大肠杆菌L-蛋氨酸合成途径的硫同化模块重构

Methionine is the only bulk amino acid that has not been produced through fermentation method until now. At present methionine is produced chemically with the highly toxic hydrocyanic acid as intermediates and DL type mixture as the product,which seriously pollutes the environment. The complicated biosynthetic network of L-methionine needs a large amount of reducing cofactor, methyl and sulfur elements. In Escherichia coli, the pathways of coenzyme and methyl regeneration are active, while the sulphur assimilation pathway is lengthy and complex, which becomes the bottleneck in the methionine synthesis network. This program intends to integrate exogenous sulphur assimilation pathway (containing 2 reactions) for replenishing the native transsulfuration pathway (containing 9 reactions), and then improve the compatibility of this pathway with metabolic environment in E. coli. The metabolic flux towards metabolic synthesis will be enhanced through screening of exogenous thiol, metabolic by-products recycling and metabolic cofactor regeneration, and fine assembly of metabolic module. The sulfur metabolic module will be reconstructed to elucidate the key metabolic mechanism of methionine synthesis.This program will provide an efficient strategy for engineering L-methionine producing strains, and estimate theoretical foundation for realizing fermentation production of methionine and other sulfur high value-added chemicals containing sulfur.

蛋氨酸是唯一尚未实现直接发酵法生产的大宗氨基酸，目前主要的生产方法为以剧毒氢氰酸为中间体的化学合成法，环境污染严重，且产物为DL型混旋体。L-蛋氨酸的生物合成消耗大量的还原力、甲基和硫元素；大肠杆菌的还原型辅酶再生和甲基供给途径活跃，但是其转硫化途径冗长且调控机制复杂，是限制L-蛋氨酸高效合成的代谢瓶颈。本研究从前期构建的产8.1g/L蛋氨酸的大肠杆菌工程菌出发，拟引入仅包含2步反应的外源直接巯基化途径，替代内源9步反应组成的转硫化途径，以提高L-蛋氨酸合成的硫同化效率。进一步针对外源直接巯基化途径消耗乙酰辅酶A并产生乙酸的问题，增强不消耗ATP的回用乙酸再生乙酰辅酶A的aldB-mhpF途径，重构零能耗的辅因子再生循环，以提高消耗9.7个ATP的蛋氨酸生物合成的转化率。本研究将阐明硫代谢和L-蛋氨酸合成之间的协同机制，构建高效生产L-蛋氨酸的工程菌，为含硫化合物的生物制造奠定基础。

蛋氨酸是唯一尚未实现直接发酵法生产的大宗氨基酸，目前主要的生产方法为以剧毒氢氰酸为中间体的化学合成法，环境污染严重，且产物为DL型混旋体。L-蛋氨酸的生物合成消耗大量的还原力、甲基和硫元素；大肠杆菌的还原型辅酶再生和甲基供给途径活跃，但是其转硫化途径冗长且调控机制复杂，是限制L-蛋氨酸高效合成的代谢瓶颈。本研究筛选了18种不同来源的MetX、MetY和MetZ，获得了摇瓶发酵可积累7.1g/L中间代谢产物乙酰高丝氨酸的MetX，以及高效积累蛋氨酸的MetX-MetZ异源直接巯基化途径。通过引入仅包含2步反应的外源直接巯基化途径，替代内源9步反应组成的转硫化途径，以提高L-蛋氨酸合成的硫同化效率。进一步针对外源直接巯基化途径消耗乙酰辅酶A并产生乙酸的问题，构建了6种辅酶再生途径，发现aldB-mhpF途径最优，该途径不消耗ATP，零能耗的辅因子再生循环可提高消耗9.7个ATP的蛋氨酸生物合成的转化率。此外，本研究还开展了菌体生长与丙酮酸关联的适应性进化研究，获得了生长表型提高且蛋氨酸合成增强的进化株，通过全基因组测序获得了9个突变位点，目前正在开展回补实验和机制分析实验。通过摇瓶发酵筛选到最优的工程菌，进一步开展发酵小试验证，发酵48小时，L-蛋氨酸产量达到21.2g/L，达到项目预期目标。本研究阐明了硫代谢和L-蛋氨酸合成之间的协同机制，构建了高效生产L-蛋氨酸的工程菌，为含硫化合物的生物制造奠定基础。