A markerless gene deletion and integration system for Thermoanaerobacter ethanolicus. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Shao, Xiongjun; Zhou, Jilai; Olson, Daniel; Lynd, Lee
• Is Part Of: Biotechnology for biofuels. Volume 9:Issue 1(2016)
• Page Start: 1
• Page End: 8
• Publication Date: 2016-12
• Subjects: Thermoanaerobacter ethanolicus -- Gene deletion -- Gene integration -- Marker removal -- Clean knockout -- tdk -- FUDR -- Natural competence; Biotechnology -- Periodicals; Biomass energy -- Periodicals; Energy-Generating Resources -- Periodicals; 662.88
• DOI: 10.1186/s13068-016-0514-1
• Languages: English
• ISSNs: 1754-6834

Abstract

Abstract Background Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. A genetic system with reusable selective markers would be useful for deleting acid production pathways as well as other genetic modifications. Results The thymidine kinase (tdk ) gene was deleted fromT. ethanolicus JW200 to allow it to be used as a selectable marker, resulting in strain X20. Deletion of thetdk gene reduced growth rate by 20 %; however, this could be reversed by reintroducing thetdk gene (strain X20C). Thetdk and high-temperature kanamycin (htk ) markers were tested by using them to delete lactate dehydrogenase (ldh ). During positive selection ofldh knockouts in strain X20 on kanamycin agar plates, six out of seven picked colonies were verified transformants. Deletion ofldh reduced lactic acid production by 90 %. Thetdk and 5-fluoro-2′-deoxyuridine (FUDR) combination worked reliably as demonstrated by successfultdk removal in all 21 colonies tested. Conclusion A gene deletion and integration system with reusable markers has been developed forThermoanaerobacter ethanolicus JW200 with positive selection on kanamycin and negative selection on FUDR. Gene deletion was demonstrated byldh gene deletion and gene integration was demonstrated by re-integration of thetdk gene. Transformation via a natural competence protocol could use DNA PCR products amplified directly from Gibson Assembly mixture for efficient genetic modification.