| As an advanced and efficient mutation technique, heavy ion radiation has been widely used in industrial microorganism breedings due to its specific characteristics, which include high linear energy transfer(LET), high relative biological effectiveness(RBE) and high mutation rate, etc. In this study, citric acid producing strain(Aspergillus niger) was irradiated by medium-energy carbon ion beams. One hand, improved mutants with high capacity of citric acid production were obtained by using high-throughput screening method, which can satisfy the needs of high-yield strains in citric acid commercial production. On the other hand, the genetic study of the mutational key genes involving in the pathway of starch bio-degradation in A. niger were investigated.The physiological characteristic alterations of A.niger induced by carbon ion beams were investigated by flow cytometry, scanning electron microscopy, MTT and solid plate assays. The citric acid accumulations of mutants and original strain were analyzed by using submerged fermentation technique. Genomic resequencing, RTPCR and enzyme activity detection assays were used to illustrate how carbon ion beam radiations affected the citric acid metabolic pathways, mainly focusing on the expression of the key genes involving in starch bio-degradation. The main results were as follows:1. The carbon ion radiations induced physiological alterations of the conidiums in A.niger. Compared with the original strain, HW2 mutant showed smaller conidium diameter and H4 mutant showed larger conidium diameter. The scanning electron microscopy results demonstrated that the proportion of conidiums with wrinkled surface in HW2 increased compared with the original strain,while H4 displayed no significant difference. According to MTT assays, the conidium vitality of H4 mutant was higher than that of original strain, whereas HW2 did not show significant difference. It can be concluded that the metabolic activity of H4 was higher than that of the original strain, and HW2 did not show obvious difference. Depending on the results of growth rate, it was found that, when using glucose, starch, or xylose as carbon source in the metabolism process, the colony diameter of HW2 became smaller than the original strain, whereas the colony diameter of H4 was larger using starch or xylose as carbon source. These results showed that there were obvious differences of the growth rates between the mutants and original strain, which means the growth rate of the H4 was faster, and the HW2 was slower.2. Carbon ion radiations induced alterations of the capacities of citric acid accumulation in A.niger. After fermentation using different carbon or nitrogen sources, it was found that HW2, H4, and H1 mutants showed obvious differences in citric acid accumulation compared with original strain. The capacity of citric acid accumulation in HW2 was enhanced, while the capacities of both H4 and H1 were weaken. Meanwhile, HW2, H4 and H1 mutants showed different colony morphologies, respectively. According to the results of 10 L fermentation experiment, HW2 showed more excellent capacity of citric acid accumulation, and lower consumption of sugar compared with original strain. It was concluded that HW2 mutant was a strain with commercial value in the industrial production of citric acid.3. Genomic comparison between H4 mutant and original strain demonstrated that carbon ion radiations induced genes mutations involving in the pathway of starch bio-degradation. In the early stage of the fermentation(24 h), RT-PCR results proved that the expression of glucoamylase gene in HW2 mutant was increased, and no difference of the expression of starch degradation regulation gene was observed. The expressions of glucoamylase gene and starch degradation regulation gene in H4 mutant decreased. However, in the later stage of fermentation(48 h), compared with original strain, the expressions of glucoamylase gene and starch degradation regulation gene in both HW2 and H4 mutants had no significant differences. The above results were confirmed via the detection assay of glucoamylase activity between mutants and original strian. Thus, it was concluded that the impact of starch bio-degradation pathway on citric acid accumulation in A.niger mainly occurred in early stage. Meanwhile, HW2 mutant displayed enhanced ability of starch degradation, while the capacity of H4 was weaken. To sum up, heavy ion radiations can induce the different expressions of key genes involving in the pathway starch degradation, which could affect the accumulation of citric acid in A.niger.In conclusion, heavy ion radiation mutation technique has been applied in screening excellent A.niger mutants for citric acid production successfully. It was the first research which reported that the physiological characteristics of A.niger can be affected by carbon ion radiations. An improved mutant with high capacity of citric acid production, named HW2, was obtained. It was also proved that the expressions of key genes involving in the pathway of starch bio-degradation had a remarkable effect on the citric acid accumulation in A.niger. |
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