原壳小球藻番茄红素ε环化酶基因的克隆和分析

摘要：【目的】番茄红素ε环化酶（lycopene epsilon cyclase）是叶黄素代谢途径中处于分支位点的关键酶。它催化线性的番茄红素选择性环化,形成胡萝卜素,再进一步合成叶黄素（lutein）。本研究的目标是克隆原壳小球藻（Chlorella protothecoides CS-41）LCYE基因的全长cDNA,通过该基因的生物学信息分析预测其表达产物的空间结构与功能位点,并验证该表达产物的生物学活性。【方法】采用cDNA末端快速扩增（rapid-amplification of cDNA ends）和RT-PCR技术克隆原壳小球藻LCYE基因的全长cDNA序列。分别采用PredictProtein、Pfam HMMs和Swiss-Model等在线分析软件分析LCYE的基本生物学信息,预测蛋白质功能位点和空间结构。利用pET28-a(+)构建LCYE基因的原核表达质粒pET-LCYE,并转入大肠杆菌BL21（DE3）,再利用IPTG诱导LCYE基因超量表达。此外,携带pAC-LYC质粒的大肠杆菌工程菌能够积累番茄红素,可用于验证LCYE基因编码蛋白的酶活。【结果】获得了原壳小球藻的LCYE基因的cDNA序列,长2107 bp,GenBank登录号为FJ752528。序列分析表明：它含有1731 bp的完整开放阅读框（ORF）,编码576个氨基酸,与高等植物和其他藻类的LCYE有很高的相似性,其中相似性最高的是莱茵衣藻（Chlamydomonas reinhardtii XM001696477.1）,达到67%。第48～459个氨基酸残基为一个典型的番茄红素环化酶蛋白（Lycopene cyclase protein）结构域（pfam05834）,第261～284个氨基酸残基为一个典型的环化酶保守的模体。SDS-PAGE检测表明,LCYE基因在大肠杆菌BL21（DE3）中得到了超量表达。携带pAC-LYC质粒的大肠杆菌工程菌在获得LCYE基因后,其颜色从粉红色变为黄色。【结论】原壳小球藻LCYE基因的全长cDNA为2107 bp,预测出番茄红素ε环化酶所特有的保守功能区域,构建了它的三维结构模型。同时,阐明了小球藻和衣藻的亲缘关系。最后,证实了本研究克隆到的LCYE基因编码的蛋白具有番茄红素ε环化酶的功能与活性。     

累积番茄红素的大肠杆菌工程菌及其培养条件的研究

噬夏孢欧文氏菌番茄红素合成相关基因crtE, crtB, crtI同时克隆进表达载体pET-15b构建pET-15bcrtIEB,将该重组质粒转化E.coliBL21(DE3)构建工程菌,IPTG诱导工程菌累积红色色素,经HPLC和吸收光谱分析,工程菌中合成的色素为番茄红素。研究了碳源、金属离子、培养温度、诱导剂浓度、诱导时间等参数对工程菌生长及色素累积的影响,确定了合适的培养条件：培养基为改良LB培养基（蛋白胨10g/L、酵母提取物5g/L、麦芽糖5g/L、MgCl2 0.1g/L,NaCl 10g/L）;起始培养温度为37℃;培养至OD600为0.6左右时加入IPTG,终浓度为0.5mmol/L,诱导温度降至30℃;诱导时间为14h。发酵完成后工程菌的生物量（干重）为3.45g/L,番茄红素的最高含量可达5.8mg/gDW。     

大肠杆菌共表达LeGGPS2、LePSY1和crtI基因合成番茄红素

利用大肠杆菌研究番茄红素的合成,不仅可以获得副产物少的高产菌株,而且可以探讨基因或基因簇的功能。文中将番茄LeGGPS2和LePSY1的cDNA序列,及欧文氏菌crtI的编码序列分别添加上核糖体结合位点后,以单独或组合的方式受控于T7启动子和终止子,在大肠杆菌菌株BL21(DE3)中进行表达和诱导番茄红素合成。结果显示,仅T7::crtI-LeGGPS2-LePSY1三价基因共表达时才能合成番茄红素,且将种子液以1∶50接种于含3%蔗糖的LB培养基(pH 6.8)中,于37℃摇8 h左右的对数生长后期加IPTG至80μmol/L,30℃诱导表达5 h的发酵条件下,获得2.124 mg/g DCW的番茄红素。该结果既验证了原核化的番茄LeGGPS2和LePSY1基因及与crtI基因协同作用的功能,又为在番茄质体中建立独立的番茄红素合成途径奠定了基础。     

番茄红素环化酶基因shRNA真核表达质粒的构建及鉴定

目的利用RNA干扰技术,构建靶向番茄红素环化酶基因(CarR)的干扰质粒,为选择性抑制番茄红素环化酶活性以提高番茄红素产量,奠定基础。方法用DNA重组技术将针对三孢布拉氏霉菌CarR的不同部位所设计的3对shRNA序列克隆到真核表达质粒mU6 pro中,构建CarR shRNA表达质粒重组体mU6 CarR shRNA1、2、3,转化DH5а菌株扩增。提取质粒行酶切鉴定后,进行测序分析。结果3个CarR shRNA表达载体mU6 CarR shRNA1、2、3经限制性酶切及部分序列分析证明基因插入正确。结论成功构建了CarR shRNA表达载体mU6 CarR shRNA,重组体的成功构建为研究CarR靶向RNA干扰番茄红素的环化打下基础。     

甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达

八氢番茄红素脱氢酶( CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用.以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸.与来自M.populi BJ001、M.chloromethanicum CM4和M.extorquens AM1的crtI一致性均为93％.将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI.测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40％.实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考.     

高效合成番茄红素酿酒酵母菌株的构建

番茄红素作为一种高附加价值的萜类化合物已受到国内外研究者的广泛关注。首先对酿酒酵母Saccharomyces cerevisiae模式菌株S288c和YPH499合成番茄红素的能力进行分析比较,结果表明YPH499更适合作为底盘细胞用于番茄红素的合成。随后比较组成型启动子GPDpr、TEF1pr和诱导型启动子GAL1pr、GAL10pr对番茄红素合成的影响,结果发现以GPDpr、TEF1pr作为番茄红素合成途径基因crtE、crt B和crtI的启动子,摇瓶发酵60 h后,番茄红素产量为15.31 mg/L;以GAL1pr和GAL10pr为启动子时,其产量为123.89 mg/L,提高8.09倍。继续改造甲羟戊酸(MVA)途径,过量表达N-末端截短的关键酶基因t HMG1(3-羟基-3-甲基戊二酸单酰辅酶A还原酶),番茄红素产量为265.68 mg/L,单位菌体产量72.79 mg/g。文中所设计构建的异源表达番茄红素合成途径的酿酒酵母菌株单位细胞产量高,可以进一步改造和优化后用于番茄红素的工业化生产。     

植物LYCs的特性功能及其相互关系

植物番茄红素环化酶（LYC）是将线性番茄红素分子转化为具环类胡萝卜素的关键酶,本文从番茄红素环化酶cDNA的核苷酸和氨基酸序列,底物特异性以及作用特点等方面,阐述了两种重要的番茄红素环化酶即β－环化酶和ε－环化酶的异同,并对这两种酶的作用特点,对植物具环类胡萝卜素种类及总量的调节作用以及对其它环化酶的相互关系进行了讨论。     

八氢番茄红素合成酶(PSY)的生物信息学分析

目的:通过生物信息学方法对八氢番茄红素合成酶基因(PSY)及氨基酸序列分析,并构建三维结构。方法:运用生物信息学方法对八氢番茄红素合成酶基因及其蛋白质序列的理化性质、亲/疏水性、信号肽、跨膜结构域、糖基化位点,磷酸化位点,二级结构,功能结构域和三级结构进行预测分析。结果:PSY基因含1239bp的开放阅读框,编码氨基酸数为412,为碱性不稳定蛋白;八氢番茄红素合成酶富含Arg、Leu、Ala、Ser、Val等氨基酸,为亲水性蛋白质;PSY为非跨膜蛋白,不含信号肽,具有多个磷酸化位点,α螺旋和无规卷曲是其主要结构元件。结论:用同源建模的方法构建其三维结构,得到合理模型,为采用生物工程提高番茄红素产量提供理论依据。     

甲羟戊酸途径限速步骤研究及其在产番茄红素重组大肠杆菌中的应用

甲羟戊酸途径(MVA途径)被引入重组大肠杆菌中,能够提高重组大肠杆菌中萜类化合物的合成能力。但因重组大肠杆菌中萜类化合物合成途径中间产物积累,导致细胞生长和萜类化合物合成受到限制。本研究在稳定表达MVA途径以及优化2-甲基-D-赤藻糖醇-4-磷酸途径(MEP途径)、番茄红素合成途径关键基因表达的重组大肠杆菌LYC103中,用质粒高表达MVA途径和番茄红素合成途径关键基因,挖掘该途径的限速步骤。结果表明,ispA、crtE、mvaK1、idi和mvaD基因过表达后,细胞生长没有明显变化,番茄红素产量依次提高了13.5%、16.5%、17.95%、33.7%和61.1%,说明这几个基因可能是合成番茄红素的限速步骤。mvaK1、mvaK2、mvaD三个基因在同一操纵子上,用mRNA稳定区(RNA stabilizing region)进行启动子文库(mRSL)调控mvaK1,相当于对3个基因同时调控。用高效基因组编辑技术(CAGO)对mvaK1基因的mRNA稳定区进行启动子文库的调控,得到菌株LYC104。番茄红素产量与对照菌株LYC103相比增加了2倍,细胞生长提高了32%。然后,利用CRISPR-Cas9技术在染色体lacZ位点整合idi基因,得到LYC105菌株。与出发菌株LYC103相比,细胞生长提高了147%,番茄红素产量增加了2.28倍。本研究在染色体上具有完整MVA途径的基础上,利用质粒高表达单个基因挖掘限速步骤,用同源重组方法整合限速基因、解除限速,为代谢工程构建高产菌株提供新策略。     

稳定表达MVA途径基因提高番茄红素产量

萜类化合物的直接前体物质异戊烯焦磷酸(IPP)和二甲基烯丙基焦磷酸酯(DMAPP)可以由2-甲基-D-赤藻糖醇-4-磷酸途径(MEP途径)和甲羟戊酸途径(MVA途径)合成。在已经优化MEP合成途径、番茄红素合成途径关键基因表达的重组大肠杆菌LYC101中,引入MVA途径基因,进一步提高重组大肠杆菌合成萜类化合物的能力。质粒pALV23和pALV145是本实验室在研究MVA途径基因协调表达时,用核糖体结合位点(RBS)文库连接MVA途径各基因构建质粒文库,而筛选到的有效提高β-胡萝卜素产量的质粒。首先比较了两个质粒分别在低产和高产番茄红素的菌株中对番茄红素合成的影响。结果表明,两个质粒在高、低产番茄红素的菌株中都可以有效提高番茄红素产量。在高产菌LYC101中pALV23比pALV145使番茄红素产量更高。然后,用CRISPR-Cas9系统辅助同源重组的方法,将MVA途经基因和启动子一共6.7kb的条带整合到LYC101菌株的染色体上,得到遗传稳定的菌株LYC102。LYC102的番茄红素产率达40.9mg/g,是出发菌株LYC101产率的2.19倍,比用质粒表达MVA途径基因的菌株提高了20%。在重组大肠杆菌中同时表达MVA途径和MEP途径,可以有效提高萜类化合物产率;文中构建了不含质粒的、遗传稳定的高产番茄红素菌株,为产业化合成番茄红素提供基础;同时构建平台菌株,可以用于其他萜类化合物合成。     

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.     

猪α-干扰素的原核表达及活性测定

目的：表达并纯化出具有生物学活性的重组猪α-干扰素。方法：根据前期合成的猪α-干扰素基因序列设计引物,用PCR方法扩增出猪α-干扰素基因,并将其定向克隆入原核表达载体pET28中,酶切及测序鉴定正确后,转化大肠杆菌BL21进行诱导表达,对表达产物通过镍琼脂糖凝胶柱亲和层析纯化、透析法复性后,采用微量细胞病变抑制法测定重组猪α-干扰素的活性。结果：测序结果表明构建了猪α-干扰素原核表达载体pET28-poIFN-α;诱导表达后经SDS-PAGE分析,在相对分子质量约21×10^3的位置出现明显的诱导蛋白条带,与目的蛋白大小相近;经分析表达产物主要以包涵体形式存在,约占菌体总蛋白的36%,纯化后的目的蛋白纯度约为90%;采用微量细胞病变抑制法测定其活性约为1.67×10^6U/mg。结论：获得了纯度较高并具有较高活性的重组猪α-干扰素,为下一步研究猪α-干扰素药物价值及其生物制剂的生产、应用奠定了基础。     

Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups

A survey is given of the lycopene cyclase genes present in bacteria, fungi and plants where two completely unrelated types exist. One is the classical monomeric bacterial beta-cyclase gene, crtY, which may be an ancestor of crtL, the gene for a beta-cyclase in cyanobacteria. From crtL a line of evolution can be drawn to plant beta- and epsilon-cyclase genes and to the gene of capsanthin/capsorubin synthase. In Gram-positive bacteria two genes crtYc and crtYd are present. They encode two proteins which have to interact as a heterodimer for lycopene beta-cyclization. From this type of lycopene cyclase gene the fungal lycopene cyclase/phytoene synthase fusion gene evolved.     

Functional characterization of sll0659 from Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that Sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types and mutants. In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cycilzation. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.     

Cloning and expression of Mycobacterium bovis secreted protein MPB83 in Escherichia coli

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using TA cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M.bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.     

棉花GhVHA A基因的原核表达及其重组大肠杆菌的抗逆性分析

为进一步验证棉花GhVHA-A基因的功能,该研究将棉花GhVHA-A基因构建到原核表达载体pET28a上,利用IPTG诱导其在大肠杆菌BL21(DE3)中高效表达,同时对重组大肠杆菌BL21(pET28a-GhVHA-A)进行抗逆性分析。结果表明:(1)半定量RT-PCR分析发现,棉花幼苗液泡膜H+-ATPase基因(GhVHA-A)表达水平受脱水和高盐胁迫诱导。(2)将1 872bp长的编码区序列连接至原核表达载体pET28a上,成功构建了原核表达载体pET28a-GhVHA-A;SDS-PAGE电泳检测结果表明,在70kD左右处有1条特异表达的蛋白质条带,与预期的目的产物大小一致。(3)重组菌BL21(pET28a-GhVHA-A)的抗逆性分析发现,重组菌对PEG6000(20%)和NaCl(0.5mol/L)的抗性明显高于对照菌株BL21(pET28a),表明GhVHA-A基因在大肠杆菌中表达后能够增强菌株的抗性。本研究结果为GhVHA-A基因在植物抗逆基因工程中的应用提供了理论依据。     

Generation of lycopene-overproducing strains of the fungus <Emphasis Type="Italic">Mucor circinelloides</Emphasis> reveals important aspects of lycopene formation and accumulation

Objectives

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.

Results

Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains.

Conclusions

A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

牛分枝杆菌MPB51基因的克隆及其在大肠杆菌中的表达

以牛分枝杆菌Vallee111染色体DNA为模板,以MPB51成熟蛋白基因特异性引物进行PCR扩增,获得约800bp的DNA片段。通过TA克隆技术,将PCR产物克隆至pGEMT Vector中,成功地构建出克隆质粒pGEMT51。以BamHⅠ和EcoRⅠ双酶切pGEMT51和pET28a(+),并将纯化的MPB51基因亚克隆至pET28a (+)中,构建出原核表达质粒pET28a51。将pET28a51转化至感受态E.coli BL21（DE3）中,经IPTG诱导和SDSPAGE分析,可见约30kD外源蛋白带。Western blot分析发现,该蛋白具有牛分枝杆菌抗原性,从而为进一步研究MPB51的亚单位疫苗及DNA疫苗奠定基础。