An alternative tetrahydrofolate pathway for formaldehyde oxidation in verrucomicrobial methanotrophs: Primer design for folD and ftfL and transformation of E. coli

ABSTRACT Methylotrophs make a living by using one-carbon compounds as energy and carbon source. Methanol (CH 3 OH) is utilized by various methylotrophs and is oxidized by a methanol dehydrogenase. The calcium-dependent methanol dehydrogenase MxaFI converts methanol to formaldehyde (CH 2 O). In addition to MxaFI, the lanthanide-dependent methanol dehydrogenase XoxF is found in a wide range of bacteria. XoxF isolated from the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV possesses an unusually high affinity for both methanol and formaldehyde and converts methanol to formate (HCOOH) in vitro . However, genomic analyses and biochemical studies on related XoxF methanol dehydrogenases have questioned whether these enzymes are dedicated to the conversion of formaldehyde to formate in vivo . Instead, the genes encoding the bifunctional enzyme FolD and the enzyme FtfL, which we detected in all verrucomicrobial methanotrophs, were proposed to form a formaldehyde oxidation pathway utilizing tetrahydrofolate as C1-carrier. folD and ftfL are expressed in M. fumariolicum SolV and most closely related to homologues found in the phyla Verrucomicrobia and Proteobacteria, respectively. Here, we designed primers targeting Mf- folD and Mf- ftfL and amplified these genes through PCR. The amplified genes were ligated into pET30a(+) vectors which were subsequently used for the successful transformation of E. coli XL1-Blue cells. The vectors can be used for heterologous expression and subsequent His-tag purification to biochemically investigate whether FolD and FtfL could form an alternative tetrahydrofolate pathway for formaldehyde oxidation in verrucomicrobial methanotrophs.