牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。     

中国红豆杉紫杉烯合酶cDNA的分离、表达和鉴定

紫杉烯合酶是一种二萜环化酶,催化牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯.利用PCR扩增同源探针筛选cDNA文库,克隆了一个编码中国红豆杉(Taxus chinensis (Pilg.) Rehd.)紫杉烯合酶3′端的2 151 bp的cDNA片段,也通过PCR扩增得到了该基因5′端的611 bp的cDNA片段,将这两个cDNA片段拼接在一起,得到长2 712 bp的cDNA片段,具有一个2 586个碱基的开放阅读框架(ORF),编码包括质体转移肽在内的共862个氨基酸残基;该酶与太平洋红豆杉紫杉烯合酶有97%的同源性(identity),与其他植物萜类环化酶也有较高的同源性.利用融合表达载体pET-32a在大肠杆菌BL21trxB中表达,所表达的融合蛋白以包含体形式存在.包含体经过变性、复性和再折叠,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶.用毛细管气相色谱-质谱联用对酶促反应产物进行分析,结果表明,融合的紫杉烯合酶能催化产生4(5),11(12)-紫杉烯.     

中国红豆杉紫杉烯合酶cDNA的分离、表达和鉴定

紫杉烯合酶是一种二萜环化酶 ,催化牛儿基牛儿基焦磷酸形成紫杉醇生物合成过程中的中间体紫杉烯。利用PCR扩增同源探针筛选cDNA文库 ,克隆了一个编码中国红豆杉 (Taxuschinensis (Pilg .)Rehd .)紫杉烯合酶 3′端的 2 15 1bp的cDNA片段 ,也通过PCR扩增得到了该基因 5′端的 6 11bp的cDNA片段 ,将这两个cDNA片段拼接在一起 ,得到长 2 712bp的cDNA片段 ,具有一个 2 5 86个碱基的开放阅读框架 (ORF) ,编码包括质体转移肽在内的共 86 2个氨基酸残基 ;该酶与太平洋红豆杉紫杉烯合酶有 97%的同源性 (identity) ,与其他植物萜类环化酶也有较高的同源性。利用融合表达载体pET_32a在大肠杆菌BL2 1trxB中表达 ,所表达的融合蛋白以包含体形式存在。包含体经过变性、复性和再折叠 ,利用His残基亲和凝胶柱层析得到融合的紫杉烯合酶。用毛细管气相色谱 质谱联用对酶促反应产物进行分析 ,结果表明 ,融合的紫杉烯合酶能催化产生 4(5 ) ,11(12 )_紫杉烯     

抗癌药物榄香烯的生物合成及关键酶吉马烯A合酶的优化

榄香烯是我国拥有自主知识产权的抗肿瘤药物之一,因其抗肿瘤活性强、作用范围广、毒副作用轻微和不易产生耐药性等优点,被广泛应用于各类恶性肿瘤的临床治疗中。榄香烯的生产主要依靠药用植物温郁金的分离提取。但温郁金的榄香烯含量低、分离纯化难度大、得率低且成本高,严重阻碍了榄香烯的大规模生产与应用。随着合成生物学的发展,利用微生物构建细胞工厂用于生物合成天然药物成为研究热点,也为榄香烯的生产提供了新的思路。近年来,对榄香烯的生物合成研究在不断深入。研究者通过代谢工程、组合生物学和基因工程等手段,阐明榄香烯生物合成途径和关键酶,已经成功克隆了榄香烯生物合成途径上的一些关键酶基因,初步实现榄香烯的异源生物合成。本文以合成生物学研究思维概述榄香烯生物合成途径及其工程菌的优化,重点综述关键酶吉马烯A合酶(germacrane A synthase, GAS)。从限速酶基因的过表达和分流基因的敲除,融合表达酶工程策略、吉马烯A合酶的体外进化几方面,对其生物合成途径的改造策略进行阐述。同时,也分析提高异源生物合成榄香烯产量所面临的问题与挑战,为榄香烯的高效生物合成提供参考。     

利用基因工程改造的大肠杆菌合成蒎烯

目的:以大肠杆菌为底盘细胞,利用合成生物学手段导入外源杂合蒎烯合成代谢途径,构建高效合成蒎烯的微生物工厂。方法:将来源于酵母和粪肠球菌的异源杂合甲羟戊酸(MVA)代谢途径和来源于北美巨冷杉的牻牛儿焦磷酸合酶(GPPS)和蒎烯合成酶(PS)基因序列共同导入大肠杆菌,构建催化蒎烯合成的大肠杆菌工程菌。首先优化合成GPPS、PS基因,再分别以pETDuet-1和pET-24a(+)载体为基础构建GPPS、PS共表达和融合表达载体,并分别转化大肠杆菌获得工程菌E.hzh01和E.hzh02,摇瓶培养后利用GC-MS技术检测E.hzh01和E.hzh02的蒎烯产量。进一步获取MVA代谢途径相关酶基因mvaE、mvaS、ERG12、ERG8、ERG19、IDI序列,分别利用多顺反子模型和BioBrick方法构建2种不同方案的异源MVA代谢途径,再将异源MVA代谢途径和GPPS、PS融合表达基因共同转化大肠杆菌,构建完整蒎烯合成代谢工程菌株E.hzh03和E.hzh05。结果:摇瓶培养E.hzh01和E.hzh02的蒎烯产量分别为0.85和1.86 mg/L,结果表明融合表达更有利于蒎烯的合成。E.hzh03和E.hzh05摇瓶培养得到的蒎烯产量分别为6.32和19.26 mg/L。结论:利用融合表达载体和多顺反子模型导入异源蒎烯代谢途径构建大肠杆菌工程菌,可以显著提高蒎烯的产量,为工业生物合成蒎烯奠定了一定的基础。     

利用Bio-BglBrick方法表达紫杉醇生物合成酶

目的:进行紫杉醇药物生物合成前五步催化酶二磷酸盐合酶(GGPPS)、紫杉二烯合酶(TS)、紫杉二烯5α羟化酶(THY5α)、紫杉二烯5α-O-乙酰转移酶(TAT)和紫杉烷10β羟化酶(TDH)基因在大肠杆菌异源生物合成途径的组建及串联,实现单个表达及串联表达,并试图通过连续生物催化获得紫杉醇中间体紫衫二烯。方法:依据合成生物学中Brick基因组装方法,通过对载体pET30a的酶切位点进行定向改造,设计独特的BglⅡ/BamHⅠ和XbaⅠ/SpeⅠ串联表达盒,在大肠杆菌BM Rosetta(DE3)中表达产物。结果:设计多基因表达盒,实现紫杉醇药物生物合成前五步催化酶的单个表达,GGPPS和TS以及THY5α、TAT和TDH的串联表达。结论:利用BglBrick/BioBrick基因组装方法,可以实现紫杉醇生物合成催化酶的快速组装及后续表达。     

紫杉醇生物合成相关酶基因的克隆与表达

以牛儿基牛儿基二磷酸为前体的紫杉醇生物合成大约有20步酶促反应,其反应过程已基本阐明,近一半的相关酶基因已得到克隆与表达。综述了编码参与紫杉醇生物合成的紫杉二烯合酶、紫杉二烯5α羟化酶、紫杉烷10β羟化酶、紫杉烷13α羟化酶、紫杉二烯5α醇O乙酰基转移酶、紫杉烷2α苯甲酰转移酶、去乙酰基巴卡亭Ⅲ10βO乙酰转移酶、3氨基3苯基丙酰转移酶和3N去苯甲酰2脱氧紫杉醇苯甲酰转移酶等9个酶基因的克隆和表达方面的研究情况,并指出随着紫杉醇生物合成的分子生物学研究的不断深入,利用分子生物技术大规模生产紫杉醇将为期不远 。     

紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用

为了构建高产的紫穗槐-4,11-二烯酵母工程菌,主要探究了含紫穗槐-4,11-二烯合酶基因的不同表达载体在酵母工程菌中是否存在协同效应。首先构建了含紫穗槐-4,11-二烯合酶基因的酵母表达载体pGADADS,分别将pGADADS和pYeDP60/G/ADS转入酿酒酵母W303-1B和WK1中,获得6种能产生紫穗槐-4,11-二烯的酵母工程菌：W303B[pGADADS]、W303B[pYGADS]、W303B[pYGADS+pGADADS]、WK1[pGADADS]、WK1[pYGADS]和WK1[pYG     

代谢工程改造酿酒酵母生产β-胡萝卜素

β-胡萝卜素在食品、药品和化妆品领域有广泛用途。为获得生产β-胡萝卜素的微生物细胞工厂,本研究首先在酿酒酵母BY4742中过表达甲羟戊酸(MVA)途径的限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因及二萜化合物合成的关键酶牻牛儿基牻牛儿基焦磷酸合酶(GGPS)基因,来提高牻牛儿基牻牛儿基焦磷酸(GGPP)的供给。在酿酒酵母底盘菌BY4742-T2的基础上整合来源于成团泛菌和红法夫酵母的β-胡萝卜素合成基因,比较酿酒酵母工程菌生产β-胡萝卜素的差别。结果表明提高酿酒酵母中HMGR和GGPS酶基因的表达能将工程菌中β-胡萝卜素的产量提高26.0倍。另外,来源于真核生物红法夫酵母的合成基因相比成团泛菌,更有利于酿酒酵母生产β-胡萝卜素。最终获得的酿酒酵母工程菌BW02能生产1.56 mg/g细胞干重的β-胡萝卜素,为进一步获得高产β-胡萝卜素细胞工厂提供基础。     

榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因的克隆与表达

牻牛儿基牻牛儿基焦磷酸合酶是产紫杉醇内生真菌紫杉醇合成下游途径中的关键步骤之一,在榛子产紫杉醇内生真菌中进行紫杉醇生物合成研究时首先要确认GGPP合酶的存在。该研究通过RT-PCR方法克隆得到了榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因Pa GGPPS(Gen Bank登录号为KM881430)的c DNA开放阅读框(Open reading frame,ORF)。利用生物信息学方法,我们分析了该基因的序列,并对其编码的氨基酸序列进行了预测。结果发现该基因ORF长度为1 113 bp,预计蛋白分子量为40.98k D,等电点为6.168,表明该蛋白呈酸性。对其进行亲疏水性分析发现肽链整体呈现为亲水性。Pa GGPPS的主要二级结构元件为α-螺旋,并且包含一个异戊二烯类化合物合酶功能域。对榛子产紫杉醇内生真菌与其他物种的GGPPS进行氨基酸同源性分析,发现其与娄地青霉、曲霉菌和费氏新萨托菌的一致性较高,分别为94%、76%和76%。进化树分析表明来自动物、真菌和酵母的GGPPS聚为一类,来自植物的GGPPS聚为一类,其中榛子产紫杉醇内生真菌的GGPPS和青霉菌的进化关系最近,与植物的GGPPS的进化关系最远。对其进行基础生物信息学分析后,我们构建了原核表达载体,成功诱导其表达并得到可溶性蛋白。该研究结果为下一步深入研究GGPPS基因在内生真菌Penicillium aurantiogriseum紫杉醇合成途径中的作用及和构建高产紫杉醇基因工程菌株奠定了一定的基础。     

Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production

Metabolic engineering in microbes could be used to produce large amounts of valuable metabolites that are difficult to extract from their natural sources and too expensive or complex to produce by chemical synthesis. As a step towards the production of Taxol in the yeast Saccharomyces cerevisiae, we introduced heterologous genes encoding biosynthetic enzymes from the early part of the taxoid biosynthetic pathway, isoprenoid pathway, as well as a regulatory factor to inhibit competitive pathways, and studied their impact on taxadiene synthesis. Expression of Taxus chinensis taxadiene synthase alone did not increase taxadiene levels because of insufficient levels of the universal diterpenoid precursor geranylgeranyl diphosphate. Coexpression of T. chinensis taxadiene synthase and geranylgeranyl diphosphate synthase failed to increase levels, probably due to steroid-based negative feedback, so we also expressed a truncated version of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMG-CoA reductase) isoenzyme 1 that is not subject to feedback inhibition and a mutant regulatory protein, UPC2-1, to allow steroid uptake under aerobic conditions, resulting in a 50% increase in taxadiene. Finally, we replaced the T. chinensis geranylgeranyl diphosphate synthase with its counterpart from Sulfolobus acidocaldarius, which does not compete with steroid synthesis, and codon optimized the T. chinensis taxadiene synthase gene to ensure high-level expression, resulting in a 40-fold increase in taxadiene to 8.7±0.85 mg/l as well as significant amounts of geranylgeraniol (33.1±5.6 mg/l), suggesting taxadiene levels could be increased even further. This is the first demonstration of such enhanced taxadiene levels in yeast and offers the prospect for Taxol production in recombinant microbes.     

Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol.

Taxadiene, the key intermediate of paclitaxel (Taxol) biosynthesis, has been prepared enzymatically from isopentenyl diphosphate in cell-free extracts of Escherichia coli by overexpressing genes encoding isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase and taxadiene synthase. In addition, by the expression of three genes encoding four enzymes on the terpene biosynthetic pathway in a single strain of E. coli, taxadiene can be conveniently synthesized in vivo, at the unoptimized yield of 1.3mg per liter of cell culture. The success of both in vitro and in vivo synthesis of taxadiene bodes well for the future production of taxoids by non-paclitaxel producing organisms through pathway engineering.     

紫杉醇合成代谢途径中紫杉烯合成酶cDNA的克隆

紫杉烯合成酶(Taxadienesynthase)被认为在紫杉醇合成代谢途径中起着限速酶的作用。为进一步研究紫杉烯合成酶的作用机理和紫杉醇生物合成代谢调控机制,采用RTPCR技术从东北红豆杉(Taxuscuspidata)愈伤组织中获得了紫杉烯合成酶基因片段,将该片段克隆在载体pGEMTEasyVector上,并转化到大肠杆菌JM109中,经EcoRI酶切检测,Southernblotting及部分cDNA序列分析证实该片段确为紫杉烯合成酶基因,与国外报道的从太平洋红豆杉(Taxus.brevifolia)幼茎中得到的紫杉烯合成酶基因序列具有很高的同源性。     

Taxol biosynthetic genes

The function and properties of heterologously expressed full-length cDNA clones, isolated from a Taxus cDNA library and specific to Taxol biosynthesis, are summarized. Recombinant enzymes are described that catalyze early steps of the pathway, including taxadiene synthase, taxadien-5alpha-ol-O-acetyltransferase and taxadien-5alpha-yl acetate 10beta-hydroxylase, and that catalyze late steps, including 10-deacetylbaccatin III-10beta-O-acetyltransferase and taxane 2alpha-O-benzoyltransferase. The properties of Taxus geranylgeranyl diphosphate synthase are also described; although this synthase does not mediate a committed step of Taxol biosynthesis, it does provide the universal plastidial diterpenoid precursor, geranylgeranyl diphosphate, for initiating Taxol biosynthesis.     

Biosynthesis of Taxadiene in Saccharomyces cerevisiae : Selection of Geranylgeranyl Diphosphate Synthase Directed by a Computer-Aided Docking Strategy

Identification of efficient key enzymes in biosynthesis pathway and optimization of the fitness between functional modules and chassis are important for improving the production of target compounds. In this study, the taxadiene biosynthesis pathway was firstly constructed in yeast by transforming ts gene and overexpressing erg20 and thmgr. Then, the catalytic capabilities of six different geranylgeranyl diphosphate synthases (GGPPS), the key enzyme in mevalonic acid (MVA) pathway catalyzing famesyl diphosphate (FPP) to geranylgeranyl diphosphate (GGPP), were predicted using enzyme-substrate docking strategy. GGPPSs from Taxus baccata x Taxus cuspidate (GGPPSbc), Erwinia herbicola (GGPPSeh), and S. cerevisiae (GGPPSsc) which ranked 1^st, 4^th and 6^th in docking with FPP were selected for construction. The experimental results were consistent with the computer prediction that the engineered yeast with GGPPSbc exhibited the highest production. In addition, two chassis YSG50 and W303-1A were chosen, and the titer of taxadiene reached 72.8 mg/L in chassis YSG50 with GGPPSbc. Metabolomic study revealed that the contents of tricarboxylic acid cycle (TCA) intermediates and their precursor amino acids in chassis YSG50 was lower than those in W303-1A, indicating less carbon flux was divided into TCA cycle. Furthermore, the levels of TCA intermediates in the taxadiene producing yeasts were lower than those in chassis YSG50. Thus, it may result in more carbon flux in MVA pathway in chassis YSG50, which suggested that YSG50 was more suitable for engineering the taxadiene producing yeast. These results indicated that computer-aided protein modeling directed isoenzyme selection strategy and metabolomic study could guide the rational design of terpenes biosynthetic cells.     

ggpps 5'-侧翼序列的克隆及其启动子功能验证

目的:研究牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl diphosphate synthase,GGPPs)基因启动子的活性;方法:从曼地亚红豆杉细胞中克隆ggpps基因5'-侧翼序列,并将该侧翼序列代替pBI121质粒上的CaMV35S启动子,以Gus基因作为报告基因构建植物表达载体,并进一步导入农杆菌LBA4404中获得阳性转化子,然后用叶盘转化法验证该侧翼序列的启动子活性;结果:本研究从曼地亚红豆杉细胞中成功克隆了ggpps基因的5'-侧翼序列,并且验证了该侧翼序列具有启动子活性;结论:ggpps基因的5'-侧翼序列的测序结果表明本实验成功克隆了该侧翼序列,启动子功能验证结果表明ggpps 5'-侧翼序列具有启动子活性,这些结果为进一步的通过缺失法进行ggpps基因启动子功能研究奠定了基础.