【作者】 田光明；

【导师】 陈守文；

【作者基本信息】 华中农业大学 ， 微生物学， 2014， 博士

【摘要】 聚Y-谷氨酸(poly-γ-glutamic acid,简称γ-PGA)是一种多功能的生物聚合物,具有可食用、无毒和可生物降解等特性。地衣芽胞杆菌WX-02是重要的聚Y-谷氨酸生产菌株,但菌体胞内谷氨酸合成能力较弱,在聚γ-谷氨酸发酵生产过程中需要添加大量谷氨酸,提高了发酵成本,不利于聚γ-谷氨酸的产业化开发。目前聚γ-谷氨酸代谢中的关键酶在地衣芽胞杆菌WX-02生物合成聚γ-谷氨酸中的作用仍不清楚。本研究通过分析胞内谷氨酸合成和聚γ-谷氨酸合成的代谢途径与关键步骤,并以此为基础,对地衣芽胞杆菌WX-02代谢途径中关键酶的酶学性质、胞内谷氨酸合成和聚γ-谷氨酸降解进行研究,同时利用HPLC、GC-MS和Q-RT-PCR等方法对所构建工程菌株的生理生化变化进行分析。主要结论如下：1.胞内谷氨酸的合成中,有三种酶参加反应：glnA基因编码的谷氨酰胺合成酶、gltAB基因编码的谷氨酸合成酶和分别由rocG和gudB基因编码的谷氨酸脱氢酶。本研究在Bacillus licheniformis WX-02(简称WX-02)中分别缺失rocG和gltA基因,替换rocG基因启动子,得到相关菌株WX-02△rocG、WX-02△gltA和WX-02P43rocG。经Q-RT-PCR分析发现,WX-02△rocG菌株中未检测到rocG基因转录,WX-02△gltA菌株中未检测到gltA基因转录,WX-02P43rocG菌株rocG基因转录量为WX-02菌株1.54倍。WX-02菌株中,未检测到gudB基因转录,WX-02△rocG中gudB基因转录量为WX-02△gltA菌株的4.72倍。在未添加外源谷氨酸的发酵培养基中,WX-02聚Y-谷氨酸产量为9.82g/L,WX-02△rocG和WX-02P43rocG的聚γ-谷氨酸产量分别为WX-02的54.7%和105%,WX-02△gltA的聚γ-谷氨酸产量与WX-02相比差异不显著。说明在WX-02中,负责胞内谷氨酸合成的酶为谷氨酸脱氢酶(RocG)。2.以WX-02基因组为模板扩增谷氨酸脱氢酶RocG编码基因,克隆至pET-28(+)表达载体后,转化大肠杆菌BL21(DE3),获得含有重组质粒pET-28b(+)-rocG的重组大肠杆菌菌株,对其进行诱导表达和Ni柱亲和纯化,得到具有均一电泳条带的谷氨酸脱氢酶RocG蛋白。RocG的酶学性质结果表明：该酶的最适反应温度为40℃,最适反应pH值为8.0,温度低于40℃、pH值6.0-8.5稳定性较好。α-酮戊二酸、NADPH和谷氨酸的米氏常数Km分别为4.727mmol/L、0.111mmol/L和23.296mmol/L,α-酮戊二酸、NADPH和谷氨酸的Kcat/Km分别为1.923mmol-1?L?min-1、100.09mmol-1?L?min-1和0.159mmol-1?L?min-1。说明谷氨酸脱氢酶RocG合成谷氨酸催化反应效率比分解谷氨酸高,本研究从体外实验证明了地衣芽胞杆菌WX-02谷氨酸脱氢酶(RocG)主要起生物合成谷氨酸的功能。3.乙醛酸循环可以部分回补TCA循环从而增强胞内谷氨酸合成。aceA基因编码的异柠檬酸裂解酶和aceB基因编码的苹果酸合成酶为乙醛酸循环中关键酶。本研究以WX-02为原始菌株,分别缺失aceA基因和替换aceA基因原有启动子后得到菌株WX-02△aceA和WX-02P43aceA。经Q-RT-PCR检测后发现,WK-02△aceA菌株中未检测到aceA基因转录,WX-02P43aceA菌株中aceA和aceB基因转录量分别提高了3.6和2.8倍。在未添加外源谷氨酸的培养基中,WX-02P43aceA和WX-02△aceA的聚γ-谷氨酸产量为WX-02的115%和66.8%,说明胞内谷氨酸合成需要乙醛酸循环的回补作用,增强乙醛酸循环可以提高聚γ-谷氨酸产量。培养基中分别添加乙醛酸循环抑制剂苹果酸和琥珀酸后,WX-02中aceA基因转录量下降了14%和28%,聚γ-谷氨酸产量分别提升了13.4%和16.5%。分析原因为添加苹果酸和琥珀酸虽然抑制了乙醛酸循环途径,但由于添加物属于TCA循环中间代谢产物,增强了Ⅸ-酮戊二酸向谷氨酸合成的代谢,从而提高了聚γ-谷氨酸合成产量。4.聚γ一谷氨酸降解酶可以降解菌株合成的聚γ-谷氨酸,使其分子量减小。本研究构建了聚Y-谷氨酸降解酶基因pgdS缺失菌株WX-02△pgdS,并以pHY300PLK质粒为基础构建了对照菌株WX-02/pHY菌株和pgdS基因过表达菌株WX-02/pHYpgdS。使用GPC检测WX-02、WX-02/pHY、WK-02△pgdS和WX-02/pKYpgdS生物合成的γ-PGA相对分子量后发现,WX-02△pgdS相对分子量最大,WX-02/pHYpgdS相对分子量最小,WX-02/pHY和WX-02相对分子量一致,说明聚γ-谷氨酸降解酶PgdS可以降低B. licheniformis WX-02生物合成的γ-PGA分子量。在外源添加谷氨酸的培养基中,WX-02和WX-02/pHY聚Y-谷氨酸产量差异不显著,WX-02△pgdS聚γ-谷氨酸产量下降为WX-02的83%,WX-02/pHYpgdS与对照菌株WX-02/pHY相比,聚γ-谷氨酸产量提高54%。经Q-RT-PCR分析,WX-02△pgdS菌株中未检测到pgdS基因转录,谷氨酸转运蛋白基因gltT转录量下降为WX-02的86%,WX-02/pHYpgdS菌株pgdS和gltT基因转录相比WX-02/pHY分别提高了9.86和1.8倍。说明聚γ-谷氨酸降解酶PgdS影响外源谷氨酸向胞内转运,引起聚γ-谷氨酸合成前体物的胞内浓度变化,改变了聚γ-谷氨酸产量。更多还原

【Abstract】 Poly-γ-glutamic acid (γ-PGA in short) is a multi-functional biopolymers, which is featured by its unique biological characteristics of edible、non-toxic and biodegradable. Bacillus licheniformis WX-02(WX-02in short) is a kind of poly-γ-glutamic acid producing strain relying on adding extracellular glutamate. In the process of producing poly-γ-glutamic acid, much glutamic acid is needed, which increases the cost of fermentation, and is not propitious to the industrial development of poly-γ-glutamic acid. At present,the function of the essential enzymeofpoly-γ-glutamic acidbiosynthesisin WX-02remains unknown.Based on the metabolic pathways,the characterization of key enzyme, intracellular glutamic acidbiosynthesisand γ-PGA degradationof Bacillus licheniformis WX-02were analyzied. Besides, HPLC, GC-MS, and Q-RT-PCR are also applied to the constructed engineering strains to examine their specific physiological and biochemical changes.The major findings of this research are listed as follows:1.Three enzymes, Glutamine synthetase, glutamate synthase and glutamate dehydrogenase (encoded by glnA, gltAB and rocG or gudB genes), are involved in intracellular glutamic acid biosynthesis. The WX-02ΔrocG and WK-02ΔgltA strains were obtained through knockout of rocG and gltA gene respectively. And the WX-02P43rocG was obtained by enhancing rocG gene in WX-02. Transcription of rocG and gltA were not detected in WX-02ΔrocG, WK-02ΔgltA respectively. Transcription of orcocG was1.54times higher in WX-02P43rocG than that in WX-02through Q-RT-PCR. Transcription of gudB was not detected in WX-02. Transcription of gudB was4.72times higher in WX-02ΔrocGthan that in WX-02ΔgltA. The γ-PGA yield was9.82g/L of WX-02in culture without external glutamate added. The γ-PGA productivity of WX-02ArocG and WX-02P43rocG were54.70%and105%, compared with WX-02. The difference between γ-PGA yield of WX-02ΔgltA and WX-02was not significant. It was implied that glutamate dehydrogenase was the major enzyme which was responsible for glutamic acid biosynthesis.2.The rocG gene was cloned based on B. licheniformis WX-02genome and was inserted into pET-28(+) vector. Recombination vector pET-28b(+)-rocG was transformed into E. coli BL21(DE3). The RocG was obtained after inducing by IPTG and purificating. Optimal conditions for activity were pH8.0and a temperature of40℃and RocG was stable in buffers ranging from pH6.0to8.5and a temperature below40℃. Km for substrate a-ketoglutarate, NADPH and glutamate were4.727mmol/L,0.111mmol/L, and23.296mmol/L. The Kcat/Km of a-ketoglutarate, NADPH and glutamate were1.923mmol-1?L?min-l,100.09mmol-1?L?min-1, and0.159mmol-1?L?min-1. It was noteworthy that catalytic reaction efficiency of glutamate synthesis was higher than that in reverse reaction. It was certified that glutamate dehydrogenase was responsible for glutamic acid biosynthesis in vitro.3.Glyoxylate cycle can partly complement TCA and thus increase intracellular glutamic acid biosynthesis. There were two key enzymes of isocitrate lyase encoded by aceA gene and malate synthase encoded aceB gene in glyoxylate cycle. WX-02AaceA and WX-02P43aceA were obtained through knockout and enhancing aceA gene based on WX-02respectively. Transcription of aceA was not detected in WX-02AaceA. Transcription of aceA and aceB were3.6and2.8times higher than that in WX-02. The y-PGA yield of WX-02PA3aceA and WX-02AaceA were115%and66.8%of WX-02respectively. It was implied that the complement of glyoxylate cycle was required in intracellular glutamic acid biosynthesis and y-PGA productivity can be improved through enhancing glyoxylate cycle. Transcription of aceA decreased by14%and28%after glyoxylate cycle inhibiter malic acid or succinate added, whereas culture without external glutamate and y-PGA yield increased by13.4%and16.5%respectively. It was noted that the metabolism from a-ketoglutarate to glutamic acid was enhanced to improve y-PGA production, although malic acid or succinate inhibit glyoxylate cycle inhibiter were added, which is due to the increasing additive of the intermediate of TCA.4.The molecular weight of poly-γ-glutamic acid can be decreased through degradation of poly-γ-glutamic acid hydrolase. WX-02ΔpgdS was constructed by knockout of pgdS gene based on WX-02and WX-02/pHYpgdS, which was obtained through enhancing pgdS gene with WX-02/pHY as CK. The relative molecular weight of WX-02ΔpgdS was the highest with GPC detection. The relative molecular weight of WX-02/pHYpgdS was lower than WX-02/pHY with same results compared with WX-02. It was implied that molecular weight of poly-γ-glutamic acid can be decreased by poly-γ-glutamic acid hydrolase. There was no difference of γ-PGA yield between WX-02and WX-02/pHY. The γ-PGA productivity of WX-02ApgdS decreased to83%in WX-02. However, the γ-PGA productivity from WX-02/pHYpgdS improved54%compared with WX-02/pHY. There was no transcription of pgdS in WX-02ΔpgdS and transcription of gltT decreased to86%compared with WX-02. The transcription of pgdS and gltT was 9.86and1.8times higher in WX-02/pHYpgdS than that in WX-02/pHY. It was certified that there was certain connection between PgdS and GltT which changed intracellular glutamic acid concentration and γ-PGA yield.更多还原