| Many diseases in marine animals are caused by pathogenic marine yeasts which have brought great loss for mariculture industry. Much research has shown that the killer yeasts can be applied to control the growth of pathogenic yeasts in humans, animals and plants. This method has received widely attention as a new method to control pathogenic yeasts. The marine yeast Williopsis saturnus WC91-2 isolated from sea sediment could kill a wide range of sensitive microorganisms and was able to produce more killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab than any other killer yeasts. It was found that the purifiedβ-1,3-glucanase from W. saturnus WC91-2 could inhibit the activity of the WC91-2 toxin produced by the same yeast. In order to make W. saturnus WC91-2 produce high activity of the WC91-2 toxin against the pathogenic yeasts in crab, it is necessary to delete the gene encoding the exo-β-1,3-glucanase.The knock-out vector for disruption of the WsEXG1gene encodingβ-1,3-glucanase was constructed by using the Zeocin gene as the maker gene . The fragments carrying the promoter of the WsEXG1 gene : Zeocin gene : the terminator of the WsEXG1 gene were transformed into W. saturnus. After determination ofβ-1,3-glucanase activity produced by different transformants, it was found that the transformants produce much lower level ofβ-1,3-glucanase than their wild type strain WC91-2. The lowest activity ofβ-1,3-glucanase produced by the transformants could reach 0.71U/mL. Furthermore, the transformants could produce much more killer toxin (the size of inhibition zone was 9±0.6mm)than its wild type W. saturnus WC91-2 (the size of inhibition zone was 13±0.5mm).In order to further reduceβ-1,3-glucanase activity and remove the chromosomes in which the WsEXG1 gene had not been deleted, the transformant strain was grown in the sporulation medium, the ascospores formed were released from the asci and each ascospore released was grown on plates. After determination ofβ-1,3-glucanase activity produced by different strains from different ascospores, it was observed that W. saturnus WC91-2-2 among the different yeast strains including W. saturnus WC91-2 had the lowestβ-1,3-glucanase activity which was 0.45U/mL and killer toxin production was further improved compared to its wild type W. saturnus. PCR results demonstrate that the zeocin gene had been inserted into the ORF of the WsEXG1 gene in W.saturnus WC91-2-2 .The optimal medium components and growth conditions for killer toxin production by the disruptant W.saturnus WC91-2-2 were studied using"one-variable-at-a-time"method, the results showed that after the disruptant grew in the medium containing 1.0% glucose, 3.0% yeast extract, 0% NaCl, pH 4.5 at 24°C,180rpm, it could produce high level of killer toxin (the size of inhibition zone was 19±0.8mm) while its wild type strain only produce 13±0.6mm, the activity ofβ-1,3-glucanase droped from 1.6 U/mL to 0.2 U/mL.The 2-L fermentation was performed to scale up killer toxin production by the disruptant strain under the conditions of the agitation speed of 250 rpm, aeration rate of 8 l/min, the temperature of 24.0°C. Within 36 h of the fermentation, W. saturnus WC91-2-2 produced the highest amount of killer toxin (the size of inhibition zone was 22±0.7 mm). After optimization of the agitation speed, it was found when the agitation speed was 250 rpm, W. saturnus WC91-2-2 produced more killer toxin. |
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