Adaptive evolution in producing microtiter cultivations generates
genetically stable Escherichia coli production hosts for
continuous bioprocessing
Abstract
The production of recombinant proteins usually reduces cell fitness and
the growth rate of producing cells. The growth disadvantage favors
faster-growing non-producer mutants. Therefore, continuous bioprocessing
is hardly feasible in Escherichia coli due to the high escape
rate. We investigated the stability of E. coli expression systems
under long-term production conditions and how metabolic load triggered
by recombinant gene expression influences the characteristics of
mutations. We conducted iterated fed-batch-like microbioreactor
cultivations under production conditions. We used the easy-to-produce
green fluorescent protein (GFP) and a challenging antigen-binding
fragment (Fab) as model proteins, and BL21(DE3) and
BL21Q strains as expression hosts. In comparative
whole genome sequencing analyses, we identified mutations that allowed
cells to grow unhindered despite recombinant protein production. A T7
RNA polymerase expression system is only conditionally suitable for
long-term cultivation under production conditions. Mutations leading to
non-producers occur in either the T7 RNA polymerase gene or the T7
promoter. The host RNA polymerase-based BL21Q
expression system remained stable in the production of GFP in long-term
cultivations. For the production of Fab, mutations in lacI of the
BL21Q derivatives had positive effects on long-term
stability. Our results indicate that adaptive evolution carried out with
genome-integrated E. coli expression systems in microtiter
cultivations under industrial relevant production conditions is an
efficient strain development tool for production hosts.