{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,18]],"date-time":"2026-10-18T11:59:09Z","timestamp":1792324749804,"version":"4.4.0"},"reference-count":67,"publisher":"Oxford University Press (OUP)","issue":"15","license":[{"start":{"date-parts":[[2022,8,3]],"date-time":"2022-08-03T00:00:00Z","timestamp":1659484800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"publisher","award":["075-15-2019-1658"],"award-info":[{"award-number":["075-15-2019-1658"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,8,26]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via \u03bbRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of \u03bb phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted \u03bbRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50\u2013100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.<\/jats:p>","DOI":"10.1093\/nar\/gkac649","type":"journal-article","created":{"date-parts":[[2022,8,3]],"date-time":"2022-08-03T06:39:59Z","timestamp":1659508799000},"page":"8947-8960","source":"Crossref","is-referenced-by-count":16,"title":["Robust counterselection and advanced \u03bbRed recombineering enable markerless chromosomal integration of large heterologous constructs"],"prefix":"10.1093","volume":"50","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6248-1909","authenticated-orcid":false,"given":"Dmitrii M","family":"Bubnov","sequence":"first","affiliation":[{"name":"Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute for Genetics and Selection of Industrial Microorganisms of National Research Center \u2018Kurchatov Institute\u2019 (NRC \u2018Kurchatov Institute\u2019 \u2013 GosNIIgenetika) , 1-st Dorozhny pr., 1, Moscow 117545, Russia"},{"name":"Kurchatov Complex of Genetic Research, NRC \u2018Kurchatov Institute\u2019, Kurchatov Square , 1, Moscow 123098, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tigran V","family":"Yuzbashev","sequence":"additional","affiliation":[{"name":"Department of Bioengineering and Imperial College Centre for Synthetic Biology, Imperial College London , London SW7 2AZ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrey A","family":"Khozov","sequence":"additional","affiliation":[{"name":"Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute for Genetics and Selection of Industrial Microorganisms of National Research Center \u2018Kurchatov Institute\u2019 (NRC \u2018Kurchatov Institute\u2019 \u2013 GosNIIgenetika) , 1-st Dorozhny pr., 1, Moscow 117545, Russia"},{"name":"Kurchatov Complex of Genetic Research, NRC \u2018Kurchatov Institute\u2019, Kurchatov Square , 1, Moscow 123098, Russia"},{"name":"Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University, Lenin's Hills \u00a01-12, Moscow 119234, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olga\u00a0E","family":"Melkina","sequence":"additional","affiliation":[{"name":"Kurchatov Complex of Genetic Research, NRC \u2018Kurchatov Institute\u2019, Kurchatov Square , 1, Moscow 123098, Russia"},{"name":"Laboratory of Bacterial Genetics, NRC \u2018Kurchatov Institute\u2019 \u2013 GosNIIgenetika, 1-st Dorozhny pr. , 1, Moscow 117545, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatiana\u00a0V","family":"Vybornaya","sequence":"additional","affiliation":[{"name":"Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute for Genetics and Selection of Industrial Microorganisms of National Research Center \u2018Kurchatov Institute\u2019 (NRC \u2018Kurchatov Institute\u2019 \u2013 GosNIIgenetika) , 1-st Dorozhny pr., 1, Moscow 117545, Russia"},{"name":"Kurchatov Genomic Center, NRC \u2018Kurchatov Institute\u2019 - GosNIIgenetika, 1-st Dorozhny pr. , 1, Moscow 117545, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guy-Bart","family":"Stan","sequence":"additional","affiliation":[{"name":"Department of Bioengineering and Imperial College Centre for Synthetic Biology, Imperial College London , London SW7 2AZ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergey P","family":"Sineoky","sequence":"additional","affiliation":[{"name":"Bioresource Center Russian National Collection of Industrial Microorganisms (BRC VKPM), State Research Institute for Genetics and Selection of Industrial Microorganisms of National Research Center \u2018Kurchatov Institute\u2019 (NRC \u2018Kurchatov Institute\u2019 \u2013 GosNIIgenetika) , 1-st Dorozhny pr., 1, Moscow 117545, Russia"},{"name":"Kurchatov Complex of Genetic Research, NRC \u2018Kurchatov Institute\u2019, Kurchatov Square , 1, Moscow 123098, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2022,8,3]]},"reference":[{"key":"2022082523580492600_B1","doi-asserted-by":"crossref","first-page":"5978","DOI":"10.1073\/pnas.100127597","article-title":"An efficient recombination system for chromosome engineering in Escherichia coli","volume":"97","author":"Yu","year":"2000","journal-title":"Proc. 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