J. Muller et al., CLONING OF HETEROLOGOUS GENES SPECIFYING DETRIMENTAL PROTEINS ON PUC-DERIVED PLASMIDS IN ESCHERICHIA-COLI, MGG. Molecular & general genetics, 252(1-2), 1996, pp. 207-211
A system is described that enables the cloning of genes specifying det
rimental proteins in Escherichia coli. The system is based on pUC plas
mids and was developed for the expression of the Bacillus subtilis csa
A gene, which is lethal when expressed at high levels. Suppressor stra
ins that tolerate the presence of plasmids for high-level expression o
f csaA were isolated, which contained small cryptic deletion variants
of the parental plasmid in high copy numbers. The cryptic plasmids con
sisted mainly of the pUC replication functions and lacked the csaA reg
ion and selectable markers. The co resident, incompatible, cryptic pla
smids enabled the maintenance of the csaA plasmids by reducing their c
opy number 20-fold, which resulted in a concomitant 3- to 7-fold reduc
tion in the expression of plasmid-encoded genes. Strains carrying thes
e cryptic endogenous plasmids proved to be useful for the construction
of pUC-based recombinant plasmids carrying other genes, such as the s
kc gene of Streptococcus equisimilis, which cannot be cloned in high c
opy numbers in E. coli. Several strategies to reduce production levels
of heterologous proteins specified by plasmids are compared.