【作者】 李家洲；

【导师】 谢明权；

【作者基本信息】 华南理工大学 ， 发酵工程， 2013， 博士

【摘要】 辅酶Q10对生物体具有多重生理功能，其在医药、化妆品、食品、保健品等领域的应用十分广泛，但由于缺乏高产辅酶Q10的菌种，长期限制了辅酶Q10及其相关产业的发展壮大。本实验克隆了辅酶Q10合成过程的关键酶基因并对各酶的保守结构进行了分析。克隆的基因包括放射型根瘤菌(R. radiobacter)聚十异二烯基焦磷酸合成酶基因（ddsA）、1-脱氧-D-木酮糖-5-磷酸合成酶基因（dxs）、对羟基苯甲酸聚异戊烯基焦磷酸转移酶基因（ubiA）和大肠杆菌（E. coli）法呢基焦磷酸合成酶基因（ispA）、异戊二烯焦磷酸异构酶基因（idi）。并在此基础上，对这些基因表达产物的保守结构进行了分析。聚十异戊二烯焦磷酸合成酶与法呢基焦磷酸合成酶同属于反式聚异戊二烯焦磷酸合成酶超家族，二者拥有相似保守结构，不同的是二者的链长决定区域的结构相差较大。R. radiobacterDXP合酶的保守结构分析表明，该酶具有两个焦硫胺素结合结构域，且该酶的N端与转酮醇酶N-端及丙酮酸脱氢酶E1亚单位具有的高度保守性。E. coli IDI属于Nudix水解酶超家族成员，含有Nudixmotif结构GHPQLGESNEDAVIRRCRYELGV。通过强化E. coli DE3Q10CoQ10合成过程中的关键酶ddsA、ubiA、ispA、idi等，可以使大肠杆菌CoQ10合成能力由出发菌株的0.52mg/g(干菌体)，提高至1.18mg/g(干菌体)，提高达126%。对R. r DPS的热稳定性进行了改造。对ddsA基因表达产物DPS催化的聚异戊二烯焦磷酸链长不专一的现象进行了重点研究。利用基因敲入技术，使ddsA置换大肠杆菌DE3ispB基因构建合成辅酶Q10的菌种E. coli DE3Q10。通过探索温度、溶氧、pH和限制性营养基质浓度（蛋白胨）四者对E. coli DE3Q10产辅酶Q种类的影响，发现R.radiobacter DPS的催化专一主要受温度影响显著。由此推测该酶的链长决定机理除受传统的“空腔”结构理论影响之外，还受其结构热稳定性的影响。在此基础上，通过同源建模分析其结构中热稳定因子（B-Factor）高的氨基酸残基，并与已知稳定的DPS序列等位对比，确定了Q64P、M92L、W342L、K343R四个潜在的热稳定性增加突变点。经过将突变体基因敲入E. coli DE3，置换其ispB，用HPLC检测其合成的辅酶Q种类发现，M92L、W342L与K343R形成的突变体，即使在高温下，其合成产物也只有辅酶Q10，而无辅酶Q9。通过结构比较发现，其热稳定性增强的原因是突变后减少了空间位阻，增加了氢键数量，使局部能量更合理。构建了受溶氧调控的辅酶Q10高产工程菌E.coli DE3104。通过同源重组的原理，以辅酶Q合成的关键酶基因ddsA、ubiA和idi、ispA依次取代nirB操纵子的nirB、nirD、nirC结构基因，相应构建菌种E. coli DE3Q101、Q102、Que103；以dxs取代E. coliDE3Q103的narG调控操纵子中的结构基因narG构建菌种E. coli DE3Q104。构建了受溶氧和硝酸盐协同诱导的辅酶Q10关键酶强化系统，以增强其辅酶Q10合成能力。并对构建的菌种DE3Q104，通过PCR扩增和A-T克隆，进行测序确认其结构正确无误。对比E. coli DE3Q10、DE3Q101、DE3Q102、DE3Q103、DE3Q104，其与各自的出发菌株相比，DE3Q101辅酶Q10产量平均提高63.8%，DE3Q102平均提高19.9%，DE3Q103平均提高50.0%，DE3Q104平均提高52.9%。与原始出发菌株DE3Q10相比，则分别提高了63.8%、96.5%、194.7%、350.7%。这表明，通过在nirB和narG两个受限制性溶氧和硝酸盐双重调控操纵子中，以辅酶Q10合成的关键酶基因替换其结构基因，并以限制性溶氧和硝酸盐进行诱导，可以正确表达这些基因的产物，并可以在后续的有氧条件下，正常催化合成辅酶Q10。确立了E. coli DE3Q104高产辅酶Q10的工艺路线和条件。工艺路线为先将构建的大肠杆菌培养至OD600至0.3左右，然后通过厌氧环境和硝酸盐对整合入nirB和narG两个操纵子的辅酶Q10关键酶基因进行诱导表达，然后再转入好氧环境中，利用诱导所产生的酶进行催化合成辅酶Q10，反复厌氧诱导与好氧合成的过程，直至获得辅酶Q10最大产量。最佳工作条件为：4mM硝酸盐浓度结合无氧条件下诱导25min，而后在溶氧充足条件下培养2h，后重复上述诱导合成过程3次。干菌体中辅酶Q10的最高产量可达4.5mg/g以上。实验同时发现，对于构建的菌株E. coli DE3Q104，温度和pH对其合成辅酶Q10的能力没有显著影响。本实验通过基因敲入方式，利用大肠杆菌的nirB和narG两个操纵子进行多酶代谢途径重组和表达构建出了辅酶Q10高产菌种，建立了改造微生物菌种、增强工程菌遗传稳定性和提高产物产量的新方法，为其它代谢产物生产菌种的构建提供了有益的借鉴，具有重要的科学和适用价值。更多还原

【Abstract】 Coenzyme Q10has multiple physiological functions of organisms, and now it is usedwidely in medicine, cosmetics, food, health products and other fields, but due to the lack ofhigh yield of coenzyme Q10strain, the developments of coenzyme Q10and its relatedindustry have been limited for a long time.In this experiment, based on cloning of decaprenyl pyrophosphate synthase gene (ddsA),1-deoxy-D-xylulose-5-phosphate synthase gene (dxs), p-hydroxybenzoatepolyprenylpyrophosphate transferase gene (ubiA) from Rhizobium radiobacter and farnesylpyrophosphate synthase gene (ispA), isopentenyldiphosphateisomerase gene (idi) fromEscherichia coli, the length uncertainty of polyprenyl pyrophosphate catalyzed by ddsA geneexpressed DPS was focused on. The enzymes conserved domain were analyzed bybioinformatics. Decaprenyldiphosphate synthase and farnesyldiphosphate synthase possessthe similar conserved domain, except for the chain length determination region. DXP synthasehas two thiamine binding regions. Its N-terminal is highly conserved with the N-terminal oftransketolase and E1subunit of pyruvate dehydrogenase. E. coli IDI belongs to Nudixdehydrogenase superfamily with the nudix motif GHPQLGESNEDAVIRRCRYELGV.Usinggene knock-in technology, thestrain E. coli DE3Q9was constructed with strain E. coli DE3ispB replaced by ddsA.After strengthened with key enzymes genes of CoQ10synthesis in E.coli DE3Q9, such as ddsA、ubiA、ispA、idi, production of CoQ10can be increased126%, to1.180.52mg·g-1DCM from0.52mg·g-1DCM.By exploring the effects of temperature, dissolved oxygen, pH and limiting nutrientsubstrate concentration (peptone) on the E. coli DE3Q9, it was found that R. radiobacterDPScatalytic specificity is mainly affected by temperature significantly.It is suggested that thepolyprenyl chain length is determined not only by the polyprenydiphosphate synthase’straditional "cavity" structure theory, but also by its structural thermal stability. Based on thehomology modeling of the R. radiobacter DPS and the analysis of the structural B-factor,four potential mutation point for increasing the thermal stability, i.e. Q64P, M92L, W342Land K343R, were determined. After the replacement ofispB in E. coli DE3by mutant genes,it was found that the mutants of M92L, W342L and K343R could synthesize only CoQ10rather than CoQ9even under high temperature. Compared with structure, the reason ofstrength of mutant thermal stability is the decreasing of stereospecific blockade and increasingof H-bond number. This made the local energy of mutant more reasonable than the wild type.According to the principle of homologous recombination, the key enzymes’ genes ofCoQ10biosynthesis, ddsA, ubiA andidi, ispA were knocked-in the genome of E. coli DE3Q10by the replacement of the structural genes ofnirB operon, namely nirB, nirDandnirC in turns,and correspondingly strains E. coli DE3Q101, E. coli DE3Q102and E. coli DE3Q103wereconstructed. Strain E. coli DE3Q104was constructed bydxsgene substituting for narGgen innarGoperon of E. coli DE3Q103. The key enzymes enhancement system of CoQ10biosynthesis under regulation of both limited dissolved oxygen and nitrate was constructed forincreasing the CoQ10yield. The structures of gene knock-in were determined by PCR andsequences analysis.Compared with them starting strain, the CoQ10products of E. coli DE3Q101, DE3Q102,DE3Q103and DE3Q104increased63.8%,19.9%,50.0%and52.9%respectively in average.Compared with the E. coli DE3Q10, the CoQ10products of E. coli DE3Q101, DE3Q102,DE3Q103and DE3Q104increased63.8%,96.5%,194.7%and350.7%respectively. Thisshows that the key enzyme genes knocked-in nirBand narGoperon could be expressed underthe induction of both anaerobic condition and nitrate correctly, and could catalyze thebiosynthesis of CoQ10in the following aerobic cultivation normally.The optimal process conditions for high-yield CoQ10of the strain E. coli DE3Q104areinduction under anaerobic condition and4mM nitrate for25min, then aerobic culture for2hr,and repeating the previous operation3times. More than4.5mg·g-1of CoQ10DCM can beobtained. At the same time, it was found that the pH and temperature had no effects on theability of E. coli DE3Q104CoQ10biosynthesis.With multi-enzymes genes integrated innirB andnarG operon of E. coliandexpressedsimultaneously through gene knock-in, high-yieldCoQ10strain was constructed. Anew technology was verified for transformation of microbial strains, strength of engineeringbacteria genetic stability, improvement of products yield. It provides a useful reference for thestrains construction for other metabolites production. It has important scientific and appliedvalue.更多还原