氧化葡萄糖酸杆菌膜结合乙醇脱氢酶亚基Ⅲ的功能鉴定及提高菌株羟基酸合成能力的研究

发布时间：2018-01-06 06:32

本文关键词：氧化葡萄糖酸杆菌膜结合乙醇脱氢酶亚基Ⅲ的功能鉴定及提高菌株羟基酸合成能力的研究　出处：《华东理工大学》2016年博士论文　论文类型：学位论文

【摘要】：氧化葡萄糖酸杆菌(Gluconobacter oxydans)的细胞膜上具有大量膜结合的乙醇脱氢酶(membrane-bound alcohol dehydrogenase, mADH),能够不完全氧化多种伯醇及伯二醇生成相应的酸。已有研究表明,mADH与G oxydan的呼吸链尤其是抗氰呼吸密切相关,还可介导其它膜结合脱氢酶的电子转移,如葡萄糖脱氢酶。本课题组前期通过分离纯化和活性测定,鉴定出G. oxydans DSM 2003中存在的mADH的两个亚基(Ⅰ和Ⅱ),分别由adhA和adhB基因编码。本论文通过生物信息学手段鉴定出G oxydans DSM 2003中mADH的亚基Ⅲ；基于对亚基Ⅲ的功能研究,理性构建高效表达mADH的G oxydans工程菌,提高其对羟基酸的合成能力。首先,mADH亚基Ⅲ的鉴定及功能研究。通过序列比对在G oxydans DSM 2003基因组中发现假定的adhS,将该基因在G oxydans DSM 2003中过表达以及与adhAB共同表达,另以adhAB表达菌作为对照,通过对这三株工程菌中mADH转录水平、蛋白表达及催化活性的比较,发现亚基Ⅲ的高表达可使更多游离于胞内的亚基Ⅰ结合于细胞膜,使其能够表现出正常的生理活性,从而提高菌株mADH的催化能力。亚基Ⅲ的表达量是决定Goxydans中mADH活性的一个重要因素。其次,mADH的过表达对G. oxydans产羟基酸能力的影响。在1,2-丙二醇的摇瓶转化中,过表达菌株G. oxydans-adhABS可将D-乳酸的产量提高53.8%,转化效率从0.65 g/(l·h)提高到1.0g/(l·h)；在乙二醇的摇瓶转化中,G. oxydans-adhABS可将羟基乙酸的产量提高164%,转化效率从0.3g/(l·h)提高到0.78g/(l.h)。由此可见,mADH的过表达极大提高了G. oxydans对羟基酸的合成能力。将乙二醇的转化过程在7L发酵罐中放大,通过在线调节pH、提高溶氧水平以及流加底物等手段,G. oxydans-adhABS河在45 h内累积生成73.3g/l羟基乙酸,转化率为93.5%,转化效率提高到1.63g/(l·h)。利用发酵法直接转化乙二醇,45 h可合成羟基乙酸113.8 g／l,转化率为92.9%,转化效率进一步提高到2.53 g／(l.h),此羟基乙酸产量和转化效率均为目前所报道的最高水平。第三,mADH的过表达对G oxydans呼吸链及其它膜结合脱氢酶的影响。过表达菌株G oxydans-adhABS中各呼吸链元件(NADH脱氢酶除外)的转录水平均显示不同程度的上调,尤其是两个呼吸链末端氧化酶(细胞色素bo3和bd醌氧化酶)。在生长对数期,菌体的摄氧能力显著增强,培养36 h的生物量比对照菌增加26%-33%,可为醇催化反应尤其是直接发酵法转化提供了更多的生物催化剂。此外,G.oxydans-adhABS中其它膜结合脱氢酶的转录水平和催化活性也有一定的提高,尤其是膜结合乙醛脱氢酶(mALDH),其相对活性提高26%,也可在一定程度上加速G. oxydans-adhABS的产酸进程。第四,mADH差异过表达对G. oxydans中甘油氧化产物选择性的影响。已知mADH是转化甘油生成甘油醛的关键酶,基于mADH的活性差异,工程菌G. oxydans-adhABS和G. oxydans-adhS对甘油的转化率降低至30%,但对甘油酸的选择性分别增加7.9倍和7.6倍；相反,G. oxydans-adhAB对甘油的转化率高达94%,对二羟基丙酮的选择性增加到92.6%。表明mADH的差异过表达可显著改变G. oxydans对甘油氧化产物的选择性。
[Abstract]:G.oxydans (Gluconobacter oxydans) has a large number of membrane-bound alcohol dehydrogenase on the cell membrane (membrane-bound alcohol dehydrogenase, mADH), can not complete the oxidation of various primary alcohols and primary glycol to produce the corresponding acid. Studies have shown that the mADH and G oxydan of the respiratory chain especially the cyanide resistant respiration is closely related, but also lies the other membrane bound electron transfer dehydrogenase, such as glucose dehydrogenase. Ourprevious through purification and activity determination, identified two subunits in G. oxydans DSM 2003 mADH (I and II), respectively by adhA and adhB gene encoding. The bioinformatics identified subunit G oxydans DSM 2003 mADH III; function research of subunit III based on the rational construction of high expression of G oxydans mADH engineering bacteria, improve the ability of synthesis of hydroxy acids. Firstly, mADH subunit III Study on the identification and function of adhS in G. We identified oxydans DSM 2003 genome by sequence alignment, the gene in G oxydans DSM 2003 expression and adhAB expression, adhAB expression in other bacteria as the control, based on the mADH transcription level of these three strains of engineering bacteria, comparison of protein expression and catalytic activity. And we found that high expression of subunit subunit III can make more free intracellular binding to the cell membrane, which can show the normal physiological activity, so as to improve the catalytic ability of strain mADH. The expression of subunit III is an important factor to determine the activity of mADH in Goxydans. Secondly, mADH effect of G. overexpression on oxydans hydroxy acid ability. In shake flask into 1,2- propylene glycol, G. oxydans-adhABS over expression strain can be D- lactic acid production increased 53.8%, the conversion efficiency from 0.65 g/ (L - H) to 1.0g/ (L - H); B two Shake flask transformation of alcohol, G. oxydans-adhABS can be of glycolic acid was increased by 164% and the conversion efficiency from 0.3g/ (L - H) to 0.78g/ (L.h). Thus, the overexpression of mADH can greatly improve the ability of G. oxydans on the synthesis of hydroxy acids. The conversion process of ethylene glycol amplification in 7L fermentation tank in the regulation of pH by means of online, increase the dissolved oxygen level and feeding substrate, the G. oxydans-adhABS River in 45 h cumulation 73.3g/l hydroxy acetic acid, the conversion rate was 93.5%, the conversion efficiency is increased to 1.63g/ (L - H). The direct conversion of ethylene glycol by fermentation method, 45 h 113.8 g / L synthesis of glycolic acid and the conversion rate was 92.9%, the conversion efficiency is further increased to 2.53 g / (L.h), the hydroxy acetic acid yield and conversion efficiency are the highest level of the current report. Third, the over expression of mADH dehydrogenase binding effect on G oxydans respiratory chain and other membrane expression strain G oxy. The respiratory chain components in dans-adhABS (except the transcription level of NADH dehydrogenase) showed varying degrees of increase, especially the two respiratory chain terminal oxidase (cytochrome BO3 oxidase and BD quinone). In logarithmic phase, significantly enhanced the oxygen uptake of bacteria, biomass of 36 h cultured bacteria than the control 26%-33%. For the alcohol catalytic reaction especially into direct fermentation provides a biological catalyst more. In addition, other G.oxydans-adhABS membrane binding transcription level and catalytic activity of dehydrogenase also increased, especially the membrane binding of acetaldehyde dehydrogenase (mALDH), the relative activity increased by 26%, but also can accelerate the acid production process G. oxydans-adhABS to a certain extent. Fourth. The effect on the expression of G. oxydans in glycerol oxidation selectivity mADH difference. Known mADH is a key enzyme in glycerol generation glyceraldehyde, activity difference based on mADH, G. oxydans-adhABS and G. oxydans-adhS engineering bacteria for glycerol conversion rate decreased to 30%, but the selectivity of glyceric acid was increased by 7.9 times and 7.6 times; on the contrary, G. oxydans-adhAB conversion of glycerol rate as high as 94%, two selectivity for acetol increased to 92.6%. showed that the expression of G. oxydans can significantly change the selectivity of glycerol oxidation products the difference in mADH.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93

【相似文献】