Mesorhizobium loti cells were cultivated at 30 °C in tryptose–yeast (TY) medium and pyridoxine (PN) synthetic medium, as described previously (Yuan et al., 2004). Plasmids pTA2 (Toyobo, Osaka, Japan) and pET-21a (Novagen) were used for cloning and expression. pK18mobsacB

and pKRP12 (National Bioresource Project) were used for disruption of the mll6786 gene. The primers shown in Table 1 were purchased from Roxadustat solubility dmso Invitrogen Japan (Tokyo, Japan). 4-Pyridoxolactone (Tamura et al., 2008), FHMPC (Yokochi et al., 2009), HMPDC (Mukherjee et al., 2007), HMPC (Yuan et al., 2006), and AAMS (Yuan et al., 2008) were prepared as described previously. A biotin-labeled marker DNA (biomarker BGB324 cost low, biotin conjugate) was purchased from BioVentures, Inc. (Murfreesboro, TN), and marker DNA fragments (λ-HindIII) from New England Biolabs Japan, Inc. (Tokyo, Japan). 5′-CATATGCCCCCAGATTTCAATTTGCGA-3 (underline, NdeI site) 5′-AAGCTTCCTCAAATCCCGTTGTCCATGGAT-3 (underline, HindIII site) 5′-TCTAGAGCGTCGCGAGATGAAGTGGT-3 (underline, XbaI site) 5′-CTGCAGCAGGCTGTCATTGCTGGAGG-3 (underline, PstI site) 5′-CTGCAGGTCATGACCGCCGCGGACTTCTATT-3 (underline, PstI site) 5′-AAGCTTAGTCCCAATCGTAGCTGCGGCCCT-3 (underline, HindIII site) 5′-CACCACCACCACCACCACTGAGAT-3 (double underline, His6-coding site) 5′-A*TGTCTGCCGCCATGTCCAT-3

(*biotin-labeled) Sinomenine A disruption plasmid was constructed as follows. A 630-bp fragment harboring 400-bp of the 5′ end of mll6786 plus its 230-bp upstream region was amplified by PCR with primers 6786-mut-1F and 6786-mut-1R. A 640-bp fragment harboring 280-bp of the 3′ end of mll6786 plus its 360-bp downstream region was amplified by PCR with primers 6786-mut-2F and 6786-mut-2R. The fragments were cloned into the pTA2 vector, separately, to construct pTA2-630 and pTA2-640. Then, the 630-bp fragment cut out from pTA2-630

with XbaI and PstI was cloned into plasmid pK18mobsacB to construct pK18-630, to which the 640-bp fragment cut out from pTA2-640 with PstI and HindIII was ligated to construct pK18-1270. The 2000-bp tetracycline resistance gene obtained from pKRP12 by digestion with PstI was inserted into pK18-1270, and the resulting plasmid pK18-1270::Tc was used as the disruption plasmid. The plasmid was transferred into M. loti MAFF303099 via conjugation with E. coli S17-1/pK18-1270::Tc (Simon et al., 1983) and transconjugants were selected as described previously (Yokochi et al., 2006). mll6786 was amplified by PCR from the chromosomal DNA of M. loti with primers 6786-F and 6786-R. The amplified 680-bp fragment was cloned into pTA2 to construct pTA2-680. pTA2-680 was digested with NdeI and HindIII, and then the digested DNA fragment was inserted into the NdeI/HindIII sites of pET21a+ to construct expression plasmid pET6786.