本文選題：γ-氨基丁酸 + 谷氨酸棒桿菌�。� 參考：《江南大學(xué)》2017年碩士論文

【摘要】：γ-氨基丁酸(γ-Aminobutyric acid,簡(jiǎn)稱GABA)是一種具有多種生理功能的氨基酸,已經(jīng)在食品和醫(yī)藥中廣泛應(yīng)用。谷氨酸棒桿菌(Corynebacterium glutamicum)是重要的氨基酸生產(chǎn)菌,工業(yè)上用來生產(chǎn)L-谷氨酸等,L-谷氨酸經(jīng)外源gad基因(如gadB1、gadB2)編碼的谷氨酸脫羧酶(GAD)催化脫羧反應(yīng)生成GABA,是GABA合成的直接前體。GABA在中性條件下經(jīng)GABA轉(zhuǎn)氨酶(GABA-T)和琥珀酸半醛脫氫酶(SSADH)連續(xù)催化兩步反應(yīng)后轉(zhuǎn)變成琥珀酸,從而發(fā)生分解。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),敲除GABA-T編碼基因gabT后,重組C.glutamicum菌ATCC13032ΔgabT/pJYW-4-gadB1-gadB2(SYN201)中GABA在中性條件下仍然會(huì)分解。本研究用代謝工程手段對(duì)重組C.glutamicum菌GABA合成和分解途徑進(jìn)一步進(jìn)行改造,并對(duì)分解途徑相關(guān)酶的酶學(xué)性質(zhì)進(jìn)行研究。研究內(nèi)容與結(jié)果如下:(1)將pJYW-4-gadB1-gadB2轉(zhuǎn)入L-谷氨酸工業(yè)生產(chǎn)菌株C.glutamicum S9114中構(gòu)建S9114/pJYW-4-gadB1-gadB2菌株。為了減少發(fā)酵副產(chǎn)物L(fēng)-丙氨酸,敲除谷丙轉(zhuǎn)氨酶編碼基因alaT,構(gòu)建S9114ΔalaT和S9114ΔalaT/pJYW-4-gadB1-gadB2。搖瓶發(fā)酵,結(jié)果表明,S9114ΔalaT的L-丙氨酸產(chǎn)量比對(duì)照減少了11.4%,S9114ΔalaT/pJYW-4-gadB1-gadB2的L-丙氨酸產(chǎn)量比對(duì)照減少了5.5%。RT-PCR表明alaT敲除菌中編碼纈氨酸轉(zhuǎn)氨酶的avtA基因轉(zhuǎn)錄水平上調(diào),造成alaT敲除菌中L-丙氨酸沒有明顯減少。并且發(fā)酵后期隨著pH上升,GABA含量降低,發(fā)生分解。(2)為了阻斷C.glutamicum中GABA分解途徑,在實(shí)驗(yàn)室前期構(gòu)建的gabT敲除菌SYN101中敲除另一個(gè)轉(zhuǎn)氨酶編碼基因NCgl2515,并轉(zhuǎn)入pJYW-4-gadB1-gadB2質(zhì)粒,構(gòu)建得到gabT和NCgl2515雙敲表達(dá)GAD菌SYN203。上罐發(fā)酵,結(jié)果表明,SYN203在中性條件下GABA含量維持在25.0 g·L-1,說明分解途徑被阻斷,因此NCgl2515很可能參與GABA分解。(3)為了確定NCgl2515是否編碼GABA-T,克隆C.glutamicum ATCC13032基因組中已知的分別編碼GABA-T和SSADH的gabT和gabD以及前面發(fā)現(xiàn)的NCgl2515基因,并在大腸桿菌(Escherichia coli)BL21(DE3)中表達(dá),純化后進(jìn)行酶學(xué)性質(zhì)研究。結(jié)果表明,NCgl2515蛋白的GABA-T活性很低,但是通過測(cè)定NCgl2515-GabD偶聯(lián)活性發(fā)現(xiàn),它不僅能以α-酮戊二酸(α-KG)還能以丙酮酸作為氨基受體催化GABA轉(zhuǎn)氨反應(yīng),活性分別為0.034和0.032 U·mg-1,因此是一種新的GABA-T,最適pH為7.5-8.0。而GabT的GABA-T活性比較高(1.29 U·mg-1),但它只能以α-KG為氨基受體,且最適pH為7.8,pH低于6.0時(shí)幾乎沒有酶活。因此這兩種GABA-T都是在中性條件下催化GABA發(fā)生轉(zhuǎn)氨反應(yīng),從而導(dǎo)致GABA分解。
[Abstract]:緯 -Aminobutyric acid (GABA) is a kind of amino acid with many physiological functions, which has been widely used in food and medicine. Corynebacterium glutamicum is an important amino acid producing bacterium. Industrial production of L- glutamic acid and L- glutamic acid is catalyzed by the decarboxylation of glutamate decarboxylase encoded by exogenous gad gene (such as gadB1gadB2) to produce GABA, which is the direct precursor of GABA synthesis through GABA transaminase GABA-T and succinic acid under neutral conditions. Hemaldehydes dehydrogenase (SSADH) was used to catalyze the two-step reaction and then convert to succinic acid. Thus decomposition occurs. It was found that GABA in the recombinant C.glutamicum strain ATCC13032 螖 gabT / pJYW-4-gadB1-gadB2 SYN201would still decompose under neutral conditions after the GABA-T coding gene gabT was knocked out. In this study, the pathway of GABA synthesis and decomposition of recombinant C.glutamicum strain was further modified by metabolic engineering, and the enzymatic properties of enzymes related to catabolism pathway were studied. The contents and results were as follows: (1) pJYW-4-gadB1-gadB2 was transformed into C.glutamicum S9114, an industrial production strain of L-glutamic acid, to construct S9114/pJYW-4-gadB1-gadB2 strain. In order to reduce the fermentation by-product L-alanine, the transglutaminase gene Ala T was knocked out, and S9114 螖 alaT and S9114 螖 Ala T / pJYW-4-Bgad1-gadB2 were constructed. The results of shaking flask fermentation showed that the L- alanine production of S9114 螖 alaT decreased by 11.4% than that of the control, and the L- alanine production of S9114 螖 alaT/pJYW-4-gadB1-gadB2 decreased compared with that of the control. The results showed that the transcription level of avtA gene encoding valine aminotransferase in alaT knockout bacteria was up-regulated. There was no significant decrease in L-alanine in alaT knockout bacteria. In order to block the GABA decomposition pathway in C.glutamicum, another transaminase encoding gene NCgl2515 was knocked out of the gabT knockout SYN101 constructed in the early stage of the laboratory and transferred into pJYW-4-gadB1-gadB2 plasmid. GabT and NCgl2515 double knockout expressing GAD strain SYN203were constructed. The results showed that the GABA content of SYN203 was maintained at 25.0 g / L ~ (-1) under neutral conditions, indicating that the decomposition pathway was blocked. In order to determine whether NCgl2515 encodes GABA-T, the known gabT and gabD encoding GABA-T and SSADH and the previously discovered NCgl2515 genes in the C.glutamicum ATCC13032 genome are cloned and expressed in Escherichia coli BL21DDE3. The enzymatic properties were studied after purification. The results showed that the GABA-T activity of NCgl2515 protein was very low, but it could not only use 偽 -ketoglutaric acid (偽 -KG) but also pyruvate as aminoreceptor to catalyze the transammonia reaction of GABA. The activity is 0.034 U mg-1 and 0.032 U mg-1, respectively, so it is a new GABA-T, and the optimum pH is 7.5-8.0. The activity of GABA-T in GabT was higher than that of 1.29 U mg-1, but only 偽 -KG was used as amino receptor, and the optimum pH was 7.8 渭 g / h, and there was almost no enzyme activity when pH was lower than 6.0. Therefore, both GABA-T catalyze the transammonia reaction of GABA under neutral conditions, which leads to the decomposition of GABA.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ922

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