组合蛋白质工程和代谢工程设计全细胞催化剂合成靛蓝和靛玉红

Biosynthesis of indigo and indirubin by whole-cell catalyst designed by combination of protein engineering and metabolic engineering

摘要:

从嗜高温放线菌Thermobifida fusca中分离得到的苯基丙酮单加氧酶主要催化芳香族化合物的Baeyer-Villiger氧化反应。对该酶的结构和功能进行研究时，发现位于底物结合口袋的Met446位点突变可以赋予突变酶催化C–H键活化的新功能，氧化吲哚合成靛蓝和靛玉红，但产量仅为1.89 mg/L。为了获得合成靛蓝和靛玉红的全细胞催化剂，直接补加吲哚并不能提高细胞合成效率，补加吲哚的前体物质L-色氨酸可以使细胞合成靛蓝和靛玉红的能力提高4.5倍，达到8.43 mg/L。为了进一步提高细胞的生物合成效率，通过代谢工程改造大肠杆菌的糖代谢途径，阻断葡萄糖异构酶基因pgi，使磷酸戊糖途径代替糖酵解途径成为葡萄糖的主要代谢通路，从而为细胞提供更多氧化吲哚所需的辅因子NADPH，导致细胞合成靛蓝和靛玉红的效率进一步提高3倍，达到25 mg/L。通过组合蛋白质工程和代谢工程设计全细胞催化剂不仅可以高效地合成靛蓝和靛玉红，而且设计理念为相关全细胞催化剂的开发提供了一种新的策略。

Abstract:

The phenylacetone monooxygenase, isolated from Thermobifida fusca, mainly catalyzes Baeyer-Villiger oxidation reaction towards aromatic compounds. Met446 plays a vital role in catalytic promiscuity, based on the structure and function of phenylacetone monooxygenase. Mutation in Met446 locus can offer enzyme new catalytic feature to activate C—H bond, oxidizing indole to finally generate indigo and indirubin, but the yield was only 1.89 mg/L. In order to further improve the biosynthesis efficiency of the whole-cell catalyst, metabolic engineering was applied to change glucose metabolism pathway of Escherichia coli. Blocking glucose isomerase gene pgi led to pentose phosphate pathway instead of the glycolytic pathway to become the major metabolic pathways of glucose, which provided more cofactor NADPH needed in enzymatic oxidation of indole. Engineering the host E. coli led to synthesis of indigo and indirubin efficiency further increased to 25 mg/L. Combination of protein and metabolic engineering to design efficient whole-cell catalysts not only improves the synthesis of indigo and indirubin, but also provides a novel strategy for whole-cell catalyst development.