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      A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

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          Summary

          Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase β – an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recβ homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus‐wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Recβ functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads – and beyond.

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          Molecular Cloning : A Laboratory Manual

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            Programming cells by multiplex genome engineering and accelerated evolution.

            The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.
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              A complementation analysis of the restriction and modification of DNA in Escherichia coli.

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                Author and article information

                Contributors
                vdlorenzo@cnb.csic.es
                Journal
                Microb Biotechnol
                Microb Biotechnol
                10.1111/(ISSN)1751-7915
                MBT2
                Microbial Biotechnology
                John Wiley and Sons Inc. (Hoboken )
                1751-7915
                02 November 2017
                January 2018
                : 11
                : 1 , Thematic Issue: Microbial Electrochemical technologies come of age ( doiID: 10.1111/mbt2.2018.11.issue-1 )
                : 176-188
                Affiliations
                [ 1 ] Systems Biology Program Centro Nacional de Biotecnología (CNB‐CSIC) Campus de Cantoblanco Madrid 28049 Spain
                [ 2 ] Synthetic and Systems Biology Unit Institute of Biochemistry Biological Research Centre of the Hungarian Academy of Sciences Szeged H‐6726 Hungary
                Author notes
                [*] [* ]For correspondence. E‐mail vdlorenzo@ 123456cnb.csic.es ; Tel. (+34) 915854536; Fax (+34) 915854506.
                Article
                MBT212846
                10.1111/1751-7915.12846
                5743808
                29094478
                b27a0a33-32d7-4fb7-b63f-4871d34f7a7f
                © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 28 June 2017
                : 27 July 2017
                : 03 August 2017
                Page count
                Figures: 5, Tables: 2, Pages: 13, Words: 9485
                Funding
                Funded by: Spanish Ministry of Economy and Competitiveness
                Award ID: RTC‐2014‐1777‐3
                Award ID: 2015‐66960‐C3‐2‐R
                Funded by: ARISYS
                Award ID: ERC‐2012‐ADG‐322797
                Funded by: EmPowerPutida
                Award ID: EU‐H2020‐BIOTEC‐2014‐2015‐6335536
                Funded by: FUTURE
                Award ID: 704410‐H2020‐MSCA‐IF‐15
                Funded by: European Research Council
                Funded by: Wellcome Trust
                Funded by: GINOP
                Award ID: TUMORDNS: GINOP‐2.3.2‐15‐2016‐00020
                Award ID: EVOMER: GINOP‐2.3.2‐15‐2016‐00014
                Funded by: Hungarian Academy of Sciences
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                mbt212846
                January 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.2.8 mode:remove_FC converted:26.12.2017

                Biotechnology
                Biotechnology

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