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

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crtY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。

Abstract:

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound 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 coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia 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 β-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 CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-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.