The Research Of Regulators ACE2 And ACE3 On The Cellulase Expression In Trichoderma Orientalis EU7-22

Lignocellulose is the most abundant renewable resource in the world.The cellulase system produced by microbial fermentation can degrade it into glucose,which can be further converted into ethanol,butanol and other liquid fuels by microorganisms.Filamentous fungi are the most important source of commercial cellulase.However,the high cost of cellulase restricts the biotransformation of lignocellulose.For this reason,reducing the production cost of cellulase has become a practical need for cellulase research.However,filamentous fungi which produce cellulase with a complex regulatory network.The prerequisite for genetic engineering is to study the mechanism of cellulase production.Trichoderma orientalis EU7-22 is a filamentous fungus of the Trichoderma genus isolated,screened and mutagenized in our laboratory.The cellulase system and hemicellulase system of Trichoderma orientalis EU7-22 are complete,and the growth rate of Trichoderma orientalis EU7-22 is fast.It has the potential to producing cellulase in industry.In this thesis,by studying the effects of transcriptional regulators ACE2 and ACE3 on the cellulase production of Trichoderma orientalis EU7-22,it provides a certain basis for strain transformation.The main results were achieved as follows:(1)ace2 gene was cloned from QM9414 from Trichoderma reesei.An ace2 expression cassette with hygromycin B resistance marker was constructed.And ace2 gene was expressed heterologously in Trichoderma orientalis EU7-22.Eight transformations were obtained by transformation,screening and culture.The filter paper activity of the strains and the transformed strains was lower than that of the original strains.Three strains with more average performance were selected for further analysis.Cultivated under the conditions of combined induction of microcrystalline cellulose and wheat bran,the filter paper activity,endocellulose activity and total protein concentration of the transformed strains were lower than those of the original bacteria,but β-glucosidase activity and xylanase activity have been improved to a certain extent.When growing on a medium with glucose,xylose,xylan,cellobiose,lactose,glycerol,CMC-Na and microcrystalline cellulose as the only carbon source,the colony of transformed strains ace2eh1-1 and EU7-22 have been measured and observed.The spore morphology is basically the same.In addition,by fluorescence quantitative PCR analysis of the transcription amounts of related genes,the bgl1 transcription amount of the heterologous ace2 strain increased,while the transcription regulator genes ace3 and xyr1 decreased.(2)By constructing geneticin G418 and hygB resistance marker ace3 expression cassettes,overexpressing ace3 in Trichoderma orientalis,transforming and screening 6 transformed strains.After the initial screening,the enzyme activity of ace3eG-5 was higher than that of the original bacteria.Under the co-induction of microcrystals and bran,the highest activity of filter paper activity,exo-cellulase activity andβ-glucosidase activity of this strain was relatively high.The highest enzyme activity of the original bacteria has been increased by 98.2%,341%,and 48.5%respectively.With glucose as the only carbon source,the growth rate of ace3eG-5 was significantly slower than that of the original bacteria.Hydrolyzing the pretreated bamboo pulp with the same volume of crude enzyme solution on the fifth day of the enzyme production medium,the reducing sugar release of ace3eG-5 was 171%of EU7-22.Through RT-PCR analysis,overexpression of ace3 also increased the transcription levels of the main cellulase gene and transcriptional regulator gene xyr1.(3)ace3 was overexpressed on the basis of ace2eh1-1 transformed strain,and study the synergistic effect of ace2 and ace3 on cellulase production by Trichoderma orientalis.After screening,six positive transformants of recombinant strain ace23-6 were obtained with the highest enzyme activity.The filter paper activity still increased after the fourth day,and the filter paper activity of the enzyme increased by 11.1%.Through RT-PCR analysis,compared with the original strain EU7-22,the transcription levels of the main cellulase genes have increased,and the transcription levels of crel and ace1 have decreased.Compared with ace2eh1-1,overexpression of ace3 can generally increase the transcription level of cellulase genes and transcription regulator genes,and overexpression of ace3 can also regulate ace2,which increases its transcription level.In summary,this article studied the effects of regulatory factors ACE2 and ACE3 on the cellulase and hemicellulase production of Trichoderma orientalis EU7-22,and analyzed the cellulase and hemicellulase production by expressing ACE2 and overexpressing ACE3 at the same time.A recombinant strain with high cellulase and hemicellulase production was obtained,which laid the foundation for the construction of an industrialized cellulase-producing strain of Trichoderma orientalis.