Expression of toxic genes in Methylorubrum extorquens with a tightly repressed, cumate-inducible promoter

Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter P_Q2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.