| Cellulase has been widely applied in food,medicine,textile,brewing and other industries.And it has great market potential.With the increasing demand for cellulase,problems such as the long production cycle,low activity and high production costs need to be solved immediately.With the rapid development of molecular biology techniques,many gene manipulation tools have been successfully applied to the fungal system.And the genetic engineering efficiency of Trichoderma reesei has been greatly improved.However,cellulase is a complex enzyme system.The increase of cellulase activity is closely related to the expression of a series of genes.Some achievements have been made in the genetic engineering of those strains,but further research is needed to increase cellulase yield.Therefore,it is necessary to screen cellulase hyperproducing mutants.In our study,atmospheric pressure room temperature plasma mutagenesis system was applied to obtain cellulase hyperproducing mutants with excellent traits,taking Trichoderma reesei RUT-C30 as parent strain.Whole genome sequencing was performed to analyze the mutant types of the hyperproducing strain Trichoderma reesei JNDY-13.Then,the fermentation optimization of JNDY-13 was carried out to further increase the yield of cellulase.Our main work are as follows:1.The basic screening method was established with the atmospheric pressure room temperature plasma mutagenesis system and Congo red swelling medium.And then,the screening medium was optimized.We found that the addition of 0.3%?w/w?lactose to the primary screening medium can significantly shorten the spore germination time,and adding0.1%?w/w?lithium chloride can significantly increase the positive mutation rate.After mutagenesis through the atmospheric pressure room temperature plasma mutagenesis system,the hyperproducing mutant Trichoderma reesei JNDY-13 with high filter paper activity of2.21 IU·m L-1 was selected,which was 2.21 times that of the parent strain Trichoderma reesei RUT-C30.2.Whole genome sequencing of cellulase hyperproducing strain JNDY-13 was performed.And mutation analysis showed that 752 mutations were identified in JNDY-13,of which 18 were hydrolases,34 were synthetases,20 were kinases,52 were related to energy metabolism,73 were related to transportation,38 were related to transcription and translation,8 were related to growth,399 were hypothetical proteins and 110 were related to other functions.In addition,105 of the 752 SNPs were identified as point mutations,336 mutations were deletion,165 insertions and 99 mutations were mixed mutations.3.Analysis of galactokinase gene mutation revealed that 18 bases?non-CDS regions?were inserted at 454 bp of the galactokinase gene.Galactokinase activity assay and real time PCR showed that galactokinase activity of JNDY-13 decreased when compared with Trichoderma reesei RUT-C30,but the transcriptional level of galactokinase gene was significantly higher than that of RUT-C30.Since the transcriptional level of galactokinase gene is closely related with secretion of cellulase,mutation in galactokinase gene may attribute to increased cellulase production.In addition,RT-PCR was applied to analyze the transcriptional levels of cellulase genes,hemicellulase genes and regulatory factors genes in the cellulase hyperproducing mutant JNDY-13.It was found that in the early log phase,6 of11 cellulase and hemicellulase related genes were upregulated compared with the parent strain.In the mid-logarithmic phase,the transcription of 8 genes was up-regulated.However,in the stationary phase,the transcription of cellulase-related genes was significantly downregulated.4.According to the characteristics of JNDY-13,an orthogonal experiment was designed to optimize the fermentation medium.The optimal fermentation medium consists of 10 g·L-1lactose,10 g·L-1 cellulose,corn steep liquor 12 g·L-1,(NH42)SO4 1.5 g·L-1,MgSO4 1.2 g·L-1,CaCl2 1.4 g·L-1,1 mL·L-1 Mandels trace element nutrient,2 m L·L-1 Tween 80,pH 4.8.And orthogonal experiments were designed to optimize the fermentation conditions.The optimal fermentation conditions obtained were:28?,stirring speed 400 r·min-1,aeration volume 2vvm,pH 5.0.Under the optimal fermentation conditions,the maximum enzyme activity reached 4.74 IU·m L-1 for batch fermentation,and the highest dry weight reached 8.91 g·L-1.5.During the fed-batch fermentation,the ingredients of the fed concentrate were optimized.The optimal feed components were lactose and ammonium sulfate?carbon to nitrogen ratio 10:1?.The highest total enzyme activity reached 5.40 IU·m L-1,and the highest dry weight reached 9.19 g·L-1 with fed-batch fermentation. |
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