【作者】 杨帆；

【导师】 李寅；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2014， 硕士

【副题名】1,2,4-丁三醇生物合成途径的创建和优化

【摘要】 1,2,4-丁三醇是一种在民用和军工上都具有重要用途的化学品。作为重要的有机合成中间体,可用于制备生物活性剂、医药用缓释剂、卷烟添加剂等。硝化产物1,2,4-丁三醇三硝酸酯,可作为飞机、火箭、导弹等军事武器的推进剂,较硝化甘油具有冲击敏感性低、热稳定性好等优点。目前,1,2,4-丁三醇的工业化生产主要依靠化学合成法,缺点是副产物多、产物产率低、对环境有危害等,工业界期待建立一条清洁、高效的1,2,4-丁三醇生物合成途径。然而,自然界不存在1,2,4-丁三醇的天然生物合成途径,生物法生产1,2,4-丁三醇的研究也进展缓慢。近几年来,国外已有利用木糖或阿拉伯糖生产1,2,4-丁三醇的生物反应途径的专利报道,但涉及军工用途,需建立自主产权的技术。本研究旨在通过酶表达量调节、新酶挖掘等策略,获得知识产权,提高重组细胞木糖的利用效率,优化1,2,4-丁三醇生物合成途径,以期提高重组菌株1,2,4-丁三醇的产量。本论文首先以来源于恶臭假单胞菌(Pseudomonas putida)的苯甲酰甲酸脱羧酶(MDLC)为1,2,4-丁三醇合成途径中催化第三步反应的2-酮酸脱羧酶,构建了能够利用D-木糖合成1,2,4-丁三醇的大肠杆菌重组细胞BW-011。再以BW-011为初始菌株,通过改变各步酶表达量,寻找整条途径中的限速酶,并通过提高限速酶的表达量、多酶表达量组合调控的方式进行一系列的菌株优化,获得1,2,4-丁三醇产量是BW-011的4倍的大肠杆菌重组细胞BW-026。根据实验结果,对重组细胞BW-026而言,催化3-脱氧-D-甘油-戊酮糖酸脱羧反应的2-酮酸脱羧酶仍然为1,2,4-丁三醇合成途径的限速酶。为优化该限速酶,本论文在KEGG等数据库中搜索类似的基团反应,筛选获得了 5种可能的2-酮酸脱羧酶,并通过外源导入的方式成功构建了 3株1,2,4-丁三醇生物合成菌株。摇瓶发酵结果显示,来自乳酸乳球菌(Lactococcus lactis)的α-酮异戊酸脱羧酶(KIVD)比MDLC有更高的催化3-脱氧-D-甘油-戊酮糖酸脱羧反应的活性,表达该酶的重组菌株BW-025的1,2,4-丁三醇产量也是初始菌株BW-011的3倍。为了进一步更加理性的对1,2,4-丁三醇生物合成途径的各步酶进行精细调控,本论文还纯化了各步酶,测定了各自的活性和稳定性。通过优化纯化条件,获得了 XDH、YJHG、KIVD、ADHP四种酶蛋白,其中XDH纯酶已证实具有较高的比酶活和稳定性,可以满足后续实验的要求,而ADHP纯酶的酶活损失严重,需进一步改良蛋白保存的条件。该研究为后续进行1,2,4-丁三醇合成途径的限速酶分析和优化提供了依据。更多还原

【Abstract】 The compound 1,2,4-butanetriol(BT)is a valuable chemical both in civil and military industry.As an important organic intermediate,BT can be used in the production of bioactivator,sustained-release drug and cigarette additive,etc.It is also known as a precursor for the production of 1,2,4-butanetriol trinitrate,an energetic plasticizer for aifcrafts,rockets and missiles,with lower impact sensitivity and better thermostability compared to nitroglycerin.The BT industrial production is mainly by chemical synthesis at present,which is not environment friendly,with several byproducts and low yield,therefore clean and efficient biosynthetic pathways are paid attention to be bulit.However,no natural pathways were found,and only a little progress was made in BT biosynthesis.In recent years,BT biosynthetic pathway utilizing xylose or arabinose as substrates have been reported.As involving military purposes,it is necessary to establish independent property rights of technology.In this study,it is aimed at improving xylose utilization efficiency of the recombinant strains and optimizing the BT biosynthetic pathway by strategies such as enzyme expression regulation and new enzyme search,consequently increasing BT production of the recombinant strains.In this paper,benzoylformate decarboxylase(MDLC)from Pseudomonas putida,which acted as the 2-keto acid decarboxylase catalyzing the third step of the pathway,was recruited into an engineered E.coli strain BW-010,resulting in the recombinant strain BW-011.The BT titer of BW-011 was 0.51 g/L.Then the strains were optimized by searching rate-limiting enzymes and fine tuning the expression of enzymes in the pathway,and strains with higher BT production were obtained subsequently.We have got a recombinant strain BW-026,producting four-fold BT than BW-011.The 2-keto acid decarboxylase catalyzing 3-deoxy-D-glycero-pentulosonic acid decarboxylation,the third step of the pathway,was found to be the rate-limiting enzyme according to analysis of experiment results.Alternative enzymes were selected by searching and analysis in protein databases like KEGG,and then transformed into the starting strain to construct new recombinants.Alpha-ketoisovalerate decarboxylase(KIVD)from Lactococcus lactis was found with higher catalytic efficiency in 3-deoxy-D-glycero-pentulosonic acid decarboxylation compared to MDLC,and improved BT production was obtained accordingly.In order to analyze and optimize the BT biosynthetic pathway,all four enzymes in the pathway have been expressed and purified in this paper.Enzyme activity of D-xylose dehydrogenase(XDH)have been veried and alcohol dehydrogenase(ADHP)were evaluated by specific activity detection.XDH was proved to have higher enzyme activity and stability,and can meet the requirements of subsequent experiments.But the enzyme activity of ADHP is loss low.We are further going to change protein preservation conditions.更多还原