Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli.
Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli.
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DOI：
10.1002/bit.22560
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发表时间：
2010-02-15
影响因子：
3.8
通讯作者：
Pfeifer, Blaine A.
中科院分区：
文献类型：
--
作者：
Zhang, Haoran;Boghigian, Brett A.;Pfeifer, Blaine A.
6-脱氧红素内酯B （6dEB）是抗生素天然产物红霉素的大环苷元前体。在大肠杆菌中，6dEB的异源生产部分是通过设计过表达天然prpE基因（编码丙酰辅酶a合成酶）和异源pcc基因（编码丙酰辅酶a羧化酶）来提供所需的丙酰辅酶a和(2S)-甲基丙酰辅酶a生物合成底物来完成的。单独的大肠杆菌代谢包括三种酶，Sbm（甲基丙二酰辅酶a变化酶），YgfG（甲基丙二酰辅酶a脱羧酶）和YgfH（丙二酰辅酶a：琥珀酸辅酶a转移酶），也参与丙二酰辅酶a和甲基丙二酰辅酶a的代谢。本研究分别删除和过表达sbm、ygfG和ygfH基因，研究它们对异源6dEB产生的影响。结果表明，sbm的缺失和过表达不影响6dEB的产生；在摇瓶实验中，ygfG过表达使6dEB的产生减少了四倍，而ygfH缺失使6dEB滴度从65 mg/L增加到129 mg/L。研究还发现，在缺乏外源丙酸和提供底物的pcc基因的情况下，原生大肠杆菌的代谢能够支持6dEB的生物合成。最后，在间歇式生物反应器培养中测试了ygfH缺失的影响，其中6dEB滴度从206 mg/L提高到527 mg/L。
6-Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over-expression of a native prpE gene (encoding a propionyl-CoA synthetase) and heterologous pcc genes (encoding a propionyl-CoA carboxylase) to supply the needed propionyl-CoA and (2S)-methylmalonyl-CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl-CoA mutase), YgfG (a methylmalonyl-CoA decarboxylase), and YgfH (a propionyl-CoA:succinate CoA transferase), also involved in propionyl-CoA and methylmalonyl-CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over-expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over-expression of sbm did not influence 6dEB production; ygfG over-expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L.
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影响因子：
3.5
作者：
CHEREPANOV, PP;WACKERNAGEL, W
通讯作者：
WACKERNAGEL, W
大肠杆菌K-12的构造框架，单基因敲除突变体：Keio Collection。
影响因子：
9.9
作者：
通讯作者：
--
影响因子：
3.7
作者：
Tsuruta H;Paddon CJ;Eng D;Lenihan JR;Horning T;Anthony LC;Regentin R;Keasling JD;Renninger NS;Newman JD
通讯作者：
Newman JD
影响因子：
5
作者：
Wang, Yong;Boghigian, Brett A.;Pfeifer, Blaine A.
通讯作者：
Pfeifer, Blaine A.
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL