High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering

Application 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 chro...

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Bibliographic Details
Published iniScience Vol. 23; no. 3; p. 100946
Main Authors Aparicio, Tomas, Nyerges, Akos, Martínez-García, Esteban, de Lorenzo, Víctor
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 27.03.2020
Elsevier
Subjects
Bioengineering
Biotechnology
Metabolic Engineering
Microbial Biotechnology
Bioengineering
Metabolic Engineering
Biotechnology
Microbial Biotechnology
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Summary:Application 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 of Pseudomononas putida. To this end high-level heat-inducible co-transcription of the rec2 recombinase and P. putida's allele mutLE36KPP was 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 of P. putida, as required for metabolic reprogramming of this important synthetic biology chassis. [Display omitted] •Pseudomonas putida is 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 of P. putida Bioengineering; Metabolic Engineering; Biotechnology; Microbial Biotechnology
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Lead Contact
Present address: Harvard Medical School, Boston, MA 02115, USA
ISSN:2589-0042
2589-0042
DOI:10.1016/j.isci.2020.100946