CONCLUSIONS
In this study, we constructed a series of integration
expression vectors for single copy or multiple
copy integration, which can be applied in RMM and
CIChE. We engineered a plasmid-free E. coli
TBW20134, using CIChE, RMM as well as deletion
of menaquinone biosynthetic pathway. The engineered
E. coli TBW20134 produced CoQ10 content of (10.7±0.6)
mg·g−1 DCM and CoQ10 concentration of (77.4±4.2)
mg·L−1. Moreover, no antibiotics were used in the
fermentation process for the strain. This can reduce
the cost and avoid the problem caused by addition of
antibiotics. The engineered strain remained stable as
determined by examination of its CoQ10-producing
capacity after 30 sequential transfers. This study
demonstrated that applying CIChE along with RMM
could flexibly control gene copy numbers. The proposed
methods can be applied to the production of
other metabolites where various copy numbers for
different genes is concerned.