Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation
Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation
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DOI：
10.1016/j.chembiol.2006.09.014
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发表时间：
2006-12-01
影响因子：
--
通讯作者：
Mueller, Rolf
中科院分区：
文献类型：
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作者：
Gross, Frank;Ring, Michael W.;Mueller, Rolf
在粘杆菌 Sorangium cellulosum So ce56 的测序项目中鉴定了一个由三个开放阅读框组成的操纵子，在计算机上注释为甲基丙二酰辅酶 A (mm-CoA) 差向异构酶、mm-CoA 变位酶 (MCM) 和 meaB。这个假定的 MCM 途径操纵子是通过 Red/ET 重组工程从细菌人工染色体亚克隆到源自 p15A 的最小复制子上的。该质粒经过修饰，可在恶臭假单胞菌中整合和异源表达，从而能够产生需要 mm-CoA 作为前体的复杂次级代谢产物。通过基于气相色谱/质谱的分析方法鉴定了重组恶臭假单胞菌菌株中的丙二酸甲酯。该工程菌株能够合成需要 mm-CoA 作为延伸单元的聚酮化合物，这通过将生物合成基因簇整合到染色体中并诱导表达后产生 myxothiazol 来证明。
An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.