Development of Intergeneric Conjugal Gene Transfer System in Streptomyces diastatochromogenes 1628 and Its Application for Improvement of Toyocamycin Production

Abstract

Streptomyces diastatochromogenes 1628, capable of producing toyocamycin (TM), has exhibited a potential biocontrol effect in inhibiting the development of phytopathogens in the agriculture field. In this study, an efficient transformation system was developed using the intergeneric conjugation. This was achieved by optimization of experimental parameters. Under optimal conditions, a maximal conjugation frequency of 4.1 × 10(-4) per recipient was obtained. In order to heterologously express the gene vgb encoding Vitreoscilla hemoglobin in S. diastatochromogenes 1628, we placed vgb under the control of the constitutive promoter PermE(*) and constructed plasmid pIB139-vgb. This plasmid was integrated into the chromosome of S. diastatochromogenes 1628 using intergeneric conjugation established above. Finally, strain 1628-VHB-23 with the highest TM production was screened. Results indicated that expression of vgb gene had always significantly promoted the cell growth and TM production in S. diastatochromogenes 1628 under different dissolved oxygen conditions. In particular, under the limited aerobic condition, strain 1628-VHB-23 obtained 33.3 % more DCW and produced 210 % more TM in 7-l fermentor as compared with the wild-type strain.

Development of an Intergeneric Conjugal Transfer System for Xinaomycins-Producing Streptomyces noursei Xinao-4

“…In contrast with the results of Streptomyces coelicolor [21] and Streptomyces nodosus [25], where the donor-to-recipient ratio did not affect the conjugal efficiency, our results indicated that the maximum efficiency was received with a certain donor-to-recipient ratio of 100:1. Similar results had been observed in Kitasatospora setae [32], Streptomyces lincolnensis [24], and Streptomyces diastatochromogenes 1628 [31], Streptomyces natalensis [33] in which a certain ratio of donor to recipient cell number was required to achieve highest frequency.…”

“…Compared to the previous studies, in which the transformation efficiency is below 10 −3 [1,26,31,33,48], our study showed high transformation efficiency. The availability of this protocol was confirmed by the 22 double-crossover mutants of glycosyltranferase gene.…”

“…Incubation of heat-shocked spores prior to mixing with E. coli was recommended for many Streptomyces strains [23,31]. Therefore, the effect of incubation time of heat-shocked spores on the conjugation efficiency was also investigated.…”

An Efficient Method To Generate Gene Deletion Mutants of the Rapamycin-Producing Bacterium Streptomyces iranensis HM 35

“…Sun et al (64) found that the ratio of donor to recipient cell numbers was the most influential factor for conjugation into Streptomyces noursei. Also, for Streptomyces diastatochromogenes, the number of spores and their use instead of mycelia, in combination with a cultivation step at 30°C subsequent to the heat shock, were found to be the most important factors (65). In contrast, Du et al (63) achieved the highest conjugation efficiencies for Streptomyces lincolnensis by using mycelia rather than spores as recipients and by addition of 10.3% sucrose to the SM medium (63).…”

“…Theoretically, the described procedure provides a promising approach for the ultimate site-specific homologous recombination between this cassette and the bacterial chromosome to generate targeted gene knockouts. However, in practice, it does not work properly for every Streptomyces species, and further optimization is often required to successfully conjugate E. coli with refractory species (62)(63)(64)(65). Various species-dependent parameters which strongly affected the transformation efficiency were identified.…”