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KMID : 0380020070220050297
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Korean Journal of Biotechnology and Bioengineering
2007 Volume.22 No. 5 p.297 ~ p.304
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Mass Screening of Lovastatin High-yielding Mutants through Statistical Optimization of Sporulation Medium and Application of Miniaturized Fungal Cell Cultures
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Jeong Yong-Seob
Kim Pyeung-Hyeun
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Abstract
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For large and rapid screening of high-yielding mutants of lovastatin produced by filamentous fungal cells of Aspergillus terreus, one of the most important stage is to test as large amounts of mutated strains as possible. For this purpose, we intended to develop a miniaturized cultivation method using culture tube instead of traditional flask (working volume ). For obtaining large amounts of conidiospores to be used as inoculums for miniaturized cultures, 4 components i.e., glucose, sucrose, yeast extract and were intensively investigated, which had been observed to show positive effect on enhancement of spore production through Plackett-Burman design experimet. When optimum concentrations of these components that were determined through application of response surface method (RSM) based on central composite design (CCD) were used, maximum spore numbers amounting to spores/plate were obtained, resulting in approximately 190 fold increase as compared to the commonly used PDA sporulation medium. Using the miniaturized cultures, intensive strain development programs were carried out for screening of lovastatin high-yielding as well as highly reproducible mutants. It was observed that, for maximum production of lovastatin, the producers should be activated through ’PaB’ adaptation process during the early solid culture stage. In addition, they should be proliferated in condensed filamentous forms in miniaturized growth cultures, so that optimum amounts of highly active cells could be transferred to the production culture-tube as reproducible inoculums. Under these highly controlled fermentation conditions, compact-pelleted morphology of optimum size (less than 1 mm in diameter) was successfully induced in the miniaturized production cultures, which proved essential for maximal utilization of the producers’ physiology leading to significantly enhanced production of lovastatin. As a result of continuous screening in the miniaturized cultures, lovastatin production levels of the 81% of the daughter cells derived from the high-yielding producers turned out to be in the range of 80%120% of the lovastatin production level of the parallel flask cultures. These results demonstrate that the miniaturized cultivation method developed in this study is efficient high throughput system for large and rapid screening of highly stable and productive strains.
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KEYWORD
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Aspergillus terreus, lovastatin, screening, sporulation medium, response surface method, miniaturized cultures
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