Skip to main content
SpringerLink
Log in

Improving cellulases production by Myceliophthora thermophila through disruption of protease genes

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Objective

To identify main protease genes for the proteolytic degradation of cellulases in M. thermophila and generate a lower-proteases fungal host that can be used for further metabolic engineering to increase cellulase production and heterologous protein expression.

Results

Systematic transcriptomic analysis were conducted on the expression of proteases genes in M. thermophila genome and five highly expressed genes encoding extracellular proteases were selected for mutation analyses. A series of single- and multi-gene mutants of these five selected genes was constructed using the CRISPR-Cas9 technique. Compared with WT, the ΔMtalp1 and the quintuple mutant showed significantly lower protease activity (decreased 52.7% and 58.4%, respectively) and at least double enhanced cellulase production.

Conclusions

The results indicated that Mtalp1 is a critical protease gene in cellulase degradation in M. thermophila and disruption of protease genes showed significantly decreased protease activity and obviously enhanced cellulase production in the fermentation broth of ΔMtalp1 and the quintuple mutant.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abad S, Kitz K, Hormann A, Schreiner U, Hartner FS, Glieder A (2010) Real-time PCR-based determination of gene copy numbers in Pichia pastoris. Biotechnol J 5:413–420

    Article  CAS  Google Scholar 

  • Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LDH, Baker SE, Magnuson J, LaBoissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–929

    Article  CAS  Google Scholar 

  • Coffman JA, Cooper TG (1997) Nitrogen GATA factors participate in transcriptional regulation of vacuolar protease genes in Saccharomyces cerevisiae. J Bacteriol Parasitol 179:5609–5613

    Article  CAS  Google Scholar 

  • Coradetti ST, Craig JP, Xiong Y, Shock T, Tian C, Glass NL (2012) Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi. Proc Natl Acad Sci 109:7397–7402

    Article  CAS  Google Scholar 

  • Delgado-Jarana J, Pintor-Toro JA, Benitez T (2000) Overproduction of beta-1,6-glucanase in Trichoderma harzianum is controlled by extracellular acidic proteases and pH. Biochim Biophys Acta 1481:289–296

    Article  CAS  Google Scholar 

  • Dogaris I, Mamma D, Kekos D (2013) Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations. Appl Microbiol Biotechnol 97:1457–1473

    Article  CAS  Google Scholar 

  • Dos Santos HB, Bezerra TMS, Pradella JGC, Delabona P, Lima D, Gomes E, Hartson SD, Rogers J, Couger B, Prade R (2016) Myceliophthora thermophila M77 utilizes hydrolytic and oxidative mechanisms to deconstruct biomass. AMB Express 6:1–12

    Article  Google Scholar 

  • Eneyskaya EV, Kulminskaya AA, Savel'ev AN, Savel'eva NV, Shabalin KA, Neustroev KN (1999) Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases. Appl Microbiol Biotechnol 52:226–231

    Article  CAS  Google Scholar 

  • Hervas-Aguilar A, Rodriguez JM, Tilburn J, Arst HN Jr, Penalva MA (2007) Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC. J Biol Chem 282:34735–34747

    Article  CAS  Google Scholar 

  • Hubbard JP, Harman G, Eckenrode CJ (1982) Interaction of a biological control agent, Chaetomium globosum, with seed coat microflora. Can J Microbiol 28:431–437

    Article  Google Scholar 

  • Jin FJ, Watanabe T, Juvvadi PR, Maruyama J, Arioka M, Kitamoto K (2007) Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae. Appl Microbiol Biotechnol 76:1059–1068

    Article  CAS  Google Scholar 

  • Katz ME, Rice RN, Cheetham BF (1994) Isolation and characterization of an Aspergillus nidulans gene encoding an alkaline protease. Gene 150:287–292

    Article  CAS  Google Scholar 

  • Kerstens W, Van Dijck P (2018) A Cinderella story: how the vacuolar proteases Pep4 and Prb1 do more than cleaning up the cell's mass degradation processes. Microb Cell 5:438–443

    Article  CAS  Google Scholar 

  • Kimura S, Maruyama J, Takeuchi M, Kitamoto K (2008) Monitoring global gene expression of proteases and improvement of human lysozyme production in the nptB gene disruptant of Aspergillus oryzae. Biosci Biotechnol Biochem 72:499–505

    Article  CAS  Google Scholar 

  • Kolbusz MA, Di Falco M, Ishmael N, Marqueteau S, Moisan MC, Baptista CDS, Powlowski J, Tsang A (2014) Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila. Fungal Genet Biol 72:10–20

    Article  CAS  Google Scholar 

  • Laskar A, Rodger EJ, Chatterjee A, Mandal C (2011) Modeling and structural analysis of evolutionarily diverse S8 family serine proteases. Bioinformation 7:239–245

    Article  Google Scholar 

  • Liu Q, Gao R, Li J, Lin L, Zhao J, Sun W, Tian C (2017) Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol Biofuels 10:1–14

    Article  Google Scholar 

  • Mandujano-Gonzalez V, Villa-Tanaca L, Anducho-Reyes MA, Mercado-Flores Y (2016) Secreted fungal aspartic proteases: a review. Rev Iberoam Micol 33:76–82

    Article  Google Scholar 

  • Marques M, Mojzita D, Amorim MA, Almeida T, Hohmann S, Moradas-Ferreira P, Costa V (2006) The Pep4p vacuolar proteinase contributes to the turnover of oxidized proteins but PEP4 overexpression is not sufficient to increase chronological lifespan in Saccharomyces cerevisiae. Microbiology 152:3595–3605

    Article  CAS  Google Scholar 

  • Nemoto T, Watanabe T, Mizogami Y, Maruyama J, Kitamoto K (2009) Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant. Appl Microbiol Biotechnol 82:1105–1114

    Article  CAS  Google Scholar 

  • Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20

    Article  CAS  Google Scholar 

  • Ravagnani A, Gorfinkiel L, Langdon T, Diallinas G, Adjadj E, Demais S, Gorton D, Arst HN Jr, Scazzocchio C (1997) Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA. EMBO J 16:3974–3986

    Article  CAS  Google Scholar 

  • Rawlings ND, Barrett AJ (1994) Families of serine peptidases. Methods Enzymol 244:19–61

    Article  CAS  Google Scholar 

  • Riley PA (1994) Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int J Radiat Biol 65:27–33

    Article  CAS  Google Scholar 

  • Singh B (2014) Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential. Crit Rev Biotechnol 36:59–69

    Article  Google Scholar 

  • Smit S, Derks MF, Bervoets S, Fahal A, van Leeuwen W, van Belkum A, van de Sande WW (2016) Genome sequence of Madurella mycetomatis mm55, isolated from a human mycetoma case in Sudan. Genome Announc 4:1–2

    Article  Google Scholar 

  • Song L, Chen Y, Du Y, Wang X, Guo X, Dong J, Xiao D (2017) Saccharomyces cerevisiae proteinase A excretion and wine making. World J Microbiol Biotechnol 33:1–12

    Article  CAS  Google Scholar 

  • Tilburn J, Sarkar S, Widdick DA, Espeso EA, Orejas M, Mungroo J, Penalva MA, Arst HN Jr (1995) The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid- and alkaline-expressed genes by ambient pH. EMBO J 14:779–790

    Article  CAS  Google Scholar 

  • vanKuyk PA, Cheetham BF, Katz ME (2000) Analysis of two Aspergillus nidulans genes encoding extracellular proteases. Fungal Genet Biol 29:201–210

    Article  CAS  Google Scholar 

  • VasquezLaslop N, Tenney K, Bowman BJ (1996) Characterization of a vacuolar protease in Neurospora crassa and the use of gene RIPing to generate protease-deficient strains. J Biol Chem 271:21944–21949

    Article  Google Scholar 

  • Visser H, Joosten V, Punt PJ, Gusakov AV, Olson PT, Joosten R, Bartels J, Visser J, Sinitsyn AP, Emalfarb MA, Verdoes JC, Wery J (2011) Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1. Ind Biotechnol 7:214–223

    Article  CAS  Google Scholar 

  • Yoon J, Kimura S, Maruyama J, Kitamoto K (2009) Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae. Appl Microbiol Biotechnol 82:691–701

    Article  CAS  Google Scholar 

  • Yoon J, Maruyama J, Kitamoto K (2011) Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 89:747–759

    Article  CAS  Google Scholar 

  • Zhu L, Maruyama J, Kitamoto K (2013) Further enhanced production of heterologous proteins by double-gene disruption (∆AosedD ∆Aovps10) in a hyper-producing mutant of Aspergillus oryzae. Appl Microbiol Biotechnol 97:6347–6357

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the key project of Ministry of Science and Technology of China (Grant No. 2018YFA0900500), National Natural Science Foundation of China (Grant Nos. 31670042 and 31771386) and Youth Innovation Promotion Association of Chinese Academy of Sciences (Grant No. 2019180).

Supporting information

Supplementary Figure S1—Distribution and classification of peptidases family in M. thermophila. A, aspartic peptidases; C, cysteine peptidases; G, glutamic peptidases; M, metallopeptidases; S, serine peptidases; T, threonine peptidases

Supplementary Figure S2—Transcriptome (a) and secretome (c) of 28 extracellular proteases in M. thermophila when grown on glucose, monocot (barley, oat, triticale) and dicot (alfalfa, canola, flax) straw. b Overlap of proteases in expression and secretion profiles in M. thermophila when grown on glucose, monocot (barley, oat, triticale) and dicot (alfalfa, canola, flax) straw. Levels of expression are described by FPKM (Fragment Per Kilobase of transcript for Milion mapped reads) while secrete protein quantitation analyses were performed using the Scaffold reported value for the averaged Total Ion Chromatogram (TIC) value of the top three peptides assigned to each protein. SP represent proteases with secretory signal peptide predicted. The specific data of related proteases can be seen in Supplemental Table S2

Supplementary Figure S3—PCR analysis of proteases genes deletion in selected transformants ΔMtalp1 (a), ΔMtpep4 (b), ΔMtgp1 (c), ΔMtcap1 (d) and ΔMtga1 (e) with one primer (protease-KO-SF) located upstream of the 5′ flanking region of the genomic DNA and the other (protease-KO-SR) located in the 3′ flanking region of the genomic DNA. The expected length of disrupted transformants was much larger than that of the host strain, WT, which used as a negative control. (f) The copy number determination of the integrated PtrpC-neo marker gene in these transformants by RT-qPCR. The actin gene was used as an internal control

Supplementary Figure S4—a Growth phenotype of mutants and WT on different medium. GM, Vogel’s minimal medium (MM) supplemented with 2% glucose; 0.4% Casein, GMN- supplemented with 0.4% casein; 0.4% YNB, GMN- supplemented with 0.4% YNB; 0.5M NaCl, GM supplemented with 0.5M NaCl; 1mM H2O2, GM supplemented with 1mM H2O2. There was no obvious growth when cultivated on 1M/ 2 M NaCl nor 2.5 mM H2O2 medium. b Colony diameter of these mutants and WT. Data are average of three replicates with standard error

Supplementary Figure S5—Sodium dodecylsulfate-polyacrylamide gel electrophoresis of secreted protein in 4-day Vogel’s MM with 2% Avicel and 0.75% yeast extract cultures

Supplementary Figure S6—Relative dried mycelia weight of the mutants versus WT grown on Vogel’s MM with 2% Avicel and 0.75% yeast extract cultures

Supplementary Table S1—Predicted peptidases annotation in M. thermophila

Supplementary Table S2—Transcriptome and secretome of 28 extracellular proteases in M. thermophila when grown on glucose, monocot (barley, oat, triticale) and dicot (alfalfa, canola, flax) straw as carbon sources

Supplementary Table S3—Primers used in this study

Author information

Authors and Affiliations

  1. State Key Laboratory of Agrobiotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China

    Xiaolin Li & Qun He

  2. Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Xiaolin Li, Qian Liu, Wenliang Sun & Chaoguang Tian

Authors
  1. Xiaolin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qian Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wenliang Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qun He
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chaoguang Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Qun He or Chaoguang Tian.

Ethics declarations

Conflict of interest

A patent involved in improving cellulases production through disruption of protease genes in filamentous fungus was submitted by the institute of authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, X., Liu, Q., Sun, W. et al. Improving cellulases production by Myceliophthora thermophila through disruption of protease genes. Biotechnol Lett 42, 219–229 (2020). https://doi.org/10.1007/s10529-019-02777-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-019-02777-0

Keywords

Search

Navigation