Abstract: The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E. coli strain for coenzyme Q₁₀ production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution (multiple copy integration) and replicon-free and markerless chromosomal integration (single copy integration), respectively. A coenzyme Q₁₀ hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway. The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass (DCM) of coenzyme Q₁₀ when supplemented with 0.075 g·L^-1 of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q₁₀ production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.

Key words: coenzyme Q₁₀, Escherichia coli, chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration, chromosomal engineering

摘要： The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E. coli strain for coenzyme Q₁₀ production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution (multiple copy integration) and replicon-free and markerless chromosomal integration (single copy integration), respectively. A coenzyme Q₁₀ hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway. The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass (DCM) of coenzyme Q₁₀ when supplemented with 0.075 g·L^-1 of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q₁₀ production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.

关键词: coenzyme Q₁₀, Escherichia coli, chemically inducible chromosomal evolution, replicon-free and markerless chromosomal integration, chromosomal engineering