【作者】 陆文强；

【导师】 于洪巍；

【作者基本信息】 浙江大学 ， 生物化工， 2014， 博士

【摘要】 辅酶Q10是参与细胞有氧呼吸作用的重要电子传递体,可以为机体提供能量物质ATP。并且其抗氧化作用可以保护细胞避免氧化剂的损伤,辅酶Q10还被证明对治疗心脑血管疾病具有良好效果,因此目前被广泛地应用于身体保健及疾病预防。为了提高辅酶Q10的产量,对辅酶Q10生产菌的育种一直以来都是研究的热点。本文通过代谢工程的手段对产辅酶Q10的细菌—类球红细菌进行遗传改造,提高其辅酶Q10的产量。为了对类球红细菌进行代谢改造,首先构建了该菌的一个表达载体,该载体包含广宿主载体骨架和一个可以组成型表达的启动子。通过实时定量PCR验证,该表达载体可以在葡萄糖存在的情况下实现组成型表达,以UbiG基因作为表征,其在该表达载体的控制下表达量可以达到野生型的133倍左右。本研究在该表达载体的基础上通过引入不同的代谢调控策略有效地提高了类球红细菌中辅酶Q10的产量,同时比较了不同的策略对不同的研究对象产生的效果。在代谢工程改造过程中需要注意对整个代谢网络的平衡控制,本文以MEP途径代谢调控为例,构建了一个自反馈调控系统。该系统是基于lacO操纵子工作原理,利用核糖体结合位点(RBS)作为调节工具,对操纵子中的基因表达进行优化调节,使之既能提高目标产物的产量又能满足代谢网络平衡的要求。本研究通过在自反馈调控系统中引入5个不同强度的RBS序列对MEP途径进行优化调控后,得到了一株辅酶Q10产量提高了87%的重组菌株。同时,通过引入新的外界干扰,证明了该系统在代谢调控中是比较稳定和灵活的。醌环修饰途径也是辅酶Q10生物合成途径中的一个重要环节,虽然不为辅酶Q1o提供前体物质,但是可以提高辅酶Q10前体物质到辅酶Q10的转化率。通过不同的表达筛选,确定了类球红细菌辅酶Q10合成途径中的关键酶UbiE、UbiH和UbiG。通过不同的表达策略,确定了将UbiE和UbiG融合到膜结合蛋白pufX上,可以将醌环修饰途径的通量最大化。接着本文对辅酶Q10进行了系统化的调控,通过系统性地对整个代谢网络进行调控可以实现不同的代谢优化结果的整合,使代谢改造的效果更加明显。本研究即在MEP调控及醌环修饰途径代谢优化的基础上,将两者通过自反馈调控系统进行整合来提高辅酶Q10的产量。本研究通过代谢工程手段,构建了类球红细菌的表达载体以及自反馈调控体系,对类球红细菌的辅酶Q10生物合成途径进行了系统地代谢调控,有效地提高了类球红细菌中辅酶Q10的产量,使之达到原来的3倍左右。更多还原

【Abstract】 Coenzyme Q10(CoQ10) is an essential electron carrier in the aerobic respiratory electron transfer system. It generates ATP for the body and has been proven effective in treating cardiovascular disease and hypertension. Also, CoQ10is an important antioxidant. Currently, CoQ10is widely used in health care and disease prevention. For improving the production of CoQ10, metabolic engineering of CoQ10producers has widely drawn the attention from the researchers. In this study, Rhodobacter sphaeroides, a natural CoQ10producer, was metabolically engineered to improve its production of CoQ10.For metabolic engineering of Rhodobacter sphaeroides, a constitutive expression plasmid was constructed. This plasmid harbors a broad-host-range plasmid backbone and a constitutive promoter. Real-time PCR verified that the plasmid could constitutively express the gene cloned in the plasmid in the presence of glucose. Taking UbiG as an example, the expression level of UbiG in the recombinant with UbiG cloned in the plasmid was133-times as high as that in the wild type. In this study, different strategies were developed based on this plasmid to improve the production of CoQ10.Metabolic balance is a key issue in metabolic engineering. In this study, we tried to improve the CoQ10production by enhancing the flux of the MEP pathway with a self-regulation system. This system was constructed based on the mechanism of Lac operator and ribosomal binding site regulation. By introducing5different ribosomal binding sites, a pathway-balanced mutant with87%CoQ10production higher than the wildtype was obtained. Also, this system was corfirmed to be robust and flexible in tuning metabolic balance.The quinone modification pathway directly influences the CoQ10production by transfor ming the precursors to CoQ10. We screened the key enzymes in the pathway and idenfied UbiE, UbiH and UbiG as the candidates. To expand the ubiquninoe modification pathway, different coexpression strategies were developed. It was found that by fusing UbiG and UbiE to the pufX peptide, which dereases the loss of intermediates diffusion, the flux of the pathway could be maximized and the production of CoQ10could be subsequently improved.Systematic engineering of the metabolic pathway is a highlight in metabolic engineering. It can integrate the positive results of the sectional pathway optimizations. In this study, the optimized MEP pathway and ubiquinone modification pathway were intergrated. As a result, a higher level of CoQ10production was achieved.In this study, we systematically engineered the CoQ10pathway in Rhodobacter sphaeroides and successfully improved the production of CoQ10by about3times.更多还原