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 Q

10

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 Q

10

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 Q

10

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 Q

10

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.