@article{MTMT:31355775,
	title = {High-Efficiency Multi-site Genomic Editing of Pseudomonas putida through Thermoinducible ssDNA Recombineering},
	url = {https://m2.mtmt.hu/api/publication/31355775},
	author = {Aparicio, Tomas and Nyerges, Ákos and Martinez-Garcia, Esteban and de, Lorenzo Victor},
	doi = {10.1016/j.isci.2020.100946},
	journal-iso = {ISCIENCE},
	journal = {ISCIENCE},
	volume = {23},
	unique-id = {31355775},
	abstract = {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 mutL(E36K)(PP) was designed under the control of the P-L/cl857 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.},
	year = {2020},
	eissn = {2589-0042},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X}
}

@article{MTMT:30927710,
	title = {Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene},
	url = {https://m2.mtmt.hu/api/publication/30927710},
	author = {Aparicio, Tomas and Nyerges, Ákos and Nagy, István and Pál, Csaba and Martinez-Garcia, Esteban and de, Lorenzo Victor},
	doi = {10.1111/1462-2920.14814},
	journal-iso = {ENVIRON MICROBIOL},
	journal = {ENVIRONMENTAL MICROBIOLOGY},
	volume = {22},
	unique-id = {30927710},
	issn = {1462-2912},
	abstract = {The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutL(E36K)). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.},
	year = {2020},
	eissn = {1462-2920},
	pages = {45-58},
	orcid-numbers = {Nyerges, Ákos/0000-0002-1581-490X; de, Lorenzo Victor/0000-0002-6041-2731}
}