通过番茄红素环化酶的优化构建β-胡萝卜素高产菌株  

Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production

作　　者：金应福 韩莉[1] 张莎莎[1,2] 李世忠 刘伟丰 陶勇[1] 

机构地区：[1]中国科学院微生物研究所,北京100101 [2]中国科学院大学,北京100049

出　　处：《生物工程学报》2017年第11期1814-1826,共13页Chinese Journal of Biotechnology

基　　金：中国科学院重点部署项目(No.ZDRW-ZS-2016-3)资助~~

摘　　要：目标产物的合成途径往往需要对关键酶的来源、表达水平等因素进行系统性优化才能实现代谢通量的最大化。β-胡萝卜素是一类具有重要应用价值的萜类化合物,其中番茄红素环化酶(Lycopene cyclase,CrtY)是β-胡萝卜素合成途径中的关键酶,能够催化FAD依赖的环化反应将番茄红素转化合成β-胡萝卜素。本研究通过对CrtY的系统优化提高β-胡萝卜素的合成水平,并确定CrtY的表达对代谢通路的影响。在大肠杆菌中以番茄红素合成模块为基础,通过引入番茄红素环化酶基因crt Y构建了β-胡萝卜素合成模块。并进一步利用寡聚接头介导的DNA组装方法 (Oligo-linker mediated assembly method,OLMA)引入一系列不同强度的人工设计的核糖体结合位点(Ribosome-binding site,RBS),对CrtY的表达强度、基因来源等因素进行高通量的优化。通过OLMA文库构建和平板筛选,获得了5株高产β-胡萝卜素的工程菌株。在摇瓶中,5株工程菌株的β-胡萝卜素产量可达15.79-18.90 mg/g DCW(Dry cell weight),比优化前提高了65%。进一步选取了其中的CP12菌株,在5 L发酵罐上,利用高密度培养技术验证工程菌株合成β-胡萝卜素的能力。最终β-胡萝卜素产量可达1.9 g/L。RBS强度分析及代谢中间体分析表明,适当地降低CrtY表达强度能够有利于β-胡萝卜素模块相关基因之间协同发挥作用。以上结果为β-胡萝卜素合成途径的优化规律提供了理论指导。To optimize key enzymes, such as to explore the gene resources and to maximize metabolic pathways of target products. β-carotene is a terpenoid compound modify the expression level, can with important application value. Lycopene cyclase （CrtY） is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide （FAD）-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase （CrtY） to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by eoexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escheriehia coli. Then we simultaneously optimized the ribosome-binding site （RBS） intensity and the species of crtY using oligo-linker mediated DNA assembly method （OLMA）. Five strains with high ]g-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW （Dry cell weight）, 65% higher than that of the original strain at shake flask level. The optimal strain CP 12 was further identified and evaluated for ]3-carotene production at 5 L fermentation level. After process optimization, the final [k-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

关 键 词：番茄红素环化酶(CrtY) 大肠杆菌 β-胡萝卜紊 寡聚接头介导的DNA组装(OLMA) 代谢工程 

分 类 号：Q55[生物学—生物化学] Q78