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KMID : 0380020050200050376
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Korean Journal of Biotechnology and Bioengineering
2005 Volume.20 No. 5 p.376 ~ p.382
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Development of Avermectin B(1a) High-yielding Mutants through Rational Screening Strategy based on Understanding of Biosynthetic Pathway
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Song Sung-Ki
Jeong Yong-Seob
Chun Gie-Taek
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Abstract
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Avermectin (AVM) produced by Streptomyces avermitilis via polyketide pathway is a secondary metabolite with powerful anthelmintic and insecticidal activities, thus being used as an efficient agent in the field of agriculture and animal health. It has been reported that a precursor for AVM biosynthesis was isoleucine and the biosynthetic pathway of AVM was closely similar to that of fatty acid. Based on understanding of the biosynthetic pathway of AVM , we intended to screen various mutants resistant against O-methyl threonine (OMT), an isoleucine-anti metabolite, and/or mutants resistant against p-fluoro phenoxy acetic acid (pFAC), an inhibitor of fatty acid biosynthesis. It was inferred that these mutants could produce AVM more efficiently, due to the acquired capability of not only overproducing isoleucine intracellularly but also channelling metabolized carbon-sources into the polyketide pathway, thus leading to enhanced biosynthesis of AVM . The resulting mutant (PFA-1 strain) resistant against 100 ppm of pFAC was able to produce approximately 42 fold higher amount of AVM compared to the parallel mother strain (4,200 vs. 100 units/l). In addition, through the process of continuous strain improvement program carried out by gradually increasing the OMT concentration, it was possible to obtain a more attractive mutant with greater AVM production capacity (9,000 units/l). Notable was that significantly higher producer (12,000 units/l) could be selected through further screening of the resistant mutants, this time, to even higher concentration of PFAC. Meanwhile, through the analysis of AVM Bla production histograms (i.e., number of strains according to their AVM biosynthetic ability) for the earlier strains in comparison with the high producers having the characteristics of resistance to OMT and pFAC, it was found that production stability of the high-yielding producers were remarkably improved, as demonstrated by the fact that larger proportion of the mutated strains had greater capability of AVM biosynthesis ( in the range between 5,000 and 7,000 units/L; in the range between 6,000 and 7,000 units/l). Based on these consequences, it was concluded that the rational screening strategy based on the understanding of the biosynthetic pathway of AVM was very effective in obtaining high-yielding mutants with the features of enhanced production stability.
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KEYWORD
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Streptomyces avermitilis, avermectin B(1a), mutation, rational screening, polyketide
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