产γ-氨基丁酸重组谷氨酸棒杆菌的构建及相关分解酶的研究

发布时间：2018-05-16 02:36

  本文选题：γ-氨基丁酸 + 谷氨酸棒杆菌　； 参考：《江南大学》2017年硕士论文

【摘要】：γ-氨基丁酸(γ-Aminobutyric acid,简称GABA)是一种具有多种生理功能的氨基酸,已经在食品和医药中广泛应用。谷氨酸棒杆菌(Corynebacterium glutamicum)是重要的氨基酸生产菌,工业上用来生产L-谷氨酸等,L-谷氨酸经外源gad基因(如gadB1、gadB2)编码的谷氨酸脱羧酶(GAD)催化脱羧反应生成GABA,是GABA合成的直接前体。GABA在中性条件下经GABA转氨酶(GABA-T)和琥珀酸半醛脱氢酶(SSADH)连续催化两步反应后转变成琥珀酸,从而发生分解。本实验室前期研究发现,敲除GABA-T编码基因gabT后,重组C.glutamicum菌ATCC13032ΔgabT/pJYW-4-gadB1-gadB2(SYN201)中GABA在中性条件下仍然会分解。本研究用代谢工程手段对重组C.glutamicum菌GABA合成和分解途径进一步进行改造,并对分解途径相关酶的酶学性质进行研究。研究内容与结果如下:(1)将pJYW-4-gadB1-gadB2转入L-谷氨酸工业生产菌株C.glutamicum S9114中构建S9114/pJYW-4-gadB1-gadB2菌株。为了减少发酵副产物L-丙氨酸,敲除谷丙转氨酶编码基因alaT,构建S9114ΔalaT和S9114ΔalaT/pJYW-4-gadB1-gadB2。摇瓶发酵,结果表明,S9114ΔalaT的L-丙氨酸产量比对照减少了11.4%,S9114ΔalaT/pJYW-4-gadB1-gadB2的L-丙氨酸产量比对照减少了5.5%。RT-PCR表明alaT敲除菌中编码缬氨酸转氨酶的avtA基因转录水平上调,造成alaT敲除菌中L-丙氨酸没有明显减少。并且发酵后期随着pH上升,GABA含量降低,发生分解。(2)为了阻断C.glutamicum中GABA分解途径,在实验室前期构建的gabT敲除菌SYN101中敲除另一个转氨酶编码基因NCgl2515,并转入pJYW-4-gadB1-gadB2质粒,构建得到gabT和NCgl2515双敲表达GAD菌SYN203。上罐发酵,结果表明,SYN203在中性条件下GABA含量维持在25.0 g·L-1,说明分解途径被阻断,因此NCgl2515很可能参与GABA分解。(3)为了确定NCgl2515是否编码GABA-T,克隆C.glutamicum ATCC13032基因组中已知的分别编码GABA-T和SSADH的gabT和gabD以及前面发现的NCgl2515基因,并在大肠杆菌(Escherichia coli)BL21(DE3)中表达,纯化后进行酶学性质研究。结果表明,NCgl2515蛋白的GABA-T活性很低,但是通过测定NCgl2515-GabD偶联活性发现,它不仅能以α-酮戊二酸(α-KG)还能以丙酮酸作为氨基受体催化GABA转氨反应,活性分别为0.034和0.032 U·mg-1,因此是一种新的GABA-T,最适pH为7.5-8.0。而GabT的GABA-T活性比较高(1.29 U·mg-1),但它只能以α-KG为氨基受体,且最适pH为7.8,pH低于6.0时几乎没有酶活。因此这两种GABA-T都是在中性条件下催化GABA发生转氨反应,从而导致GABA分解。
[Abstract]:纬 -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 伪 -ketoglutaric acid (伪 -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 伪 -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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ922

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