的方法生产，这些困难阻碍了通过现有的工业生物技术生产部分药品、精细化学物质以及聚合物，如提取一剂紫杉醇需要一棵100年的红豆杉(Taxus brevifola)的整棵树皮[6]。然而目前的基因组学、转录组学和合成生物学等新兴技术在分子和细胞水平上对理解和调控代谢途径提供了新工具。因此从环境和经济等多角度考虑，类异戊二烯化合物的微生物生产越来越受到关注。
一项相关研究表明，在酿酒酵母中构建了具有GPP/FPP合成酶和香叶醇合成酶活性的双功能酶Erg20p(F96W-N127W)-t3CrGES，Erg20ww/ t3CrGES与另一个Erg20ww蛋白间的相互作用增强了GPP的积累以及可利用性，使香叶醇含量达到523.96 mg/L，最终在2 L发酵体系中得到1.68 g/L香叶醇，这是目前报道的真核细胞中的最高含量[88]。法尼醇(farnesol, FOH)是一类具有芳香气味的脂肪醇类，Wang等[89]在无FOH合成酶的情况下，通过使用MVA途径和过表达大肠杆菌中法尼基焦磷酸合酶基因ispA增强FPP的合成，获得了135.5 mg/L的FOH产量，这是关于大肠杆菌直接产生FOH的首次报道。
7 小结与展望类异戊二烯化合物结构复杂、功能多样，在食品、化妆品以及医药等行业都具有广阔的应用前景和重要的商业价值。以异戊烯基转移酶作为切入点来提高类异戊二烯化合物的质量与产量具有很大的研究价值。因此，类异戊二烯合成途径及途径中限速酶的基础研究十分必要。目前对于类异戊二烯合成以及异戊烯基转移酶分子机制的研究更多偏向于植物与微生物中相关基因的克隆以及功能分析，研究人员根据大量组学等生物信息综合分析，利用分子生物学及生物信息学等手段探索并验证编码异戊烯基转移酶的关键基因对下游支路中的基因以及次生代谢物的调控机制，但各个通路之间的互作机制尚不清楚。虽然人们对异戊烯基转移酶基因参与的途径在分子生物学水平上已有大致的了解并取得了进展，但对于通路上相关基因的作用机理仍缺乏深入研究。
了解异戊烯基转移酶家族关键基因对异戊二烯化合物合成途径的调控，将是未来另一个研究重点，况且微生物是一个很好的研究对象，可以为相关研究提供一定程度的参考。通过对挖掘到的关键基因采用分子生物学技术进行定向编辑，改变酶活性来进一步阐明关键限速酶对类异戊二烯化合物合成的作用机制，探索下游代谢支路或合成途径中的关键基因调控对途径中异戊二烯类物质含量的影响，结合基因工程等分子生物学技术研究编码限速酶基因的调控机制，深入开发利用这一类古老又神秘的化合物家族。
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