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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 from  T. ethanolicus  JW200 to allow it to be used as a selectable marker, resulting in strain X20. Deletion of the  tdk  gene reduced growth rate by 20 %; however, this could be reversed by reintroducing the  tdk  gene (strain X20C). The  tdk  and high-temperature kanamycin ( htk ) markers were tested by using them to delete lactate dehydrogenase ( ldh ). During positive selection of  ldh  knockouts in strain X20 on kanamycin agar plates, six out of seven picked colonies were verified transformants. Deletion of  ldh  reduced lactic acid production by 90 %. The  tdk  and 5-fluoro-2′-deoxyuridine (FUDR) combination worked reliably as demonstrated by successful  tdk  removal in all 21 colonies tested.    Conclusion  A gene deletion and integration system with reusable markers has been developed for  Thermoanaerobacter ethanolicus  JW200 with positive selection on kanamycin and negative selection on FUDR. Gene deletion was demonstrated by  ldh  gene deletion and gene integration was demonstrated by re-integration of the  tdk  gene. Transformation via a natural competence protocol could use DNA PCR products amplified directly from Gibson Assembly mixture for efficient genetic modification.

A markerless gene deletion and integration system for Thermoanaerobacter ethanolicus