@article{MTMT:34331356,
	title = {Protective mechanism of milk fat globule membrane proteins on Lactobacillus acidophilus CICC 6074 under acid stress based on proteomic analysis},
	url = {https://m2.mtmt.hu/api/publication/34331356},
	author = {Zhang, Tao and Yang, Yujie and Zeng, Xiaoqun and Wu, Zhen and Pan, Daodong and Luo, Haibo and Tao, Mingxuan and Guo, Yuxing},
	doi = {10.1016/j.foodchem.2023.137297},
	journal-iso = {FOOD CHEM},
	journal = {FOOD CHEMISTRY},
	volume = {434},
	unique-id = {34331356},
	issn = {0308-8146},
	abstract = {The prerequisite for lactic acid bacteria to perform their probiotic function is that they could survive the acidstressed environment of production and application. In this experiment, the protective mechanism of milk fat globule membrane (MFGM) proteins on lactic acid bacteria under acid stress was investigated. Scanning electron microscopy and fluorescence probe were used to analyze the condition of the acid-treated bacteria, which showed that MFGM proteins could enhance the survival ability of Lactobacillus acidophilus CICC 6074 under acid stress by maintaining cell morphology, elevating intracellular pH and H+-ATPase activity. Furthermore, Tandem Mass Tags (TMT) proteomic analysis revealed that MFGM protein could exert protective effects on L. acidophilus CICC 6074 by regulating amino acid metabolism, ATPase activity, peptidoglycan synthesis, gene repair and heritage, etc. The results will provide a new approach for the protection and development of functional lactic acid bacteria.},
	keywords = {proteome; Acid tolerance; MFGM proteins; Lactobacillus acidophilus CICC 6074},
	year = {2024},
	eissn = {1873-7072}
}

@article{MTMT:34410308,
	title = {Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species},
	url = {https://m2.mtmt.hu/api/publication/34410308},
	author = {Asin-Garcia, Enrique and Garcia-Morales, Luis and Bartholet, Tessa and Liang, Zhuobin and Isaacs, Farren J and Martins dos Santos, Vitor A P},
	doi = {10.1093/nar/gkad1024},
	journal-iso = {NUCLEIC ACIDS RES},
	journal = {NUCLEIC ACIDS RESEARCH},
	volume = {52},
	unique-id = {34410308},
	issn = {0305-1048},
	abstract = {The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.},
	year = {2023},
	eissn = {1362-4962},
	orcid-numbers = {Asin-Garcia, Enrique/0000-0001-5568-345X; Isaacs, Farren J/0000-0001-8615-8236; Martins dos Santos, Vitor A P/0000-0002-2352-9017}
}

@article{MTMT:34617900,
	title = {Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species},
	url = {https://m2.mtmt.hu/api/publication/34617900},
	author = {Kalb, Madison J. and Grenfell, Andrew W. and Jain, Abhiney and Fenske-Newbart, Jane and Gralnick, Jeffrey A.},
	doi = {10.1128/spectrum.03176-23},
	journal-iso = {MICROBIOL SPEC},
	journal = {MICROBIOLOGY SPECTRUM},
	unique-id = {34617900},
	issn = {2165-0497},
	abstract = {Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 x 10-4, 3 x 10-4, and 5 x 10-5, respectively, in these strains using lambda-Red Beta recombinase from Escherichia coli. We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus.},
	keywords = {PSEUDOMONAS; Homologous recombination; recombineering; Plant growth promoting rhizobacteria; SSAPs},
	year = {2023},
	eissn = {2165-0497}
}

@article{MTMT:34308918,
	title = {An expanded CRISPR-Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates},
	url = {https://m2.mtmt.hu/api/publication/34308918},
	author = {Pankratz, Debbie and Gomez, Nicolas Oswaldo and Nielsen, Agnes and Mustafayeva, Ayten and Guer, Melisa and Arce-Rodriguez, Fabian and Nikel, Pablo Ivan and Haeussler, Susanne and Arce-Rodriguez, Alejandro},
	doi = {10.1038/s41596-023-00882},
	journal-iso = {NAT PROTOC},
	journal = {NATURE PROTOCOLS},
	unique-id = {34308918},
	issn = {1754-2189},
	abstract = {Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.},
	year = {2023},
	eissn = {1750-2799}
}

@{MTMT:32900618,
	title = {High-Efficiency Multi-site Genomic Editing (HEMSE) Made Easy},
	url = {https://m2.mtmt.hu/api/publication/32900618},
	author = {Aparicio, T. and de, Lorenzo V. and Martínez-García, E.},
	booktitle = {Vaccine Design},
	doi = {10.1007/978-1-0716-2233-9_4},
	volume = {2479},
	unique-id = {32900618},
	abstract = {The ability to engineer bacterial genomes in an efficient way is crucial for many bio-related technologies. Single-stranded (ss) DNA recombineering technology allows to introduce mutations within bacterial genomes in a very simple and straightforward way. This technology was initially developed for E. coli but was later extended to other organisms of interest, including the environmentally and metabolically versatile Pseudomonas putida. The technology is based on three pillars: (1) adoption of a phage recombinase that works effectively in the target strain, (2) ease of introduction of short ssDNA oligonucleotide that carries the mutation into the bacterial cells at stake and (3) momentary suppression of the endogenous mismatch repair (MMR) through transient expression of a dominant negative mutL allele. In this way, the recombinase protects the ssDNA and stimulates recombination, while MutLE36KPP temporarily inhibits the endogenous MMR system, thereby allowing the introduction of virtually any possible type of genomic edits. In this chapter, a protocol is detailed for easily performing recombineering experiments aimed at entering single and multiple changes in the chromosome of P. putida. This was made by implementing the workflow named High-Efficiency Multi-site genomic Editing (HEMSE), which delivers simultaneous mutations with a simple and effective protocol. © 2022, Springer Science+Business Media, LLC, part of Springer Nature.},
	keywords = {metabolism; GENETICS; GENOMICS; GENOMICS; Escherichia coli; Escherichia coli; single stranded DNA; Pseudomonas putida; Pseudomonas putida; Pseudomonas putida; DNA, Single-Stranded; Synthetic biology; procedures; recombinase; Gene editing; Gene editing; ssDNA; multiplex genome editing; recombinases; Cycled recombineering; HEMSE},
	year = {2022},
	pages = {37-52}
}

@article{MTMT:33550799,
	title = {Recombineering in Non-Model Bacteria},
	url = {https://m2.mtmt.hu/api/publication/33550799},
	author = {Corts, A. and Thomason, L.C. and Costantino, N. and Court, D.L.},
	doi = {10.1002/cpz1.605},
	journal-iso = {CURR PROT},
	journal = {CURRENT PROTOCOLS},
	volume = {2},
	unique-id = {33550799},
	issn = {2691-1299},
	abstract = {The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species. Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.},
	keywords = {Humans; GENETICS; BACTERIA; human; Base Sequence; nucleotide sequence; bacterium; single stranded DNA; DNA, Single-Stranded; Homologous recombination; Homologous recombination; recombineering; Gene editing; Gene editing; RecET; λ Red; annealase; non-model bacteria},
	year = {2022}
}

@article{MTMT:33056492,
	title = {Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing},
	url = {https://m2.mtmt.hu/api/publication/33056492},
	author = {Volke, D.C. and Martino, R.A. and Kozaeva, E. and Smania, A.M. and Nikel, P.I.},
	doi = {10.1038/s41467-022-30780-z},
	journal-iso = {NAT COMMUN},
	journal = {NATURE COMMUNICATIONS},
	volume = {13},
	unique-id = {33056492},
	issn = {2041-1723},
	abstract = {CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far. © 2022, The Author(s).},
	keywords = {metabolism; PHENOTYPE; PHENOTYPE; PHENOTYPE; GENETICS; BACTERIA; RNA; GENOME; bacterium; bacterium; CYTIDINE; CYTIDINE; procedures; CRISPR-CAS SYSTEMS; Gene editing; Gene editing; CRISPR Cas system},
	year = {2022},
	eissn = {2041-1723}
}

@article{MTMT:32603427,
	title = {ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida},
	url = {https://m2.mtmt.hu/api/publication/32603427},
	author = {Asin-Garcia, E. and Martin-Pascual, M. and Garcia-Morales, L. and Van, Kranenburg R. and Martins, Dos Santos V.A.P.},
	doi = {10.1021/acssynbio.1c00297},
	journal-iso = {ACS SYNTH BIOL},
	journal = {ACS SYNTHETIC BIOLOGY},
	volume = {10},
	unique-id = {32603427},
	issn = {2161-5063},
	abstract = {Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5′-NNG-3′ of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond. © 2021 The Authors. Published by American Chemical Society.},
	keywords = {Pseudomonas putida; Multiplexing; Recoding; recombineering; CRISPR-ScCas9-mediated counterselection; minimal-PAM},
	year = {2021},
	pages = {2672-2688}
}

@article{MTMT:32235702,
	title = {Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A},
	url = {https://m2.mtmt.hu/api/publication/32235702},
	author = {Cook, T.B. and Jacobson, T.B. and Hofstetter, H. and Amador-Noguez, D. and Thomas, M.G. and Pfleger, B.F.},
	doi = {10.1016/j.ymben.2021.06.004},
	journal-iso = {METAB ENG},
	journal = {METABOLIC ENGINEERING},
	volume = {67},
	unique-id = {32235702},
	issn = {1096-7176},
	abstract = {Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A. © 2021 International Metabolic Engineering Society},
	keywords = {PEPTIDES; POINT MUTATION; TRANSCRIPTION; ARTICLE; BACTERIA; DNA MODIFICATION; Plants (botany); high performance liquid chromatography; Genetic engineering; Genetic engineering; controlled study; nonhuman; biosynthesis; cell density; particle size; KETONES; Serratia marcescens; plasmid DNA; fatty acid metabolism; genomic DNA; bacterial strain; drug synthesis; Liquid chromatography-Mass spectrometry; respiratory chain; Prodigiosin; Pseudomonas putida; Pseudomonas putida; TRANSLATION INITIATION; start codon; DNA base composition; heterologous expression; heterologous expression; heterologous expression; bacterial genome; Synthetic biology; Synthetic biology; metabolic engineering; bacterial chromosome; mismatch repair; carbon sink; Genome editing; malonyl coenzyme A; Gene editing; guide RNA; polyketide; Chromosomal integration; Biosynthetic gene cluster; Heterologous production; Genetic modifications; natural product synthesis; Photorhabdus luminescens; pyoverdine; nonribosomal peptide synthetases; Non-ribosomal peptide; Gram negative soil bacterium; glidobactin A},
	year = {2021},
	eissn = {1096-7184},
	pages = {112-124}
}

@article{MTMT:32021979,
	title = {Spatiotemporal Manipulation of the Mismatch Repair System of Pseudomonas putida Accelerates Phenotype Emergence},
	url = {https://m2.mtmt.hu/api/publication/32021979},
	author = {Fernández-Cabezón, L. and Cros, A. and Nikel, P.I.},
	doi = {10.1021/acssynbio.1c00031},
	journal-iso = {ACS SYNTH BIOL},
	journal = {ACS SYNTHETIC BIOLOGY},
	volume = {10},
	unique-id = {32021979},
	issn = {2161-5063},
	abstract = {The development of complex phenotypes in industrially relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed toward increasing mutation frequencies, yet the precise control of mutagenesis processes in cell factories continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in the bacterial domain. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in DNA mutation frequencies and a heritable fixation of mutations in the genome. Following such an accelerated mutagenesis-followed by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin (either individually or combined) and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate the evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes toward the desired phenotype(s). ©},
	keywords = {EVOLUTION; MUTAGENESIS; Pseudomonas putida; Synthetic biology; metabolic engineering; mismatch repair system},
	year = {2021},
	pages = {1214-1226}
}

@article{MTMT:32636028,
	title = {The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa},
	url = {https://m2.mtmt.hu/api/publication/32636028},
	author = {On, Y.Y. and Welch, M.},
	doi = {10.1099/MIC.0.001120},
	journal-iso = {MICROBIOL-SGM},
	journal = {MICROBIOLOGY-SGM},
	volume = {167},
	unique-id = {32636028},
	issn = {1350-0872},
	abstract = {Over the last 70 years, we’ve all gotten used to an Escherichia coli-centric view of the microbial world. However, genomics, as well as the development of improved tools for genetic manipulation in other species, is showing us that other bugs do things differently, and that we cannot simply extrapolate from E. coli to everything else. A particularly good example of this is encountered when considering the mechanism(s) involved in DNA mismatch repair by the opportunistic human pathogen, Pseudomonas aeruginosa (PA). This is a particularly relevant phenotype to examine in PA, since defects in the mismatch repair (MMR) machinery often give rise to the property of hypermutability. This, in turn, is linked with the vertical acquisition of important pathoadaptive traits in the organism, such as antimicrobial resistance. But it turns out that PA lacks some key genes associated with MMR in E. coli, and a closer inspection of what is known (or can be inferred) about the MMR enzymology reveals profound differences compared with other, well-characterized organisms. Here, we review these differences and comment on their biological implications. © 2021 The Authors.},
	keywords = {PHENOTYPE; ARTICLE; METHYLATION; Escherichia coli; nonhuman; enzymology; Pseudomonas aeruginosa; Pseudomonas aeruginosa; Antibiotic resistance; mismatch repair; mismatch repair; Hypermutation; Hypermutation; MUTL; MutS},
	year = {2021},
	eissn = {1465-2080}
}

@article{MTMT:31961368,
	title = {Recombineering and MAGE},
	url = {https://m2.mtmt.hu/api/publication/31961368},
	author = {Wannier, Timothy M. and Ciaccia, Peter N. and Ellington, Andrew D. and Filsinger, Gabriel T. and Isaacs, Farren J. and Javanmardi, Kamyab and Jones, Michaela A. and Kunjapur, Aditya M. and Nyerges, Akos and Pál, Csaba and Schubert, Max G. and Church, George M.},
	doi = {10.1038/s43586-020-00006-x},
	journal-iso = {NATURE REV METHOD PRIM},
	journal = {NATURE REVIEWS METHODS PRIMERS},
	volume = {1},
	unique-id = {31961368},
	year = {2021},
	eissn = {2662-8449},
	orcid-numbers = {Ciaccia, Peter N./0000-0002-5745-6338; Ellington, Andrew D./0000-0001-6246-5338; Javanmardi, Kamyab/0000-0002-6449-6709; Nyerges, Akos/0000-0002-1581-490X}
}

@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:31499525,
	title = {Expanding the Reach of Recombineering to Environmental Bacteria},
	url = {https://m2.mtmt.hu/api/publication/31499525},
	author = {Borrero-de Acuna, Jose Manuel and Poblete-Castro, Ignacio},
	doi = {10.1016/j.tibtech.2020.03.014},
	journal-iso = {TRENDS BIOTECHNOL},
	journal = {TRENDS IN BIOTECHNOLOGY},
	volume = {38},
	unique-id = {31499525},
	issn = {0167-7799},
	abstract = {Broadening the application of recombineering technologies in biotechnologically important bacteria poses significant challenges. Aparicio et al. present a vital breakthrough for efficient singles-tranded recombineering by utilizing a thermoinducible system in Pseudomonas putida. This offers a simple genome-editing tool towards creating superior biocatalysts for the synthesis of chemicals and for bioremediation endeavors.},
	year = {2020},
	eissn = {1879-3096},
	pages = {684-685}
}

@article{MTMT:31598543,
	title = {Targeted mutagenesis of multiple chromosomal regions in microbes},
	url = {https://m2.mtmt.hu/api/publication/31598543},
	author = {Csörgő, Bálint and Nyerges, Ákos and Pál, Csaba},
	doi = {10.1016/j.mib.2020.05.010},
	journal-iso = {CURR OPIN MICROBIOL},
	journal = {CURRENT OPINION IN MICROBIOLOGY},
	volume = {57},
	unique-id = {31598543},
	issn = {1369-5274},
	year = {2020},
	eissn = {1879-0364},
	pages = {22-30},
	orcid-numbers = {Csörgő, Bálint/0000-0003-0397-6845; Nyerges, Ákos/0000-0002-1581-490X}
}

@article{MTMT:31383836,
	title = {Improved bacterial recombineering by parallelized protein discovery},
	url = {https://m2.mtmt.hu/api/publication/31383836},
	author = {Wannier, Timothy M. and Nyerges, Ákos and Kuchwara, Helene M. and Czikkely, Márton Simon and Balogh, Dávid and Filsinger, Gabriel T. and Borders, Nathaniel C. and Gregg, Christopher J. and Lajoie, Marc J. and Rios, Xavier and Pál, Csaba and Church, George M.},
	doi = {10.1073/pnas.2001588117},
	journal-iso = {P NATL ACAD SCI USA},
	journal = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
	volume = {117},
	unique-id = {31383836},
	issn = {0027-8424},
	abstract = {Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a highthroughput method for SSAP discovery that we call "serial enrichment for efficient recombineering" (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.},
	year = {2020},
	eissn = {1091-6490},
	pages = {13689-13698},
	orcid-numbers = {Wannier, Timothy M./0000-0002-4165-4751; Nyerges, Ákos/0000-0002-1581-490X; Lajoie, Marc J./0000-0002-0477-5511}
}

@article{MTMT:31501199,
	title = {Industrial biotechnology ofPseudomonas putida: advances and prospects},
	url = {https://m2.mtmt.hu/api/publication/31501199},
	author = {Weimer, Anna and Kohlstedt, Michael and Volke, Daniel C. and Nikel, Pablo I. and Wittmann, Christoph},
	doi = {10.1007/s00253-020-10811-9},
	journal-iso = {APPL MICROBIOL BIOT},
	journal = {APPLIED MICROBIOLOGY AND BIOTECHNOLOGY},
	volume = {104},
	unique-id = {31501199},
	issn = {0175-7598},
	abstract = {Pseudomonas putidais a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications ofP. putidaas a cell factory.},
	keywords = {Biotransformation; LIGNIN; BIOCATALYSIS; Pseudomonas putida; Synthetic biology; metabolic engineering; PHA; bioeconomy; Bacterial chassis; Microbial cell factory; KT2440; EDEMP cycle},
	year = {2020},
	eissn = {1432-0614},
	pages = {7745-7766},
	orcid-numbers = {Wittmann, Christoph/0000-0002-7952-985X}
}