HPLC analysis scanning was performed using a diode array detector model L-2450 (Hitachi, Japan) under the following conditions: ODS-80TM, i.d.=150 × 4.6 mm (Toso Co., Japan); MeCN, 0.06% TFA (30 : 70); flow rate, 0.8 mL min−1; and UV wavelength, 200–300 nm. LC-MS analysis was performed on LCMS2010 (Shimadzu) using reverse-phase INK 128 molecular weight HPLC [STR ODS-II, i.d.=150 × 2.0 mm; MeCN, 0.06% TFA (35 : 65); flow rate, 0.2 mL min−1; and UV wavelength, 220 nm]. Standard compounds of M-II, M-III, and M-VI were obtained from the fermentation broth of M. griseorubida A11725. The disruption cassette FRT-neo-oriT-FRT-attB was used to obtain the mycE disruption mutant of M. griseorubida. In previous
studies, the transconjugant of M. griseorubida has never been isolated with pSET152 as an intergeneric conjugation vector. Therefore, we estimated that M. griseorubida would not possess the bacteriophage φC31 attB site on the chromosome. The mycE-deleted plasmid pMG502, which had the mycinose biosynthetic gene cluster
region in which mycE was replaced with the disruption cassette, was generated with pSAN-lac as the suicide vector. pSAN-lac DAPT cell line was constructed with pUC18 and pIJ350 as an E. coli–Streptomyces shuttle vector, but the plasmid has never been amplified in M. griseorubida cells (data not shown). Plasmid pMG502 was transferred from E. coli to M. griseorubida A11725 by intergeneric conjugation, and some neomycin-resistant (neor) and thiostrepton-sensitive (thios) transconjugants were isolated. PCR was used to verify that the chromosomal copy of A11725 mycE was deleted by double cross-over. Using the primers mycEF and mycERBam annealing
outside the disruption cassette, the 1.4- and 1.2-kb amplified fragments were observed in TPMA0014 and the wild strain A11725, respectively (Fig. 2b). The size difference indicated that TPMA0014 was the mycE disruption mutant. M-VI was detected in the EtOAc extract from the FMM culture broth of TPMA0014 at 7.63 min (Fig. 3). PI-1840 However, the productivity of M-VI by TPMA0014 was very low (0.08 μg mL−1), and it was estimated that the direction of neo gene transcription had a negative effect on the productivity. We also isolated another mycE disruption mutant in which the direction of the neo gene was opposite to the mycinose biosynthesis gene cluster. The neor and thios transconjugant TPMA0003, which was isolated by the introduction of pMG503 into A11725, was confirmed to be a mycE disruption mutant by PCR (Fig. 2b); the M-VI productivity (13.8 μg mL−1) of TPMA0003 was higher than that of TPMA0014 (Fig. 3). Furthermore, three unknown peaks E-1, E-2, and E-3 were observed in the chromatogram of the extract of TPMA0003 at 5.62, 6.95, and 6.28 min, respectively. LC-MS was performed for the extract to measure the molecular weight of these metabolites (E-1; m/z 684, E-2; m/z 684, and E-3; m/z 698).