利用重组大肠杆菌生产L-色氨酸的研究
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摘要
L-色氨酸作为一种非常重要的芳香性氨基酸,是人体必需的八种氨基酸之一在生物体内,L-色氨酸可以合成5-羟基色胺、烟酸、色素、生物碱、辅酶、吲哚乙酸等重要的生物活性物质,对人和动物的生长发育起重要作用,广泛应用于食品、医药、饲料等方面。目前,世界市场色氨酸年需求量在万吨以上,并且以每年10%的速度增长。在医学领域,L-色氨酸广泛应用于氨基酸注射液、必需氨基酸药品及水解蛋白质的添加剂等。L-色氨酸的转化产物5-羟色胺,具有抗抑郁症、提高睡眠质量、抗高血压及镇痛作用。作为食品添加剂,L-色氨酸可以强化机体对植物蛋白的利用效率。在饲料中添加L-色氨酸,可以调节饲料中氨基酸的平衡,促进家禽家畜的生长。
L-色氨酸的生产最早主要依靠化学合成法和蛋白质水解法,但是这些方法存在着材料来源有限、周期长、工艺复杂等缺点,因而逐渐被淘汰。由于具有的成本低廉、原料来源广泛、环境污染小等特点,微生物生产L-色氨酸已经得到了广泛的应用。微生物法大体上可以分为直接发酵法、微生物转化法和酶法。
微生物转化法采用糖类作为碳源,同时添加L-色氨酸的前体物如邻氨基苯甲酸、吲哚等,利用微生物的L-色氨酸合成酶系来合成L-色氨酸。但是当转化液中前体物浓度较高时,转化率会出现下降。另外由于前体物的价格昂贵,不利于降低成本。酶法是利用微生物中L-色氨酸生物合成酶系催化功能生产L-色氨酸。它能够利用化工合成的前体为原料,同时又具有产物浓度高、纯度高、副产物少、操作简便等优点,是一种成本较低的生产L-色氨酸的工业化生产方法。但是该方法需要筛选高活力的酶和较高的底物浓度推动反应的进行,反应平衡不容易掌握。直接发酵法是以葡萄糖等廉价原料为碳源,利用筛选的如谷氨酸棒杆菌、大肠杆菌等优良的L-色氨酸生产菌种,生产L-色氨酸。对这种方法的研究起步比较早,但是在相当长的一段时间内达不到工业化生产的要求。主要原因是从葡萄糖到L-色氨酸的生物合成途径较长,在正常情况下代谢流也比较弱。而且L-色氨酸的合成需要多种前体物,生物合成途径中的调控机制比较复杂,种种因素限制了L-色氨酸产量的进一步提高。
近年来,随着代谢工程、转录组学和合成生物学的发展,人们通过分析代谢途径,过量表达内源基因或引入外源基因,消除竞争支路,成功构建了合成特定目标化合物并用于工业化生产的工程菌株。目前代谢工程在微生物合成氨基酸、有机酸、萜类化合物、聚羟基脂肪酸酯、生物燃料等领域得到了广泛的应用。大肠杆菌因其易于培养、遗传背景清楚、遗传操作简便等优点被大规模应用于代谢工程改造用于合成各种有价值的化合物。
针对L-色氨酸合成途径中的众多限制性因素,本论文首先利用基因的敲除、定点突变及过量表达技术,对野生型大肠杆菌W3110进行了代谢工程改造。通过敲除阻遏蛋白TrpR,解除了其对L-色氨酸合成途径中的关键酶的阻遏;通过敲除色氨酸酶TnaA,降低了已经合成的L-色氨酸在胞内的降解；通过敲除编码葡萄糖依赖的磷酸转移酶系统中酶IIBC组分的ptsG基因,使大肠杆菌依赖于磷酸烯醇式丙酮酸的葡萄糖转运系统部分失活,降低了细胞内磷酸烯醇式丙酮酸(PEP)的消耗,从而能更多的流入L-色氨酸合成途径。为了进一步提高L-色氨酸的产量,在低拷贝质粒pCL1920上过量表达了定点突变解除反馈抑制的邻氨基苯甲酸合成酶trpE3、3-脱氧-D-阿拉伯庚酮酸-7磷酸(DAHP)合酶aroGl以及磷酸戊糖途径中编码转酮酶的基因tktA,构建了质粒pTAT,并且转入重组大肠杆菌中构建了L-色氨酸基本产生菌株GPT1001。摇瓶发酵显示该菌株的L-色氨酸可以达到1.2g1-1,为下一步的改造奠定了基础。
色氨酸衰减子是重要的L-色氨酸合成调控元件,能够精细控制细胞内L-色氨酸的合成。另外,色氨酸操纵子的转录水平对L-色氨酸的产量也有着十分重要的影响。为了消除大肠杆菌色氨酸衰减作用并且提高色氨酸操纵子的表达水平,我们利用五个拷贝串联重复的tac核心启动子组成的5CPtacs启动子,一步法完成了色氨酸衰减子的敲除及色氨酸操纵子野生型启动子的替换,成功构建了重组大肠杆菌GPT1002。通过RT-PCR检测发现五个色氨酸操纵子基因的转录均出现上调,上调幅度为1.67-9.21倍不等。其中trpE基因的转录水平提高约9.21倍,而其他四个基因转录水平的只提高了约2倍。另外,aroG基因转录水平也上调了9.29倍。摇瓶发酵实验表明,GPT1002的生长未受到影响,经过36h培养,可以积累1.7g1-1的L-色氨酸,比对照菌株GPT1001高出31%。最后,对GPT1002进行了5-1发酵罐发酵, L-色氨酸产量可以达到10.15g1-1,证明了我们代谢途径改造是有效的。
在大肠杆菌中W3110中,存在着Mtr、TnaB和AroP三个透过酶参与L-色氨酸的吸收。为了研究透过酶的敲除对L-色氨酸产量的影响,我们在L-色氨酸生产菌株GPT1002的基础上,构建了三个L-色氨酸透过酶的单敲除菌株,并进行了L-色氨酸的吸收能力进行了检测,结果表明TnaB的缺失能够有效降低细胞的L-色氨酸吸收能力。摇瓶发酵检测中,TnaB缺失菌株L-色氨酸的产量最高,能够达到2.05g1-1。在单敲除菌株的基础上我们又构建了L-色氨酸透过酶双敲除菌株GPT1014、GPT1015和GPT1016,摇瓶发酵显示,所有的双敲除菌株生长都受到了严重影响,最大OD600约为初始菌株GPT1002的一半左右。其中AroP和TnaB双敲除菌株GPT1014呈现出了最高的L-色氨酸产量,2.44g1-1。另外两个双敲除菌株L-色氨酸产量极低,只有50-60mg1-1。最后,我们构建了L-色氨酸透过酶三敲除菌株GPT1017。 GPT1017在摇瓶发酵中的生长得到了恢复,L-色氨酸产量也超过了所有的突变菌株和初始菌株,达到2.79g1-1,比GPT1002高出51.6%。在5-1发酵罐中,L-色氨酸产量可以进一步提高,达到16.3g1-1。另外,我们还对构建的敲除突变菌株的三个中心代谢途径的关键基因,柠檬酸合成酶、葡萄糖-6-磷酸异构酶、葡萄糖-6-磷酸脱氢酶的转录水平进行了检测。在突变菌株中,三个基因的转录水平均明显下降,特别是在GPT1015和GPT1016中,三个基因的转录水平下降到对照菌株的0.01-0.08倍左右,这说明透过酶的敲除能够直接或者间接的影响代谢流量的分布。而在GPT1017中,这三个基因的转录水平有不同程度的恢复,这也与该菌体的生长和L-色氨酸产量恢复的结果也是一致的。
聚β-羟基丁酸(PHB)是研究的最为透彻的一类PHA,在细菌体内主要被用作碳源和能源的储备物。为了减少重组菌株GPT1002的乙酸分泌,提高细胞对碳源的利用效率,我们将来自真氧产碱杆菌的phaCAB操纵子转入GPT1002,构建了L-色氨酸和PHB联产菌株GPT2000。在摇瓶发酵的条件下,成功检测到了PHB的积累,并且L-色氨酸的产量比对照提高了11.6%。RT-PCR结果表明,PHB的积累能够显著提高色氨酸操纵子基因的转录,最高可达4倍左右,这可能是L-色氨酸产量提高的原因。考虑到木糖的添加可能有利于L-色氨酸前体物E4P的合成,同时可以节约葡萄糖运输过程中消耗的PEP,我们检测了以不同比例的葡萄糖和木糖作为碳源对两种产物及副产物乙酸的影响。结果表明,PHB的积累和乙酸的分泌随着木糖比例的的增加而增加,当以木糖为唯一碳源时两者均达到最大值。而对于L-色氨酸,在16g1-1葡萄糖和4g1-1木糖条件下,产量达到最高2.24g1-1。在最优的葡萄糖和木糖比例条件下,我们对重组菌株GPT2000进行了5-1发酵罐发酵,L-色氨酸和PHB的产量分别可以达到14.4g1-1和9.7%(w／w)。
最后我们建立了一种能够在基因组上整合随机拷贝数基因片段的方法。利用条件复制子oriR6Ky,构建了整合质粒pTKG,并利用卡那霉素抗性基因和绿色荧光蛋白作为报告基因进行筛选。实验结果表明,我们成功在基因组上随机整合了1-12个拷贝的基因片段。考虑到恢复培养时间的长短可能会影响整合的最大拷贝数,我们检测了不同恢复培养时间的影响。1h的恢复培养时间对我们的随机拷贝数的整合是最佳的,增加或减少恢复培养时间反而会降低整合的最大拷贝数。接下来,通过在重组大肠杆菌GPT102T基因组上整合不同拷贝数的aroK基因并分别转入质粒pTAT进行发酵检测,我们发现在基因组上存在3个拷贝的aroK基因时,单位菌体的L-色氨酸产量最高。
本论文首先构建了L-色氨酸基本产生菌株GPT1001,并在此基础上通过启动子替换等手段,提高了L-色氨酸的产量。本论文系统研究了大肠杆菌L-色氨酸透过酶的敲除对L-色氨酸产量的影响,并首次构建了L-色氨酸和PHB的联产菌株,检测了PHB积累对L-色氨酸合成及色氨酸操纵子转录水平的影响。最后,本论文创新性的设计了在基因组上整合随机拷贝数基因的方法,该方法在代谢工程等领域有广阔的应用。本论文的工作为L-色氨酸的工业化生产提供了重要的研究基础。
L-tryptophan is an essential aromatic amino acid for humans and animals which can be used as food additive, infusion liquids, pellagra treatment, sleep induction and nutritional therapy. It can synthesize serotonin, nicotinic acid, pigment, alkaloid, coenzyme, indoleacetic acid and so on. Nowadays, the worldwide demand for L-tryptophan is above ten thousand tons and is increased by almost10%every year. In the medical and pharmacological field, L-tryptophan is widely used in amino acid injection, essential amino acid Pharmaceuticals and hydrolyzed protein additives. The conversion product of L-tryptophan, serotonin, could improve sleep quality, and can be used for antihypertensive, antidepressant and treatment of pellagra. As a food additive, L-tryptophan can enhance the utilization efficiency of the vegetable protein. It can balance the amino acids in the feed and promote the growth of livestock by adding L-tryptophan in the feed.
In the past, L-tryptophan was mainly produced by the chemical synthesis and protein hydrolysis, but these methods were limited for the sources of material, the long and complex process, which were gradually eliminated. With the advantage of low-cost and environmental friendly, microbial L-tryptophan production method has been widely used. The microbial production method comprises microbial transformation, enzymatic methods and direct fermentation.
For microbial transformation method, sugars are used as carbon source, and precursors of L-tryptophan, such as anthranilate and indole are simultaneously added into the medium. However, the conversion rate will decreased when the concentration of precursors in the conversion solution are too high. In addition, it is not conducive to lower the cost due to the utilization of expensive precursors. In the method of enzymatic synthesis, L-tryptophan is produced using L-tryptophan biosynthetic enzymes of microorganism. It can take advantage of the chemical synthesis precursors as the raw materials, and has the advantages of high product concentration, high purity, less by-product, and easily operation, and therefore it is a low-cost L-tryptophan industrialized production method. However, this method requires high activity of enzymes and high concentration of the substrates to promote the reaction, as a result, the reaction balance is not easy to manipulate. Direct fermentation method uses excellent L-tryptophan-producing strains such as Corynebacterium glutamicum and Escherichia coli to produce L-tryptophan with cheap raw material such as glucose as carbon source. This method has been studied for a long time, but it had not been reached the titer of industrial requirement until now. High production of L-tryptophan is limited by the long biosynthetic pathway from glucose to L-tryptophan, the relatively low metabolic flow in normal circumstances, the requirements of a variety of precursors and the relatively complex regulation mechanism in L-tryptophan biosynthetic pathway.
In recent years, with the development of metabolic engineering, transcription genomics and synthetic biology, through the analysis of metabolic pathways, overexpression of an endogenous gene or the introduction of exogenous genes, and elimination of competition branch, several engineered strains for industrial production of the particular target compound have been constructed. Metabolic engineering has been widely used in microbial synthesis of amino acids, organic acids, terpenoids, polyhydroxyalkanoate and biofuels. E. coli, because of its easy cultivation, clear genetic background and simple genetic manipulation, has been widely used in large-scale synthesis of valuable compounds.
According to the limiting factors in L-tryptophan synthesis pathway, to generate an E. coli that overproduces and excretes L-tryptophan, the following manipulation was done:First, trpR gene, which encodes a tryptophan transcriptional repressor, was knocked out to eliminate transcription regulation of the genes in L-tryptophan pathway. Second, trpE and aroG, encoding component I of anthranilate synthase and DAHP synthase, respectively, were overexpressed after site-directed mutations to remove the feedback inhibited. Third, tktA gene, encoding a transketolase in pentose phosphate pathway. Otherwise, we knocked out ptsG, which encodes the11BC component of glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase (PTS) system, to provide more PEP. Finally, we knocked out the gene tnaA, which encodes a tryptophanase that catalyzes the reaction of L-tryptophan back into indole. The resulting L-tryptophan-synthetic strain GPT1001was able to produce1.3g l-1L-tryptophan in batch cultivation and was therefore used as base strain for further experiment.
The expression of tryptophan biosynthesis operon was negatively regulated by the attenuator. However, simply removal of the attenuator probably cannot reach a sufficient expression of the tryptophan operon genes. Therefore it is essential to improve the expression of genes in tryptophan operon at the same time of inactivating the attenuator. Therefore we constructed recombinant E. coli GPT1002by inactivating the tryptophan attenuator and replacing the original trppromoter of tryptophan operon with a novel promoter cluster consisted of five core-tac-promoters aligned in tandem (5CPtacs promoter cluster) in one step. Strain GPT1002exhibited1.67-9.29times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated1.70g l-1L-tryptophan after36h batch cultivation. Bioreactor fermentation experiments showed that GPT1002could produce10.15g l-1L-tryptophan.
In E. coli, there are three tryptophan permeases, AroP, TnaB, and Mtr. To study the function of individual permease, we knocked out these three genes aroP, tnaB, and mtr separately in L-tryptophan producing strain GPT101. Then three mutants were subjected to L-tryptophan uptake assay, and it showed knocking out of tnaB decreased the L-tryptophan utilization from0.058g l-1h-1to0.054l-1h-1per OD600. And then the L-tryptophan permease mutants were compared with respect to their growth under L-tryptophan accumulating conditions. The result is consistent with the L-tryptophan uptake assay, and proved that tnaB is the main transporter responsible for the L-tryptophan uptake in E. coli.To further understand the tryptophan permease function, we constructed the double mutants of L-tryptophan permeases. Cultivation of these mutants showed that they all grew poorly comparing to the control. GPT1014, which possesses aroP and tnaB double inactivation, showed highest L-tryptophan production of2.44g l-1,19.02%higher than tnaB single mutant GPT1012, and32.6%higher than the control. And then, we constructed the triple mutant of L-tryptophan permeases GPT1017. In spite of containing mtr, GPT1017exhibited restored cell growth compare to the double mutant of tryptophan permeases. The L-tryptophan production of GPT1017was2.79g l-1in batch cultivation,51.6%higher than the control strain GPT1002. In fermentor, the maximum L-tryptophan production of strain GPT1017reached16.3g l-1at66h. Finally, we performed RT-PCR analysis of three key genes, citrate synthase, glucose-6-phosphate dehydrogenase, and glucosephosphate isomerase in E. coli, respectively. These genes showed decreased transcription in all mutants, especially in GPT1015and GPT1016, in which they were down-regulated to0.01-0.08fold of the level in GPT1002. However, the transcription of gltA, zwf and pgi were restored in tryptophan permeases deficient mutant GPT1017.
Polyhydroxybutyrate (PHB), the best known polyhydroxyalkanoates (PHA) has been believed to change intracellular metabolic flow and oxidation/reduction state, as well as enhance stress resistance of the host. In this study, a PHB biosynthesis pathway, which contains phaCAB operon genes from Ralstonia eutropha, was introduced into an L-tryptophan producing Escherichia coli strain GPT1002. The expression of the PHB biosynthesis genes resulted in PHB accumulation inside the cells and improved the L-tryptophan production. RT-PCR analysis showed that the transcription of tryptophan operon genes in GPT2000increased by1.9-4.3times compared with the control. Xylose was then added into the medium as co-substrate to enhance the precursor supply for PHB biosynthesis. The PHB accumulation in this strain reached17.25%(w/w), the highest polymer accumulation among all tested strains. For L-tryptophan production, the mixture of16g l-1glucose and4g1-1xylose was the best. Under this condition, the highest L-tryptophan production,2.24±0.41g l-1was obtained. In addition, the secretion of acetate in the medium was also increased with the increased xylose proportion in the medium. Moreover, we obtained14.4g l-1L-tryptophan production and9.7%PHB (w/w) accumulation in GPT2000 via fed-batch cultivation.
Finally, a method of integrating random-copy genes into E. coli genome was carried out. By utilizing the condition-replicon oriR6Ky, and the kanamycin resistance gene and the GFP as a reporter gene, integration plasmid pTKG was constructed. After FLP/FRT recombination, it showed that1-12copies of the kan-trc-gfp gene were successfully integrated into the genome. Considering the recovery incubation time may affect the integration copy number, we examined the impact of different recovery incubation time on random integration copy numbers. It showed1h is the best incubation time, and increasing or decreasing the time would reduce the maximum integration copy number. By integration random copies of aroK gene encoding shimikate kinase in the genome of the L-tryptophan-producing strain GPT102T and performed batch fermentation, we found that three copies of aroK in the genome result in the highest production of L-tryptophan per unit of cell dry weight (CDW).
In this study, we firstly construct a base L-tryptophan production strains GPT1001. By use of one-step of tryptophan attenuator inactivation and promoter swapping to generate E. coli GPT1002, the L-tryptophan production was improved further. And then, the knocking out analysis of L-tryptophan permease on the production of L-tryptophan was carried out carefully. In addition, an L-tryptophan and PHB co-producing strain was constructed for the first time. The PHB accumulation was verified to improve L-tryptophan production and tryptophan transcription level. Finaly, we designed a novel method of random copies of gene integration into genome which could be widely used in metabolic engineering.
L-色氨酸的生产最早主要依靠化学合成法和蛋白质水解法,但是这些方法存在着材料来源有限、周期长、工艺复杂等缺点,因而逐渐被淘汰。由于具有的成本低廉、原料来源广泛、环境污染小等特点,微生物生产L-色氨酸已经得到了广泛的应用。微生物法大体上可以分为直接发酵法、微生物转化法和酶法。
微生物转化法采用糖类作为碳源,同时添加L-色氨酸的前体物如邻氨基苯甲酸、吲哚等,利用微生物的L-色氨酸合成酶系来合成L-色氨酸。但是当转化液中前体物浓度较高时,转化率会出现下降。另外由于前体物的价格昂贵,不利于降低成本。酶法是利用微生物中L-色氨酸生物合成酶系催化功能生产L-色氨酸。它能够利用化工合成的前体为原料,同时又具有产物浓度高、纯度高、副产物少、操作简便等优点,是一种成本较低的生产L-色氨酸的工业化生产方法。但是该方法需要筛选高活力的酶和较高的底物浓度推动反应的进行,反应平衡不容易掌握。直接发酵法是以葡萄糖等廉价原料为碳源,利用筛选的如谷氨酸棒杆菌、大肠杆菌等优良的L-色氨酸生产菌种,生产L-色氨酸。对这种方法的研究起步比较早,但是在相当长的一段时间内达不到工业化生产的要求。主要原因是从葡萄糖到L-色氨酸的生物合成途径较长,在正常情况下代谢流也比较弱。而且L-色氨酸的合成需要多种前体物,生物合成途径中的调控机制比较复杂,种种因素限制了L-色氨酸产量的进一步提高。
近年来,随着代谢工程、转录组学和合成生物学的发展,人们通过分析代谢途径,过量表达内源基因或引入外源基因,消除竞争支路,成功构建了合成特定目标化合物并用于工业化生产的工程菌株。目前代谢工程在微生物合成氨基酸、有机酸、萜类化合物、聚羟基脂肪酸酯、生物燃料等领域得到了广泛的应用。大肠杆菌因其易于培养、遗传背景清楚、遗传操作简便等优点被大规模应用于代谢工程改造用于合成各种有价值的化合物。
针对L-色氨酸合成途径中的众多限制性因素,本论文首先利用基因的敲除、定点突变及过量表达技术,对野生型大肠杆菌W3110进行了代谢工程改造。通过敲除阻遏蛋白TrpR,解除了其对L-色氨酸合成途径中的关键酶的阻遏;通过敲除色氨酸酶TnaA,降低了已经合成的L-色氨酸在胞内的降解；通过敲除编码葡萄糖依赖的磷酸转移酶系统中酶IIBC组分的ptsG基因,使大肠杆菌依赖于磷酸烯醇式丙酮酸的葡萄糖转运系统部分失活,降低了细胞内磷酸烯醇式丙酮酸(PEP)的消耗,从而能更多的流入L-色氨酸合成途径。为了进一步提高L-色氨酸的产量,在低拷贝质粒pCL1920上过量表达了定点突变解除反馈抑制的邻氨基苯甲酸合成酶trpE3、3-脱氧-D-阿拉伯庚酮酸-7磷酸(DAHP)合酶aroGl以及磷酸戊糖途径中编码转酮酶的基因tktA,构建了质粒pTAT,并且转入重组大肠杆菌中构建了L-色氨酸基本产生菌株GPT1001。摇瓶发酵显示该菌株的L-色氨酸可以达到1.2g1-1,为下一步的改造奠定了基础。
色氨酸衰减子是重要的L-色氨酸合成调控元件,能够精细控制细胞内L-色氨酸的合成。另外,色氨酸操纵子的转录水平对L-色氨酸的产量也有着十分重要的影响。为了消除大肠杆菌色氨酸衰减作用并且提高色氨酸操纵子的表达水平,我们利用五个拷贝串联重复的tac核心启动子组成的5CPtacs启动子,一步法完成了色氨酸衰减子的敲除及色氨酸操纵子野生型启动子的替换,成功构建了重组大肠杆菌GPT1002。通过RT-PCR检测发现五个色氨酸操纵子基因的转录均出现上调,上调幅度为1.67-9.21倍不等。其中trpE基因的转录水平提高约9.21倍,而其他四个基因转录水平的只提高了约2倍。另外,aroG基因转录水平也上调了9.29倍。摇瓶发酵实验表明,GPT1002的生长未受到影响,经过36h培养,可以积累1.7g1-1的L-色氨酸,比对照菌株GPT1001高出31%。最后,对GPT1002进行了5-1发酵罐发酵, L-色氨酸产量可以达到10.15g1-1,证明了我们代谢途径改造是有效的。
在大肠杆菌中W3110中,存在着Mtr、TnaB和AroP三个透过酶参与L-色氨酸的吸收。为了研究透过酶的敲除对L-色氨酸产量的影响,我们在L-色氨酸生产菌株GPT1002的基础上,构建了三个L-色氨酸透过酶的单敲除菌株,并进行了L-色氨酸的吸收能力进行了检测,结果表明TnaB的缺失能够有效降低细胞的L-色氨酸吸收能力。摇瓶发酵检测中,TnaB缺失菌株L-色氨酸的产量最高,能够达到2.05g1-1。在单敲除菌株的基础上我们又构建了L-色氨酸透过酶双敲除菌株GPT1014、GPT1015和GPT1016,摇瓶发酵显示,所有的双敲除菌株生长都受到了严重影响,最大OD600约为初始菌株GPT1002的一半左右。其中AroP和TnaB双敲除菌株GPT1014呈现出了最高的L-色氨酸产量,2.44g1-1。另外两个双敲除菌株L-色氨酸产量极低,只有50-60mg1-1。最后,我们构建了L-色氨酸透过酶三敲除菌株GPT1017。 GPT1017在摇瓶发酵中的生长得到了恢复,L-色氨酸产量也超过了所有的突变菌株和初始菌株,达到2.79g1-1,比GPT1002高出51.6%。在5-1发酵罐中,L-色氨酸产量可以进一步提高,达到16.3g1-1。另外,我们还对构建的敲除突变菌株的三个中心代谢途径的关键基因,柠檬酸合成酶、葡萄糖-6-磷酸异构酶、葡萄糖-6-磷酸脱氢酶的转录水平进行了检测。在突变菌株中,三个基因的转录水平均明显下降,特别是在GPT1015和GPT1016中,三个基因的转录水平下降到对照菌株的0.01-0.08倍左右,这说明透过酶的敲除能够直接或者间接的影响代谢流量的分布。而在GPT1017中,这三个基因的转录水平有不同程度的恢复,这也与该菌体的生长和L-色氨酸产量恢复的结果也是一致的。
聚β-羟基丁酸(PHB)是研究的最为透彻的一类PHA,在细菌体内主要被用作碳源和能源的储备物。为了减少重组菌株GPT1002的乙酸分泌,提高细胞对碳源的利用效率,我们将来自真氧产碱杆菌的phaCAB操纵子转入GPT1002,构建了L-色氨酸和PHB联产菌株GPT2000。在摇瓶发酵的条件下,成功检测到了PHB的积累,并且L-色氨酸的产量比对照提高了11.6%。RT-PCR结果表明,PHB的积累能够显著提高色氨酸操纵子基因的转录,最高可达4倍左右,这可能是L-色氨酸产量提高的原因。考虑到木糖的添加可能有利于L-色氨酸前体物E4P的合成,同时可以节约葡萄糖运输过程中消耗的PEP,我们检测了以不同比例的葡萄糖和木糖作为碳源对两种产物及副产物乙酸的影响。结果表明,PHB的积累和乙酸的分泌随着木糖比例的的增加而增加,当以木糖为唯一碳源时两者均达到最大值。而对于L-色氨酸,在16g1-1葡萄糖和4g1-1木糖条件下,产量达到最高2.24g1-1。在最优的葡萄糖和木糖比例条件下,我们对重组菌株GPT2000进行了5-1发酵罐发酵,L-色氨酸和PHB的产量分别可以达到14.4g1-1和9.7%(w／w)。
最后我们建立了一种能够在基因组上整合随机拷贝数基因片段的方法。利用条件复制子oriR6Ky,构建了整合质粒pTKG,并利用卡那霉素抗性基因和绿色荧光蛋白作为报告基因进行筛选。实验结果表明,我们成功在基因组上随机整合了1-12个拷贝的基因片段。考虑到恢复培养时间的长短可能会影响整合的最大拷贝数,我们检测了不同恢复培养时间的影响。1h的恢复培养时间对我们的随机拷贝数的整合是最佳的,增加或减少恢复培养时间反而会降低整合的最大拷贝数。接下来,通过在重组大肠杆菌GPT102T基因组上整合不同拷贝数的aroK基因并分别转入质粒pTAT进行发酵检测,我们发现在基因组上存在3个拷贝的aroK基因时,单位菌体的L-色氨酸产量最高。
本论文首先构建了L-色氨酸基本产生菌株GPT1001,并在此基础上通过启动子替换等手段,提高了L-色氨酸的产量。本论文系统研究了大肠杆菌L-色氨酸透过酶的敲除对L-色氨酸产量的影响,并首次构建了L-色氨酸和PHB的联产菌株,检测了PHB积累对L-色氨酸合成及色氨酸操纵子转录水平的影响。最后,本论文创新性的设计了在基因组上整合随机拷贝数基因的方法,该方法在代谢工程等领域有广阔的应用。本论文的工作为L-色氨酸的工业化生产提供了重要的研究基础。
L-tryptophan is an essential aromatic amino acid for humans and animals which can be used as food additive, infusion liquids, pellagra treatment, sleep induction and nutritional therapy. It can synthesize serotonin, nicotinic acid, pigment, alkaloid, coenzyme, indoleacetic acid and so on. Nowadays, the worldwide demand for L-tryptophan is above ten thousand tons and is increased by almost10%every year. In the medical and pharmacological field, L-tryptophan is widely used in amino acid injection, essential amino acid Pharmaceuticals and hydrolyzed protein additives. The conversion product of L-tryptophan, serotonin, could improve sleep quality, and can be used for antihypertensive, antidepressant and treatment of pellagra. As a food additive, L-tryptophan can enhance the utilization efficiency of the vegetable protein. It can balance the amino acids in the feed and promote the growth of livestock by adding L-tryptophan in the feed.
In the past, L-tryptophan was mainly produced by the chemical synthesis and protein hydrolysis, but these methods were limited for the sources of material, the long and complex process, which were gradually eliminated. With the advantage of low-cost and environmental friendly, microbial L-tryptophan production method has been widely used. The microbial production method comprises microbial transformation, enzymatic methods and direct fermentation.
For microbial transformation method, sugars are used as carbon source, and precursors of L-tryptophan, such as anthranilate and indole are simultaneously added into the medium. However, the conversion rate will decreased when the concentration of precursors in the conversion solution are too high. In addition, it is not conducive to lower the cost due to the utilization of expensive precursors. In the method of enzymatic synthesis, L-tryptophan is produced using L-tryptophan biosynthetic enzymes of microorganism. It can take advantage of the chemical synthesis precursors as the raw materials, and has the advantages of high product concentration, high purity, less by-product, and easily operation, and therefore it is a low-cost L-tryptophan industrialized production method. However, this method requires high activity of enzymes and high concentration of the substrates to promote the reaction, as a result, the reaction balance is not easy to manipulate. Direct fermentation method uses excellent L-tryptophan-producing strains such as Corynebacterium glutamicum and Escherichia coli to produce L-tryptophan with cheap raw material such as glucose as carbon source. This method has been studied for a long time, but it had not been reached the titer of industrial requirement until now. High production of L-tryptophan is limited by the long biosynthetic pathway from glucose to L-tryptophan, the relatively low metabolic flow in normal circumstances, the requirements of a variety of precursors and the relatively complex regulation mechanism in L-tryptophan biosynthetic pathway.
In recent years, with the development of metabolic engineering, transcription genomics and synthetic biology, through the analysis of metabolic pathways, overexpression of an endogenous gene or the introduction of exogenous genes, and elimination of competition branch, several engineered strains for industrial production of the particular target compound have been constructed. Metabolic engineering has been widely used in microbial synthesis of amino acids, organic acids, terpenoids, polyhydroxyalkanoate and biofuels. E. coli, because of its easy cultivation, clear genetic background and simple genetic manipulation, has been widely used in large-scale synthesis of valuable compounds.
According to the limiting factors in L-tryptophan synthesis pathway, to generate an E. coli that overproduces and excretes L-tryptophan, the following manipulation was done:First, trpR gene, which encodes a tryptophan transcriptional repressor, was knocked out to eliminate transcription regulation of the genes in L-tryptophan pathway. Second, trpE and aroG, encoding component I of anthranilate synthase and DAHP synthase, respectively, were overexpressed after site-directed mutations to remove the feedback inhibited. Third, tktA gene, encoding a transketolase in pentose phosphate pathway. Otherwise, we knocked out ptsG, which encodes the11BC component of glucose-specific phosphoenolpyruvate: carbohydrate phosphotransferase (PTS) system, to provide more PEP. Finally, we knocked out the gene tnaA, which encodes a tryptophanase that catalyzes the reaction of L-tryptophan back into indole. The resulting L-tryptophan-synthetic strain GPT1001was able to produce1.3g l-1L-tryptophan in batch cultivation and was therefore used as base strain for further experiment.
The expression of tryptophan biosynthesis operon was negatively regulated by the attenuator. However, simply removal of the attenuator probably cannot reach a sufficient expression of the tryptophan operon genes. Therefore it is essential to improve the expression of genes in tryptophan operon at the same time of inactivating the attenuator. Therefore we constructed recombinant E. coli GPT1002by inactivating the tryptophan attenuator and replacing the original trppromoter of tryptophan operon with a novel promoter cluster consisted of five core-tac-promoters aligned in tandem (5CPtacs promoter cluster) in one step. Strain GPT1002exhibited1.67-9.29times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated1.70g l-1L-tryptophan after36h batch cultivation. Bioreactor fermentation experiments showed that GPT1002could produce10.15g l-1L-tryptophan.
In E. coli, there are three tryptophan permeases, AroP, TnaB, and Mtr. To study the function of individual permease, we knocked out these three genes aroP, tnaB, and mtr separately in L-tryptophan producing strain GPT101. Then three mutants were subjected to L-tryptophan uptake assay, and it showed knocking out of tnaB decreased the L-tryptophan utilization from0.058g l-1h-1to0.054l-1h-1per OD600. And then the L-tryptophan permease mutants were compared with respect to their growth under L-tryptophan accumulating conditions. The result is consistent with the L-tryptophan uptake assay, and proved that tnaB is the main transporter responsible for the L-tryptophan uptake in E. coli.To further understand the tryptophan permease function, we constructed the double mutants of L-tryptophan permeases. Cultivation of these mutants showed that they all grew poorly comparing to the control. GPT1014, which possesses aroP and tnaB double inactivation, showed highest L-tryptophan production of2.44g l-1,19.02%higher than tnaB single mutant GPT1012, and32.6%higher than the control. And then, we constructed the triple mutant of L-tryptophan permeases GPT1017. In spite of containing mtr, GPT1017exhibited restored cell growth compare to the double mutant of tryptophan permeases. The L-tryptophan production of GPT1017was2.79g l-1in batch cultivation,51.6%higher than the control strain GPT1002. In fermentor, the maximum L-tryptophan production of strain GPT1017reached16.3g l-1at66h. Finally, we performed RT-PCR analysis of three key genes, citrate synthase, glucose-6-phosphate dehydrogenase, and glucosephosphate isomerase in E. coli, respectively. These genes showed decreased transcription in all mutants, especially in GPT1015and GPT1016, in which they were down-regulated to0.01-0.08fold of the level in GPT1002. However, the transcription of gltA, zwf and pgi were restored in tryptophan permeases deficient mutant GPT1017.
Polyhydroxybutyrate (PHB), the best known polyhydroxyalkanoates (PHA) has been believed to change intracellular metabolic flow and oxidation/reduction state, as well as enhance stress resistance of the host. In this study, a PHB biosynthesis pathway, which contains phaCAB operon genes from Ralstonia eutropha, was introduced into an L-tryptophan producing Escherichia coli strain GPT1002. The expression of the PHB biosynthesis genes resulted in PHB accumulation inside the cells and improved the L-tryptophan production. RT-PCR analysis showed that the transcription of tryptophan operon genes in GPT2000increased by1.9-4.3times compared with the control. Xylose was then added into the medium as co-substrate to enhance the precursor supply for PHB biosynthesis. The PHB accumulation in this strain reached17.25%(w/w), the highest polymer accumulation among all tested strains. For L-tryptophan production, the mixture of16g l-1glucose and4g1-1xylose was the best. Under this condition, the highest L-tryptophan production,2.24±0.41g l-1was obtained. In addition, the secretion of acetate in the medium was also increased with the increased xylose proportion in the medium. Moreover, we obtained14.4g l-1L-tryptophan production and9.7%PHB (w/w) accumulation in GPT2000 via fed-batch cultivation.
Finally, a method of integrating random-copy genes into E. coli genome was carried out. By utilizing the condition-replicon oriR6Ky, and the kanamycin resistance gene and the GFP as a reporter gene, integration plasmid pTKG was constructed. After FLP/FRT recombination, it showed that1-12copies of the kan-trc-gfp gene were successfully integrated into the genome. Considering the recovery incubation time may affect the integration copy number, we examined the impact of different recovery incubation time on random integration copy numbers. It showed1h is the best incubation time, and increasing or decreasing the time would reduce the maximum integration copy number. By integration random copies of aroK gene encoding shimikate kinase in the genome of the L-tryptophan-producing strain GPT102T and performed batch fermentation, we found that three copies of aroK in the genome result in the highest production of L-tryptophan per unit of cell dry weight (CDW).
In this study, we firstly construct a base L-tryptophan production strains GPT1001. By use of one-step of tryptophan attenuator inactivation and promoter swapping to generate E. coli GPT1002, the L-tryptophan production was improved further. And then, the knocking out analysis of L-tryptophan permease on the production of L-tryptophan was carried out carefully. In addition, an L-tryptophan and PHB co-producing strain was constructed for the first time. The PHB accumulation was verified to improve L-tryptophan production and tryptophan transcription level. Finaly, we designed a novel method of random copies of gene integration into genome which could be widely used in metabolic engineering.
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