摘要：SummaryApplication of single-stranded DNA recombineering for genome editing of species other than enterobacteria is limited by the efficiency of the recombinase and the action of endogenous mismatch repair (MMR) systems. In this work we have set up a genetic system for entering multiple changes in the chromosome of the biotechnologically relevant strain EM42 ofPseudomononas putida. To this end high-level heat-inducible co-transcription of therec2recombinase andP. putida's allelemutLE36KPPwas designed under the control of the PL/cI857 system. Cycles of short thermal shifts followed by transformation with a suite of mutagenic oligos delivered different types of genomic changes at frequencies up to 10% per single modification. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—e.g., ribosomal-binding sites. These results enabled multiplexing of genome engineering ofP. putida, as required for metabolic reprogramming of this important synthetic biology chassis.Graphical AbstractDisplay OmittedHighlights•Pseudomonas putidais a useful Synthetic Biology chassis for metabolic engineering•Co-expression of Rec2 recombinase and mutLE36K allele empowers ssDNA recombineering•Cyclic DNA replication fork invasion causes up to 10% single-site mutation frequency•The experimental HEMSE pipeline eases multi-site genome editing ofP. putidaBioengineering; Metabolic Engineering; Biotechnology; Microbial Biotechnology
