| Citric acid is an important organic acid, which was widely used in food, medicine, cosmetics and chemical industries. Aspergillus niger is a major filamentous fungi for citric acid fermentation. The mycelial morphology is important for the synthesis of citric acid during submerged fermentation. We can improve the morphology of mycelia to increase the production of citric acid by regulating the expression of morphological genes. This paper mainly studies the impact of chitin synthase gene chsC on the mycelial morphology and the production of citric acid.In this paper, we choose pJL43-RNAi as the vector for gene silencing. The fragments of chsC gene were amplified by PCR, which was ligated to plasmid pJL43-RNAi to generate the pchsC-RNAi vector. In order to increase the transformation frequency, the conditions of the preparation, regeneration and transformation of protoplasts were optimized. The results showed that the mycelia incubated for 16 hours at 36.5℃were most suitable for protoplasts release, which digested by enzyme combination of 5 mg/mL lysozyme,10 mg/mL snailase and 10 mg/mL cellulase for 3 hours as 100 r/min at 30℃, and a high yield of protoplasts (5.11×06/mL) were obtained. In addition, the maximum regeneration rate was 36.97%, while the complete medium as the regeneration media.The pchsC-RNAi vector was mediated by polyethylene glycol into the protoplasts. After transformation, seven morphological mutants were obtained. The results of RT-PCR and qRT-PCR analysis showed that all of the transformants showed different degree of reduction in the chsC gene mRNA steady-state levels in comparison with the original strain. The chitin content of cell wall was positively related with the chsC mRNA transcript levels.All transformants exhibited the improvement of citric acid production, especially chsC-2 and chsC-5 whose yields were 42.57% and 36.73% higher than that in the original strain. Although the interference effect is remarkable, the citric acid yields of chsC-1 rose just 0.37%. The results suggest that maintaining the expression of chsC gene at a certain level plays an important role in the accumulation of citric acid. The mycelial morphology of all transformants and the original strain in submerged fermentation were investigated by light microscopy. The branching frequency and length of the hypha both had a clear reduction in the morphological mutants, whose dispersed mycelia were less than the origin strain.The decrease of viscosity that improves the transfer efficiency of oxygen and mass, which is conducive to the accumulation of citric acid.The optimum culture conditions of chsC-3 were investigated by single factor experiments. The results of optimization showed that the optimum medium volune was 60 mL/500mL, then inoculated with 5×104 spores/mL and fermented as 300 r/min at 37 ℃. The results showed that, after 72 hours fermentation, the citric acid production of chsC-3 increased by 7.32% and 21.74%, respectively. Moreover, after 108 hours fermentation, the citric acid yields of chsC-3 had been improved 5.35% than CK, which no longer increased. The high-yield strain of chsC-3, which the branching frequency and length of the hypha both had a clear reduction in the process of fermentation tank culture compared with CK. Moreover, the citric acid yields of chsC-3 had been improved 3.21% than CK. In addition, in submerged citric acid fermentation, the conversion rate of glucose was increased by 3.20% comparing to the original strain, as well as the fermentation period was shorten from 64 hours to 63 hours. The high-yield strain of chsC-3 exhibited the excellent production potential in the scale-up process. |
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