平衡磷酸烯醇式丙酮酸节点通量强化筑波链霉菌合成他克莫司

摘要

Tacrolimus(FK506),as one of the widely used immunosuppressants produced by Streptomyces species,has drawn much attention on clinic application. However,the low FK506 fermentation titer restricts its industrial production,which is mainly due to the insufficient precursor metabolism of the producing strain. In this work,balancing carbon flux rebalancing around phosphoenolpyruvate(PEP)node for enhancement of FK506 production were carried on. Firstly,the genes fkbO and fkbL were overexpressed in S.tsukubaensis D852,achieving S.tsukubaensis-OL1,of which FK506 production changed only slightly from 158.7mg/L to 163.9mg/L. Then,two precursor metabolic pathways,the anaplerotic and shikimate pathways emanating from PEP node,were fine-tuned for eliminating the inefficient supply of precursors of DHCHC and pipecolate. The genes encoding PPC and DAHPS were cloned from various species and expressed in S.tsukubaensis-OL1,respectively,and the FK506 production was separately increased by 40%(ppc,S.tsukubaensis)and 47%(dahP,S.roseosporus).Subsequently,the expression levels of the genes ppc and dahP were modulated under the control of four constitutive promoters(PermE*,Psco4503,Psco3410 and Psco5768)in nine engineering strains,resulting in the final increase of FK506 production from 163.9mg/L to 350.3mg/L. This work demonstrated that the optimization of the branches emanating from PEP node was a viable strategy to strengthen FK506 production in S.tsukubaensis.%他克莫司(FK506)是最重要的免疫抑制剂之一,然而前体代谢供应不足制约着工业化生产.通过优化平衡磷酸烯醇式丙酮酸(PEP)节点支路通量可提高FK506产量.本文首先在S.tsukubaensis D852中过表达基因fkbO(编码分支酸水合还原酶)和 fkbL(编码赖氨酸环化酶)得到 S.tsukubaensis-OL1,FK506 的产量仅从158.7mg/L提高到163.9mg/L.随后调节PEP节点支路回补途径和莽草酸途径通量强化FK506的合成:先分别将不同菌株中编码磷酸烯醇式丙酮酸羧化酶(PPC)和3-脱氧-D-阿拉伯糖基-heptulosonate-7-磷酸合酶(DAHPS)的基因在S.tsukubaensis-OL1中过表达,FK506的产量分别提高40%(ppc,S.tsukubaensis)和47%(dahP,S. roseosporus);然后采用4个不同强度的组成型启动子(PermE*,Psco4503,Psco3410 and Psco5768)平衡ppc和dahP的表达水平获得9株工程菌,最终使FK506的产量由163.9mg/L显著提高到350.3mg/L.这个结果说明优化平衡PEP节点竞争支路通量是提高FK506产量的有效策略.