Genetic Improvement Of Siderophore-producing Marine Yeast Aureobasidium Pullulans HN6.2

Siderophore is a kind of iron-specific chelators, which are low-molecular weightcompounds possessing a high affinity for ferric ion. It played an important role insupplying iron to the cell of micorobes. They are produced by bacteria, fungi andgraminaceous plants for scavenging iron from their living environment. They are notonly secreted to transport extracellular iron but also produced intracellularly primarilyfor iron storage. It has been known thatthebiosynthesisofthesiderophoreis influencedby iron concentration, which can greatly inhibit the synthesis of siderophore when itbeyonds a certain range. Siderophores have been biochemically characterised fromvarious microorganism and they can be divided into three groups based on theirchemical structures: catechols, carboxylates and hydroxamates.There are a number ofpotential applications for siderophores in medicine, agriculture, reprocessing ofnuclear fuel, remediation of metal contaminated sites and the treatment of petroleumwaste.In our previous studies, Aureobasidium pullulans strain HN6.2is isolated fromthe offshore ooze by our lab. It can produce high level of siderophore andextracellular polysaccharide, consisting chiefly of pullulan, which can negativelyaffect siderophore production and extraction. In this study, the genetic improvementof A. pullulans HN6.2is carried out by chemical mutation and gene disruption.A. pullulans HN6.2is mutated by the ethylmethane sulfonate (EMS), which is akind of chemical mutagens. After that, the mutant strain HN18is screened by itslower-level production of extracellular polysaccharide, and a higher-level productionin siderophore biosynthesis. Then the optimal medium ingredients and growthconditions in flask and5-l fermentor for the mutant strain HN18in siderophoreproduction were studied using “one-variable-at-a-time” method. The resultsdemonstrated that the optimal medium ingredients for the siderophore production by strain HN18were sucrose2.5%(w/v), NH4NO30.5%(w/v), K2HPO40.3%(w/v), citricacid0.1%(w/v), MgSO40.008%(w/v), ZnSO40.0002%(w/v),10mM L-ornithine,initial pH5.5and the optimal growth conditions in flask were shaking speed180rpm,temperature28℃, cultivation time120h. Under this condition, the siderophore in theoptimal medium reached the highest yields0.24g/L. The optimal growth conditionsin5-l fermentor were aeration level5L/min, agitation speed200rpm, fermentationtime156h, under this condition, the siderophore reached the highest yields0.51g/L.Then, the pullulan synthetase gene (PUL1) in the siderophore-producing A.pullulans HN6.2was disrupted after the accomplishment of knockout vector’sconstruction and the disruptant DPS1produced much less polysaccharide, butproduce more siderophore than A. pullulans HN6.2. Due to the significant reductionof polysaccharide, ethanol addition for removal of pullulan could be omitted duringthe chemical extraction from the supernatant, indicating that the siderophoreextraction could be simplified, and the anticipated goal was attained.