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CN-121975777-A - UORF for regulating and controlling fungal cellulase gene eg1 expression and application thereof

CN121975777ACN 121975777 ACN121975777 ACN 121975777ACN-121975777-A

Abstract

The invention discloses an application of a 5 '-untranslated region (5' -UTR) of an endo-beta-1, 4-glucanase gene eg1 or an upstream open reading frame (uORF) or a mutant of the upstream open reading frame (uORF) thereof in regulating and controlling fungal cellulase gene eg1 expression or regulating and controlling cellulase yield. Studies show that uORF1, uORF3 and uORF7 have core regulation and control effects, the uORF1 and the uORF3 have negative regulation and control effects in the biosynthesis process of penicillium oxalicum endo-beta-1, 4-glucanase, and the initial codon mutation can remarkably improve the cellulase yield, and the uORF7 has positive regulation and control effects. Therefore, the uORF and the mutant thereof have great application potential in precisely regulating and controlling the generation of plant polysaccharide degrading enzyme or the expression of related genes, can be used for constructing genetic engineering strains of high-yield cellulase, and have important theoretical guidance and practical values for improving the yield of industrial enzyme preparations through a molecular breeding means by identifying and controlling the regulatory element of the uORF of the penicillium oxalicum endo-beta-1, 4-glucanase gene.

Inventors

  • ZHAO SHUAI
  • FENG JIAXUN
  • SHEN XIN
  • ZHOU TINGYU

Assignees

  • 广西大学

Dates

Publication Date
20260505
Application Date
20251222

Claims (10)

  1. 1. Use of the 5' untranslated region of an endo-beta-1, 4-glucanase gene eg1 or an upstream open reading frame or a mutant of an upstream open reading frame thereof for regulating fungal cellulase gene eg1 expression or for regulating cellulase yield.
  2. 2. The use according to claim 1, wherein the gene eg1 or cellulase is derived from Penicillium oxalicum.
  3. 3. The method according to claim 1, wherein the cellulase is an endo-beta-1, 4-glucanase.
  4. 4. The method according to claim 1, wherein the cellulase yield is characterized by carboxymethyl cellulase and/or filter paper enzyme yield.
  5. 5. The method of claim 4, wherein the control of cellulase production is an increase or decrease in the production of carboxymethyl cellulase and/or filter paper enzyme under conditions induced by crystalline cellulose.
  6. 6. The uORF for regulating and controlling the expression of the fungal cellulase gene eg1 is characterized by comprising a base sequence of a sequence table SEQ ID NO.33, SEQ ID NO.35 and SEQ ID NO.39 or a derivative sequence of the base sequence by substitution, deletion or addition of one or more bases.
  7. 7. The uORF as claimed in claim 6, wherein the derivative sequence is obtained by mutating an ATG from an initiation codon in the base sequence of the uORF to AAG.
  8. 8. An expression cassette or recombinant microorganism having a uORF regulating expression of a fungal cellulase gene eg1 as claimed in claim 6 or 7.
  9. 9. The recombinant microorganism according to claim 8, wherein the recombinant microorganism is prepared from Penicillium oxalicum strain Del PoxKu70 as a starting strain.
  10. 10. The recombinant microorganism according to claim 9, wherein the strain Delta PoxKu.oxalicum is obtained by knocking out the gene PoxKu from the wild-type strain HP7-1 of Penicillium oxalicum.

Description

UORF for regulating and controlling fungal cellulase gene eg1 expression and application thereof Technical Field The invention belongs to the technical field of microbial genetic engineering and enzyme molecular regulation, and particularly relates to a uORF for regulating and controlling fungal cellulase gene eg1 expression and application thereof. Background Lignocellulose mainly comprises agricultural waste (such as rice straw, wheat straw, corn straw, sugarcane straw and the like), forestry or wood waste, cellulose waste (such as paper) and the like. The lignocellulose has the characteristics of rich resources, low price, sufficient supply and the like, and has wide application prospect. In the biological production process of lignocellulose, the degradation of cellulose and hemicellulose in lignocellulose into monosaccharide for fermentation by using cellulase and hemicellulase is a key step of biological production. Filamentous fungi are capable of secreting a large number of high-activity plant polysaccharide degrading enzymes, which are currently commonly used in the industrial production of cellulases. Therefore, research on the regulation mechanism of the synthesis of the cellulase of the filamentous fungi has important significance for molecular breeding of the fungi. The filamentous fungus Penicillium oxalicum (Penicillium oxalicum) is capable of secreting a complete cellulase system, wherein endo-beta-1, 4-glucanase EG is a key member, and cellooligosaccharides are produced by randomly cleaving beta-1, 4-glycosidic bonds within the cellulose chain. At present, the research on the expression regulation of cellulase genes is focused on transcription factors and promoter regions, but the research on the function of an upstream open reading frame (uORF) located in a 5' -untranslated region of mRNA in filamentous fungi has not been reported. Disclosure of Invention The invention aims to provide a uORF for regulating and controlling fungal cellulase gene eg1 expression and application thereof. In order to solve the technical problems, the invention adopts the following technical scheme: Use of the 5 'untranslated region (5' -UTR) of an endo- β -1, 4-glucanase gene eg1 or an upstream open reading frame (uORF) or a mutant of an upstream open reading frame (uORF) thereof for regulating fungal cellulase gene eg1 expression or for regulating cellulase yield. The gene eg1 or cellulase is derived from Penicillium oxalicum. The cellulase is endo-beta-1, 4-glucanase. Cellulase yields were characterized by carboxymethyl cellulase (CMC) and/or filter paper enzyme (FPase) yields. Regulating cellulase production is to increase or decrease the production of carboxymethyl cellulase and/or filter paper enzyme under crystalline cellulose induction conditions. The uORF for regulating and controlling the expression of the fungal cellulase gene eg1 has the base sequences of SEQ ID NO.33, SEQ ID NO.35 and SEQ ID NO.39 of a sequence table or derivative sequences of the base sequences through substitution, deletion or addition of one or more bases. The derivative sequence is obtained by mutating the ATG from the initiation codon in the base sequence of uORF to AAG. An expression cassette or recombinant microorganism having the above-described uORF that regulates expression of the fungal cellulase gene eg 1. The recombinant microorganism is prepared by taking penicillium oxalicum strain delta PoxKu as a starting strain. Penicillium oxalicum strain Delta PoxKu70 is obtained by knocking out gene PoxKu from wild type strain HP7-1 of penicillium oxalicum. Aiming at the problems of low yield of the filamentous fungal cellulase, insufficient utilization of a regulation mechanism and the like, the inventor identifies and discovers that functional uORF exists in the 5' -UTR of the penicillium oxalicum key endoglucanase gene eg1 through the analysis of a uORF-GFP report system, the construction of deletion mutant strains and overexpression strains, the measurement of cellulase yield and the like, and accordingly proposes the application of a mutant of a 5' -untranslated region (5 ' -UTR) of the endo-beta-1, 4-glucanase gene eg1 or an upstream open reading frame (uORF) thereof in regulating the expression of the fungal cellulase gene eg1 or regulating the cellulase yield. Studies show that uORF1, uORF3 and uORF7 have core regulation and control effects, the uORF1 and the uORF3 have negative regulation and control effects in the biosynthesis process of penicillium oxalicum endo-beta-1, 4-glucanase, and the initial codon mutation can remarkably improve the cellulase yield, and the uORF7 has positive regulation and control effects. Therefore, the uORF and the mutant thereof have great application potential in precisely regulating and controlling the generation of plant polysaccharide degrading enzyme or the expression of related genes, can be used for constructing genetic engineering strains of high-yield cellulase, and have important t