Avermectin Production Research Articles

Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis.

The Tn3-like Streptomyces transposon Tn4560 was used to mutagenize Streptomyces avermitilis, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. Tn4560 transposed in this strain from a temperature-sensitive plasmid to the chromosome and from the chromosome to a plasmid with an apparent frequency of about 10(-4) to 10(-3) at both 30 and 39 degrees C. Auxotrophic and antibiotic nonproducing mutations were, however, obtained only with cultures that were kept at 37 or 39 degrees C. About 0.1% of the transposon inserts obtained at 39 degrees C caused auxotrophy or abolished antibiotic production. The sites of insertion into the S. avermitilis chromosome were mapped. Chromosomal DNA fragments containing Tn4560 insertions in antibiotic production genes were cloned onto a Streptomyces plasmid with temperature-sensitive replication and used to transport transposon mutations to other strains, using homologous recombination. This technique was used to construct an avermectin production strain that no longer makes the toxic oligomycin.

Instability of the production of avermectins, sporulation, and pigmentation in Streptomyces avermitilis.

Production of avermectins, sporulation ability, and colony pigmentation were followed in Streptomyces avermitilis C-18/6 cultures during serial transfer (for eight subcultures) in four different liquid media. These phenotypes were found to be unstable and independent of each other. A procedure was established whereby depressed avermectin production could be reversed but the degree of reversion was dependent on the history of the isolate, i.e., the type of medium used. The presence of isoleucine as the sole N source caused an immediate loss of avermectin production and this could not be reversed.

Branched-chain fatty acid requirement for avermectin production by a mutant of Streptomyces avermitilis lacking branched-chain 2-oxo acid dehydrogenase activity.

The eight natural avermectins produced by Streptomyces avermitilis have the carbon skeleton of either isobutyric or S-2-methylbutyric acid incorporated into their structures. A mutant of S. avermitilis has been isolated that contains no functional branched-chain 2-oxo acid dehydrogenase activity. The mutant, in contrast to its parent, is unable to grow with isoleucine, valine and leucine as carbon sources. In medium lacking both S(+)-2-methylbutyric and isobutyric acid, the mutant is also incapable of making the natural avermectins, while supplementation with either one of these compounds restores production of the corresponding four natural avermectins. These facts indicate that in S. avermitilis the branched-chain 2-oxo acid dehydrogenase enzyme functions not only to catabolize the cellular branched-chain amino acids in order to meet energy and growth requirements but also to provide the small branched-chain organic acid precursor molecules necessary for avermectin biosynthesis. Supplementation of the mutant strain with R(-)-2-methylbutyric acid yields novel isomeric avermectins unseen in the (unsupplemented) wild-type strain. It was also concluded that acetate and propionate production by branched-chain 2-oxo acid degradation is not absolutely essential for avermectin production.

Involvement of glucose catabolism in avermectin production by Streptomyces avermitilis.

The addition of glucose in the early stage of fermentation suppressed not only avermectin production but also the activity of 6-phosphogluconate dehydrogenase in the pentose phosphate pathway. On the other hand, when glucose was added at the late stage of fermentation, suppression of avermectin formation and 6-phosphogluconate dehydrogenase activity was not observed but avermectin formation was increased and about a twofold-higher content of avermectins than that of the control fermentation was accumulated.

Avermectin B2 O-methyltransferase activity in "Streptomyces avermitilis" mutants that produce increased amounts of the avermectins.

The level of activity of avermectin B O-methyltransferase, the enzyme which catalyzes the conversion of avermectin B components to avermectin A components, was analyzed in a series of "Streptomyces avermitilis" mutants selected for increased production of the avermectins. In all of the mutants, increased avermectin production was accompanied by increased avermectin B O-methyltransferase activity. Both the average specific activity and the maximum observed specific activity of avermectin B O-methyltransferase increased in direct proportion to avermectin production. The level of avermectin B O-methyltransferase alone did not determine the extent of conversion of avermectin B components to avermectin A components, since a constant ratio of B components to A components was maintained throughout the fermentation even though avermectin B O-methyltransferase specific activity varied three- to fivefold. These results indicate that avermectin B O-methyltransferase is not rate limiting. The correlation between avermectin B O-methyltransferase specific activity and avermectin production is compatible with the hypothesis that genes coding for successive steps in the same secondary metabolite biosynthetic pathway are coordinately regulated.