Establishment of the host-vector system for Micromonospora griseorubida.

Abstract

Micromonosporastrains produce some important antibiotics, such as gentamicins1*, sisomicins2), sagamicins3), fortimicin A4) and mycinamicins5). We discovered mycinamicins, 16-membered macrolide antibiotics with a strong antimicrobial activity against Gram-positive bacteria, and reported biosynthetic pathway of mycinamicin II6). It has been our interest to clarify the mechanismsof regulation and expression of the genes for mycinamicin biosynthesis in Micromonospora griseorubida in order to improve the producing strains. For this purpose, it was necessary to establish a system to express the cloned genes in M. griseorubida. hours. Protoplasts were filtered through cotton wool, centrifuged and resuspended in 1ml of P medium15). To obtain a good yield of protoplasts, growth phase turned out to be effective as pointed out in Streptomyces16). A dramatic increase in yield was observed using mycelia for 44 hours incubation (early stationary phase). While the number of protoplasts obtained from 36 hours incubated culture was 108/ml, that from 44hours culture provided 1010/ml.For regeneration of M. griseorubida protoplasts, R2YE17) generally used for regeneration of Streptomyces protoplasts, gave the regeneration frequency as low as < 0.1 %. Therefore, we designed MR0.3S by modifying the M agar, which was used for the cultivation of M. griseorubida. MR0.3S medium contained sucrose 103.0 g, soluble starch 20.0 g, soy bean meal 7.5 g, Polypepton 5.0 g, CaCO3 1.0 g, MgSO4 à" 7H2O 5.0 g, trace elements solution15) 10.0ml, FeSO4-7H2O 40.0mg and Bacto-agar 22.0g, per liter of deionized water (adjusted to pH 7.2 with 1 n NaOH

The biosynthetic genes encoding for the production of the dynemicin enediyne core inMicromonospora chersinaATCC53710

“…Notably, these results validate the genetic system and highlight the essential nature of dynE8 (and nonessential nature of orf8) for 8 biosynthesis. Given the lack of validated plasmids for complementation experiments in enediyne producing Micromonospora (Takada et al, 1994;Ahlert et al, 2002;Li et al, 2004;Hosted et al, 2005), the orf8 and orf23 replacement mutants are particularly noteworthy examples as they illustrate M. chersina genotype alterations without abolishing metabolite production.…”