An Efficient Intergeneric Conjugation of DNA from Escherichia coli to Mycelia of the Lincomycin-Producer Streptomyces lincolnensis

Abstract

Streptomyces lincolnensis is a producer of lincomycin, which is a lincosamide antibiotic for the treatment of infective diseases caused by Gram-positive bacteria. S. lincolnensis is refractory to introducing plasmid DNA into cells because of resistance of foreign DNAs and poor sporulation. In this study, a simple and efficient method of transferring plasmids into S. lincolnensis through the intergeneric Escherichia coli-mycelia conjugation was established and optimized for the first time. The recipient mycelia of S. lincolnensis were prepared in liquid SM medium containing 10.3% sucrose for three days. The dispersed mycelia were conjugated with competent E. coli donor cells. The exconjugants were regenerated efficiently on solid mannitol soya flour (MS) medium containing 20 mM MgCl2. The average conjugation frequency was observed at 1.1 × 10−4 per input donor cell and validated functionally by transferring two types of vectors containing lincomycin resistance genes lmrA, lmrB and lmrC into S. lincolnensis mycelia. The data of fermentation in shaking flasks showed the lincomycin yield of the exconjugants increased by 52.9% for the multiple copy vector and 38.3% for the integrative one, compared with the parental strain. The efficient and convenient method of intergeneric E. coli-mycelia conjugation in this study provides a promising procedure to introduce plasmid DNA into other refractory streptomycetes.

Co-overexpression of lmbW and metK led to increased lincomycin A production and decreased byproduct lincomycin B content in an industrial strain of Streptomyces lincolnensis

“…The spores of the S. lincolnensis were cultivated at 30°C on the modified Gauze's Medium No.1 (MGM 1, containing 2% soluble starch, 0Á5% soybean, 0Á1% KNO 3 , 0Á05% NaCl, 0Á05% MgSO 4 , 0Á05% K 2 HPO 4 , 0Á001% FeSO 4 , 2% agar, pH 7Á0) for 7 days. Conjugation between E. coli and Streptomyces was conducted as previously described (Du et al 2012). Then 2 ml of the seed culture were transferred into 25 ml of fermentation medium (10% glucose, 2% soybean, 0Á15% cream corn, 0Á8% NaNO 3 , 0Á5% NaCl, 0Á6% (NH 4 ) 2 SO 4 , 0Á03% K 2 HPO 4 , 0Á8% CaCO 3 , pH 7Á1) in 250 ml flask and cultivated at 30°C and 250 rev min À1 for 7 days.…”

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

“…This is still low compared to those obtained for Streptomyces coelicolor (1.1 ϫ 10 Ϫ1 ) when the methylation-deficient donor E. coli ET12567/pUB307 was applied for the first time (55) or compared to those achieved for S. lincolnensis mycelia exconjugants (2.8 ϫ 10 Ϫ5 ) when regenerated on solid MS medium containing 10.3% sucrose (63).…”

“…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). Due to the fact that several streptomycetes degrade methylated DNA, a nonmethylating E. coli strain can be preferable as the DNA donor in intergeneric conjugation (55).…”

“…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.…”

Immobilization of Streptomyces thermotolerans 11432 on polyurethane foam to improve production of Acetylisovaleryltylosin

“…The gene was cloned into plasmid pUWL201apr [9], and then the recombinant plasmid carrying acyB1-B2 was transferred into S. thermotolerans ATCC 11416 by intergeneric conjugal transfer. Through screening of a variety of recombinant strains, strain S. thermotolerans 11432, with high production capacity, was obtained.…”