摘要
目的通过突变生物合成获得31-去甲氧基-他克莫司。方法针对大环内酯类免疫抑制剂他克莫司的聚酮链起始底物4,5-二羟基-1-烯-环己酸(DHCHC)合成酶fkbO基因,构建基因敲除载体pFIM412-fkbO并对他克莫司产生菌Streptomyces sp.FCZ-0311 fkbO基因进行框内缺失。结果获得突变株Streptomyces sp.OD14-1,经摇瓶发酵和产物HPLC分析,该突变株完全丧失了合成他克莫司的能力。在OD14-1摇瓶发酵36h后添加DHCHC类似物反式-4-羟基环己烷羧酸,发酵6d后经HPLC-MS检测发现分子量为773的他克莫司衍生物31-去甲氧基-他克莫司。结论构建了可用于他克莫司突变生物合成的基因工程菌Streptomyces sp.OD14-1,并通过外源物质的添加获得31-去甲氧基-他克莫司。
Objective To obtain 31-desmethoxytacrolimus by mutational biosynthesis. Methods FkbO is on enzyme encoding 4,5-dihydroxycyclohex-l-enecarboxylic acid （DHCHC）. In order to achieve 31-desmethoxytacrolimus, we make the fkbO in-frame deletion mutant Streptomyces sp. OD14-1 from a tacrolimus industrial strains FCZ-0311 by the gene knock-out vector pFIM412-fkbO. Results By shake flask fermentation and HPLC analysis, the mutant strain OD 14-1 lost the ability of producing tacrolimus. When DHCHC analog trans-4-hydroxycy-clohexanecarboxylic acid was added to the fermentation culture of OD 14-1 after cultivation for 36h, 31-desmethoxytacrolimus with molecular weight of 773, was detected by LC-MS after being cultured for six days. Conclusion Genetically engineered strain Streptomyces sp OD14-1 which could be used for mutational biosynthesis was constructed, and 31-desmethoxytacrolimus was obtained by addition of exogenous substances.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2017年第8期652-657,共6页
Chinese Journal of Antibiotics
基金
福建省公益类科研院所专项(No.2014R1009-5)
福建省化药技术重大研发平台(No.2014Y2001)
福州市生物制药行业技术创新中心(No.2016-PT-36)
福建省微生物分析检测技术公共服务平台建设(No.2013Y2003)
作者简介
方志锴，男，生于1986年，硕士，助理研究员，主要从事放线菌的遗传改造研究，E-mail：fangzk1986@126．com
通讯作者，E-mail：yylianfim@139．com
