【作者】 赵志军；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 L-色氨酸（L-tryptophan, L-Trp）作为人体内的一种必需氨基酸,广泛应用于医药、食品、饲料等行业。而微生物发酵法生产L-Trp由于具有原料廉价、产物纯度高、易提取等优点受到了越来越多的关注。近年来,国外通过代谢工程的手段,逐渐培育出一批高产的L-Trp生产菌株,但其在发酵过程中仍存在发酵后期L-Trp生产速率下降明显,副产物积累等问题,此外,上述工程菌的构建过程均涉及多轮的化学诱变,很难确认诱变过程中次级突变对菌株的影响,进而阻碍了菌种的进一步改良。而在国内,利用代谢工程手段选育L-Trp生产菌株的研究在2000年以后才逐渐得到展开,已构建菌株的L-Trp生产能力与国外同类菌株相比差距较大,直接限制了我国L-Trp生产行业的快速发展。针对以上国内外的研究现状,本论文运用代谢工程的研究策略,以E. coli W3110为出发菌株,在不涉及化学诱变的情况下,通过一系列的基因操作构建L-Trp的生产菌株,同时通过直接测定菌株20余种胞内代谢产物的浓度,分析了L-Trp合成代谢的流量分布特征,为进一步菌种改良提供了依据。主要的研究内容及结果如下:（1）通过定点突变删除3-脱氧-α-阿拉伯庚酮糖酸-7-磷酸（DAHP）合酶AroF第11位氨基酸残基Ile11,获得抗反馈抑制突变体AroF^fbr;通过置换邻氨基苯甲酸（ANTA）合酶第40位氨基酸残基Ser40为Phe,获得抗反馈抑制突变体TrpE^fbrD;利用Red重组技术,通过敲除E. coli W3110基因组上的trpR基因,解除了TrpR蛋白对trp操纵子转录的反馈阻遏调控;将基因aroF^fbr和trpE^fbrD克隆至低拷贝、具有PR、PL双启动子的原核表达载体pSV中,构建重组质粒pSV04,并转化至trpR敲除菌,获得工程菌FB-01/pSV04。发酵结果表明,发酵30 h,L-Trp产量为2.8 g/L,同时发酵液中有副产物积累,分别为:L-Phe 1.8 g/L,L-Tyr 1.4 g/L和ANTA 2.3 g/L。（2）在基因trpR敲除的基础上,通过敲除tnaA基因,切断菌体内的L-Trp分解途径,构建trpR.tnaA双基因敲除菌FB-02,获得工程菌FB-02/pSV04,发酵结果表明L-Trp产量大幅上升至7.8 g/L,但同时积累了较多的副产物,分别为L-Phe 5.3 g/L,L-Tyr 3.6 g/L,ANTA 5.4 g/L;为了阻止副产物L-Phe的积累,在菌株FB-02的基因组上敲除L-Phe分支途径的关键酶基因pheA,构建trpR.tnaA.pheA多基因敲除菌FB-03,工程菌FB-03/pSV04的发酵结果表明,发酵液中L-Phe积累量不足1 g/L,达到预期目的,但同时L-Tyr的积累量上升至5.1 g/L,而L-Trp产量提高至10.8 g/L;在菌株FB-03的基因组中进一步敲除L-Tyr分支途径中的关键酶基因tyrA,构建trpR.tnaA.pheA.tyrA多基因敲除菌FB-04,获得工程菌FB-04/pSV04;在基因pheA和tyrA均被敲除以后,需要在培养基中添加适量的L-Phe（2 g/L）和L-Tyr（3 g/L）,菌体FB-04/pSV04才能正常发酵,发酵结果表明L-Trp产量上升至13.3 g/L,而副产物ANTA的积累量在pheA和tyrA基因敲除前后基本没有变化。（3）以菌株FB-03为出发菌株,分别构建L-Trp吸收系统调控基因mtr、tnaB和aroP的缺失突变菌FB-T1、FB-T2和FB-T3;L-Trp吸收活性的测定分析表明,mtr、tnaB和aroP三个基因敲除菌的L-Trp吸收活性与对照菌FB-03相比,分别降低了48%、34%和17%;将重组质粒pSV04转化至上述三个突变菌株中,发酵结果表明,mtr、tnaB和aroP三个敲除菌的L-Trp产量分别为14.7、13.1、和12.3 g/L,与对照菌FB-03/pSV04相比分别提高了34%,19%和12%;此外,吸收基因mtr、tnaB和aroP的敲除,还有利于减少副产物的积累,其中, L-Tyr的积累量与对照菌相比,分别下降了40%、21%、15%; ANTA的积累量与对照菌相比,分别下降了25%、11%、6%;通过测定mtr敲除菌FB-T1/pSV04胞内的L-Trp浓度,发现在发酵过程中,mtr敲除菌胞内L-Trp浓度的增长速率远小于对照菌FB-03/pSV04,在发酵中期的16 h和24 h,mtr敲除菌的胞内L-Trp浓度分别为27.4和50.9μmol/L,与对照菌相比分别下降了42%和35%,由此探讨了L-Trp吸收系统基因敲除促进L-Trp生产的作用机理。（4）在大肠杆菌里,trp操纵子结构基因trpEDCBA的转录受到操纵子内部前导肽trpL中的一段衰减子序列的调控,利用RNA structure软件对前导肽trpL序列的RNA二级结构进行预测分析,表明在利用Red重组系统敲除trpL内部的10-118位核苷酸序列后, trpL的RNA二级结构自由能下降了32%,但trpL 10-118位序列敲除菌FB-L1/pSV04的发酵结果表明,在trpL 10-118位序列敲除前后,L-Trp的产量以及副产物的积累量均没有明显变化,分析原因可能是相对于借助重组质粒pSV04增强trpED的表达量而言,通过削弱衰减子对结构基因trpEDCBA的转录调控,增加结构基因表达量的作用有限;利用DNAMAN软件对结构基因trpEDCBA内部的限制性酶切位点进行分析,选择Eag I、Sca I和Hpa I三个酶切位点将基因trpDCBA分割成trpD*C*、trpC*B*和trpB*A（“*”表示为相应基因的部分序列）三个DNA片段,并按先后次序将其克隆至重组质粒pSV04中,完成结构基因trpE^fbrDCBA的克隆,获得重组质粒pSV05;对工程菌FB-T1/pSV05进行发酵,结果表明,L-Trp产量为17.7 g/L,与菌株FB-T1/pSV04相比提高了21%,葡萄糖转化率为0.13;同时发酵液中ANTA的积累量为1.9 g/L,与菌株FB-T1/pSV04相比下降了51%。（5）将菌株E.coli FB-02/pSV04、FB-03/pSV04和FB-T1/pSV04分别发酵培养至稳定期中期,借助三重四级杆串联质谱仪LC-MS-MS分别测定三个菌株胞内20余种代谢产物的浓度,同时通过RP-HPLC测定三个菌株主要胞外代谢产物L-Trp、L-Phe、L-Tyr和ANTA在发酵液中的浓度;根据KEGG网站（http://www.genome.jp/kegg/）上的微生物代谢网络信息绘制大肠杆菌以葡萄糖为碳源合成L-Trp的代谢网络图谱,根据测定的胞内代谢物浓度,计算三个菌株L-Trp合成代谢的流量分布,并在此基础上分别对菌体的葡萄糖吸收速率、主途径碳流量和关键中间产物的碳流量进行分析,确定提高前体物E4P和L-Ser的合成量,是菌株FB-T1/pSV05进一步改良的重点。更多还原

【Abstract】 L-tryptophan （L-Trp） is widely used in food, animal feed and pharmaceutical industries as an essential amino acid for humans. The production of L-Trp by microbial fermentation has the virtues of cheap sugar and simple extraction technology. Accompanied by the application of metabolic engineering in strain improvement, some L-Trp hyper-production strains was developed, however, there have existed some problems such as the obvious decrease of L-Trp production rate at later-stage of fermentation, the accumulation of byproducts etc.. In addition, the development of these strains is all involved in multiple rounds of random mutagenesis. Previous studies showed that random mutagenesis often produces unexpected mutations at some locations in genome together with desirable ones. Since it is difficult to ascertain the influence of these unidentified mutations, further strain improvement would be affected. In China, the relevant research of metabolic engineering of L-Trp has been only carried out since 2000, and the L-Trp production of constructed strains was much less than world’s advanced level, which hindered the development of domestic industry of L-Trp.In the present study, an L-Trp production strain E.coli FB-T1/pSV05 was developed by a series of defined genetic manipulations based on known regulatory and metabolic information. In addition, metabolic flux of L-Trp synthesis pathway was analyzed by determining the concentrations of multiple intracellular metabolites, which provided the basis for further improvement of L-Trp production. The main contents and results of this thesis are as following:（1） The feedback resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate （DAHP） synthase encoded by aroF was achieved by deleting its residue Ile11. The feedback resistant anthranilate （ANTA） synthase encoded by trpED was achieved by replacing the residue Ser40 of TrpE with Phe. The trpR gene, encoding trp repressor, was knocked out from the genome of E. coli W3110 and the trpR mutant was named as FB-01. Both aroF^fbr and trpE^fbrD were cloned into the expression vector pSV next to the PR and PL promoter, resulting in the plasmids pSV04, and the recombinant plasmid was transformed into FB-01. The Fermentation results showed that the production of L-Trp of FB-01/pSV04 was 2.8 g/L. while, the concentrations of byproducts of L-Phe, L-Tyr, and ANTA in the culture medium were 1.8 g/L, 1.4 g/L, and 2.3 g/L, respectively（2） The degradation pathway of L-Trp was cut off by knocking out its critical gene tnaA based onΔtrpR background, resulting in the strain FB-02. Strain FB-02/pSV04 produced 7.8 g/L L-Trp. However, more byproducts were accumulated in the culture and the concentrations of L-Phe, L-Tyr, and ANTA reached 5.3 g/L, 3.6 g/L, and 5.4 g/L, respectively. In order to prevent the accumulation of L-Phe and L-Tyr, L-Phe and L-Tyr synthesis pathways were blocked. Based onΔtrpR.tnaA background, pheA, which encodes a bifunctional enzyme catalyzing the first two reaction steps of the L-Phe branch pathway in E. coli, was further knocked out and the resulting strain was named as FB-03. The fermentation results of FB-03/pSV04 showed that less than 1 g/L L-Phe was accumulated, but more L-Tyr （5.1 g/L） was observed in the culture media. Based onΔtrpR.tnaA.pheA background, tyrA was further knocked out on genome and resulted in FB-04. When pheA and tyrA were both knocked out, FB-04/pSV04 grew normally only by adding appropriate amount of L-Phe （2 g/L） and L-Tyr （3 g/L） into the medium. The fermentation results indicated that L-Trp production was further increased to 13.3 g/L, and no obvious change in the concentration of ANTA was observed.（3） The three genes of mtr, tnaB, and aroP of L-Trp uptake system were respectively knocked out from the genome of FB-03, resulting in the strains FB-T1, FB-T2 and FB-T3. The results showed that the L-Trp uptake activities of mtr, tnaB, and aroP knockout mutants was decreased by 48%, 34% and 17% when compared to that of the parent FB-04 （2.9 nmol min-1 （mg dry weight） -1）. The fermentation results of FB-T1/pSV04, FB-T2/pSV04 and FB-T3/pSV04 indicated that the L-Trp production of the mtr, tnaB, and aroP knockout mutants was 14.7, 13.1, and 12.3 g/L, respectively, which were increased by 34%, 19%, and 12% when compared to that of FB-03/pSV04. Furthermore, The L-Tyr production of mtr, tnaB, and aroP mutants was decreased by 40%, 21%, and, 15% as compared to the parent strain. The ANTA production of mtr, tnaB, and aroP mutants was decreased by 25%, 11%, and 6% as compared to the parent strain. In addition, it was found that the increase rate of intracellular concentration of L-Trp in mtr mutant was much lower than that in the parent in the process of fermentation. At the culture time of 16 h and 24 h, which represented the middle-stage of fermentation, the intracellular concentration of L-Trp of mtr mutant was 27.4 and 50.9μmol/L, respectively, which was decreased by 42% and 35% when compared to that of the parent. Based on these characteristics, the mechanism of improvement of L-Trp production caused by gene knockouts of L-Trp uptake system was analyzed.（4） The prediction of RNA secondary structure of E. coli trp leader of trp operon was performed, the resulted indicated that the free energy of trp leader （trpL） was -36.5 kcal/mol and was decreased by 32% when the sequences from 10 to 118 of trpL was knockout by using the Red recombination system. However, the fermentation results indicated that the accumulations of L-Trp, L-Tyr, and ANTA have no changes occurred after the 10-118th sequences were knocked out. The restriction endonucleases analysis of trpEDCBA was analyzed by using DNAMAN software. The sequences of trpDCBA was divided into three DNA fragments, trpD*C*, trpC*B*,trpB*A by using its only three restriction sites of Eag I、Sca I and Hpa I. The three DNA fragments were cloned into pSV04 step by step. Finally, the plasmid pSV-aroF^fbr-trpE^fbrDCBA （pSV05） was constructed and transformed into strain FB-T1. The fermentation results indicated that the L-Trp production of FB-T1/pSV05 was 17.1 g/L with 13% conversion ratio from glucose, which were increased by 21% when compared to that of FB-T1/pSV04. In addition, The ANTA production of FB-T1/pSV05 was 1.9 g/L, which was decreased by 51%, as compared to strain FB-T1/pSV04. （5） Metabolic network of L-Trp biosynthesis from glucose as carbon source in E. coli was constructed based on known metabolic information from the KEGG database resource （http://www.genome.jp/kegg/）. More than twenty kinds of intracellular metabolites concentrations were respectively determined by high performance liquid chromatography-tandem mass spectrometry （LC-MS/MS） when the cells of E. coli FB-02/pSV04, FB-03/pSV04, and FB-T1/pSV04 were grown up to the mid-exponential phase. Then the Flux distribution of metabolic network of L-Trp biosynthesis was computed. Finally, the characteristics of metabolic flux of L-Trp biosynthesis pathway, including uptake rate of glucose, main pathways, and critical products, were analyzed and the improvement of synthesis of erythrose 4-phosphate （E4P） and L-serine （L-Ser） was identified as the limited step of L-Trp sysnthesis pathway in FB-T1/pSV05.更多还原