High-efficiency multi-site genomic editing (HEMSE) ofPseudomonas putidathrough thermoinducible ssDNA recombineering

SUMMARY While single-stranded DNA recombineering is a powerful strategy for higher-scale genome editing, its application to species other than enterobacteria is typically limited by the efficiency of the recombinase and the action of native mismatch repair (MMR) systems. By building on [i] availability of the Erf-like single-stranded DNA-annealing protein Rec2, [ii] adoption of tightly-regulated thermoinducible device and [iii] transient expression of a MMR-supressing mutL allele, we have set up a coherent genetic platform for entering multiple changes in the chromosome of Pseudomononas putida with an unprecedented efficacy and reliability. The key genetic construct to this end is a broad host range plasmid encoding co-transcription of rec2 and P. putida ’s mutL E36K PP at high levels under the control of the P L / c I857 system. Cycles of short thermal shifts of P. putida cells followed by transformation with a suite of mutagenic oligos delivered different types of high-fidelity genomic changes at frequencies up to 10% per single change—that can be handled without selection. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—as shown in this case with ribosomal binding sites. These results enable the multiplexing of genome engineering of P. putida , as required for metabolic engineering of this important biotechnological chassis.