Construction of ma4551 knockout plasmids

Approximately 1 kb up- and downstream regions flanking the gene ma4551 were PCR-amplified using the Q5® High-Fidelity DNA polymerase and oligonucleotide primers containing 15 nucleotide overhangs listed in Supplementary Table ³. The obtained PCR fragments were cloned into pJK301⁵⁸ using the In-Fusion® HD Cloning Kit (Takara Bio USA, Inc.) according to the manufacturer’s instructions. The upstream region of ma4551 was PCR-amplified using the primer pair UP-fw und UP-rev and subsequently cloned into the plasmid pJK301, digested with HindIII and XhoI restriction enzymes, yielding the plasmid pJK_UP. The downstream region of ma4551 was PCR-amplified with the primers DOWN-fw and DOWN-rev and cloned into the pJK_UP vector digested with BamHI and SpeI. The resulting plasmid pJK301_ma4551 was linearized using ApaI and AleI. The linear fragment containing the up- and downstream regions of ma4551, the pac-hpt cassette and the FRT sites was purified using the GeneJET PCR Purification Kit (Thermo Fisher Scientific) and transferred into M. acetivorans WWM1 for double homologous recombination.