Molecular Biology Reports

, Volume 46, Issue 2, pp 2363–2370 | Cite as

Enhancement of the enzymatic cellulose saccharification by Penicillium verruculosum multienzyme cocktails containing homologously overexpressed lytic polysaccharide monooxygenase

  • Margarita V. Semenova
  • Alexander V. GusakovEmail author
  • Pavel V. Volkov
  • Veronika Yu. Matys
  • Vitaly A. Nemashkalov
  • Vadim D. Telitsin
  • Aleksandra M. Rozhkova
  • Arkady P. Sinitsyn
Original Article


The gene lpmo1 encoding Penicillium verruculosum lytic polysaccharide monooxygenase (PvLPMO9A) was sequenced and homologously overexpressed in P. verruculosum B1-537 (ΔniaD) auxotrophic strain under the control of the cbh1 gene promoter in combination with either the cbh1 signal sequence (sCBH1-X series of samples) or the native lpmo1 signal sequence (sLPMO1-X series). Three enzyme samples of the sCBH1-X series were characterized by a lower overall content of cellobiohydrolases (CBHs: 26–45%) but slightly higher content of endoglucanases (EGs: 17–23%) relative to the reference B1-537 preparation (60% of CBHs and 14% of EGs), while the PvLPMO9A content in them made up 9–21% of the total secreted protein. The PvLPMO9A content in four enzyme preparations of the sLPMO1-X series was much higher (30–57%), however the portion of CBHs in most of them (except for sLPMO1-8) decreased even to a greater extent (to 21–42%) than in the samples of the sCBH1-X series. Two enzyme preparations (sCBH1-8 and sLPMO1-8), in which the content of cellulases was substantially retained and the portion of PvLPMO9A was 9–30%, demonstrated the increased yields of reducing sugars in 48-h saccharification of Avicel and milled aspen wood: 19–31 and 11–26%, respectively, compared to the reference cellulase cocktail.


Lytic polysaccharide monooxygenase Homologous expression Penicillium verruculosum Cellulase Saccharification 



This work was supported by the fundamental research program of the Presidium of the Russian Academy of Sciences No. 33 “Carbon energy: chemical aspects”.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Gusakov AV (2013) Cellulases and hemicellulases in the 21st century race for cellulosic ethanol. Biofuels 4:567–569CrossRefGoogle Scholar
  2. 2.
    dos Santos LV, de Barros Grassi MC, Gallardo JCM, Pirolla RAS, Calderón LL, de Carvalho-Netto OV, Parreiras LS, Camargo ELO, Drezza AL, Missawa SK, Teixeira GS, Lunardi I, Bressiani J, Pereira GAG (2016) Second-generation ethanol: the need is becoming a reality. Ind Biotechnol 12:40–57CrossRefGoogle Scholar
  3. 3.
    Payne CM, Knott BC, Mayes HB, Hansson H, Himmel ME, Sandgren M, Ståhlberg J, Beckham GT (2015) Fungal cellulases. Chem Rev 115:1308–1448CrossRefPubMedGoogle Scholar
  4. 4.
    Vaaje-Kolstad G, Westereng B, Horn SJ, Liu Z, Zhai H, Sørlie M, Eijsink VGH (2010) An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330:219–222CrossRefPubMedGoogle Scholar
  5. 5.
    Phillips CM, Beeson WT, Cate JH, Marletta MA (2011) Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem Biol 6:1399–1408CrossRefPubMedGoogle Scholar
  6. 6.
    Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VGH (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5:45CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B (2013) Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels 6:41CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Müller G, Várnai A, Johansen KS, Eijsink VGH, Horn SJ (2015) Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. Biotechnol Biofuels 8:187CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Bulakhov AG, Gusakov AV, Chekushina AV, Satrutdinov AD, Koshelev AV, Matys VY, Sinitsyn AP (2016) Isolation of homogeneous polysaccharide monooxygenases from fungal sources and investigation of their synergism with cellulases when acting on cellulose. Biochemistry 81:530–537PubMedGoogle Scholar
  10. 10.
    Hemsworth GR, Johnston EM, Davies GJ, Walton PH (2015) Lytic polysaccharide monooxygenases in biomass conversion. Trends Biotechnol 33:747–761CrossRefPubMedGoogle Scholar
  11. 11.
    Hu J, Chandra R, Arantes V, Gourlay K, van Dyk JS, Saddler J (2015) The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings. Biores Technol 186:149–153CrossRefGoogle Scholar
  12. 12.
    Cannella D, Jørgensen H (2014) Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production? Biotechnol Bioeng 111:59–68CrossRefPubMedGoogle Scholar
  13. 13.
    Morozova VV, Gusakov AV, Andrianov RM, Pravilnikov AG, Osipov DO, Sinitsyn AP (2010) Cellulases of Penicillium verruculosum. Biotechnol J 5:871–880CrossRefPubMedGoogle Scholar
  14. 14.
    Gusakov AV, Sinitsyn AP (2012) Cellulases from Penicillium species for producing fuels from biomass. Biofuels 3:463–477CrossRefGoogle Scholar
  15. 15.
    Bulakhov AG, Volkov PV, Rozhkova AM, Gusakov AV, Nemashkalov VA, Sinitsyn AP (2017) Using an inducible promoter of a gene encoding Penicillium verruculosum glucoamylase for production of enzyme preparations with enhanced cellulase performance. PLoS ONE 12:e0170404CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Dotsenko GS, Gusakov AV, Rozhkova AM, Korotkova OG, Sinitsyn AP (2015) Heterologous β-glucosidase in a fungal cellulase system: comparison of different methods for development of multienzyme cocktails. Process Biochem 50:1258–1263CrossRefGoogle Scholar
  17. 17.
    Denisenko YA, Gusakov AV, Rozhkova AM, Osipov DO, Zorov IN, Matys VY, Uporov IU, Sinitsyn AP (2017) Site-directed mutagenesis of GH10 xylanase A from Penicillium canescens for determining factors affecting the enzyme thermostability. Int J Biol Macromol 104:665–671CrossRefPubMedGoogle Scholar
  18. 18.
    Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucl Acids Res 18:6069–6074CrossRefPubMedGoogle Scholar
  19. 19.
    Sambrook J, Russell D (2001) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  20. 20.
    Sanger F, Nicklen S, Chase AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467CrossRefPubMedGoogle Scholar
  21. 21.
    Aleksenko A, Makarova N, Nikolaev I, Clutterbuck A (1995) Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate-reductase gene. Curr Genet 28:474–478CrossRefPubMedGoogle Scholar
  22. 22.
    Gusakov AV, Semenova MV, Sinitsyn AP (2010) Mass spectrometry in the study of extracellular enzymes produced by filamentous fungi. J Anal Chem 65:1446–1461CrossRefGoogle Scholar
  23. 23.
    Sinitsyna OA, Bukhtoyarov FE, Gusakov AV, Okunev ON, Bekkarevitch AO, Vinetsky YP, Sinitsyn AP (2003) Isolation and properties of major components of Penicillium canescens extracellular enzyme complex. Biochemistry 68:1200–1209PubMedGoogle Scholar
  24. 24.
    Gusakov AV, Sinitsyn AP, Salanovich TN, Bukhtojarov FE, Markov AV, Ustinov BB, van Zeijl C, Punt P, Burlingame R (2005) Purification, cloning and characterisation of two forms of thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial strain of Chrysosporium lucknowense. Enzyme Microbiol Technol 36:57–69CrossRefGoogle Scholar
  25. 25.
    Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of sugars. J Biol Chem 153:375–379Google Scholar
  26. 26.
    Peterson GL (1979) Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 100:201–220CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Markov AV, Gusakov AV, Kondratyeva EG, Okunev ON, Bekkarevich AO, Sinitsyn AP (2005) New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei. Biochemistry 70:657–663PubMedGoogle Scholar
  28. 28.
    Volkov PV, Rozhkova AM, Gusakov AV, Sinitsyn AP (2014) Homologous cloning, purification and characterization of highly active cellobiohydrolase I (Cel7A) from Penicillium canescens. Prot Expr Purif 103:1–7CrossRefGoogle Scholar
  29. 29.
    Chekushina AV, Dotsenko GS, Kondratieva EG, Sinitsyn AP (2013) Enzyme preparations from Penicillium verruculosum for bioconversion of plant raw materials is an alternative to commercial preparations obtained using Trichoderma fungi species. Biotekhnologiya 3:69–80Google Scholar
  30. 30.
    Dotsenko AS, Gusakov AV, Volkov PV, Rozhkova AM, Sinitsyn AP (2016) N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity. Biotechnol Bioeng 113:283–291CrossRefPubMedGoogle Scholar
  31. 31.
    Merzlov DA, Zorov IN, Dotsenko GS, Denisenko YA, Rozhkova AM, Satrutdinov AD, Rubtsova EA, Kondratieva EG, Sinitsyn AP (2015) Properties of enzyme preparations and homogeneous enzymes—endoglucanases EG2 Penicillium verruculosum and LAM Myceliophthora thermophila. Biochemistry 80:473–482PubMedGoogle Scholar
  32. 32.
    Volkov PV, Gusakov AV, Rubtsova EA, Rozhkova AM, Matys VY, Nemashkalov VA, Sinitsyn AP (2019) Properties of a recombinant GH49 family dextranase heterologously expressed in two recipient strains of Penicillium species. Biochimie 157:123–130CrossRefPubMedGoogle Scholar
  33. 33.
    Gouka RJ, Punt PJ, van den Hondel CA (1997) Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11CrossRefPubMedGoogle Scholar
  34. 34.
    Tanghe M, Danneels B, Camattari A, Glieder A, Vandenberghe I, Devreese B, Stals I, Desmet T (2015) Recombinant expression of Trichoderma reesei Cel61A in Pichia pastoris: optimizing yield and N-terminal processing. Mol Biotechnol 57:1010–1017CrossRefPubMedGoogle Scholar
  35. 35.
    Yang Y, Li J, Liu X, Pan X, Hou J, Ran C, Zhou Z (2017) Improving extracellular production of Serratia marcescens lytic polysaccharide monooxygenase CBP21 and Aeromonas veronii B565 chitinase Chi92 in Escherichia coli and other synergism. AMB Expr 7:170CrossRefGoogle Scholar
  36. 36.
    Low KO, Muhammad MN, Illias RM (2013) Optimization of signal peptide for recombinant protein secretion in bacterial hosts. Appl Microbiol Biotechnol 97:3811–3826CrossRefPubMedGoogle Scholar
  37. 37.
    Martin C, Volkov PV, Rozhkova AM, Puls J, Sinitsyn AP (2015) Comparative study of the enzymatic convertibility of glycerol- and dilute acid-pretreated sugarcane bagasse using Penicillium- and Trichoderma-based cellulase preparations. Ind Crops Prod 77:382–390CrossRefGoogle Scholar
  38. 38.
    Guo Z, Duquesne S, Bozonnet S, Nicaud J-M, Marty A, O’Donohue MJ (2017) Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose. Biotechnol Biofuels 10:298CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Chylenski P, Forsberg Z, Ståhlberg J, Várnai A, Lersch M, Bengtsson O, Sæbø S, Horn SJ, Eijsink VGH (2017) Development of minimal enzyme cocktails for hydrolysis of sulfite-pulped lignocellulosic biomass. J Biotechnol 246:16–23CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Margarita V. Semenova
    • 1
  • Alexander V. Gusakov
    • 1
    • 2
    Email author
  • Pavel V. Volkov
    • 1
  • Veronika Yu. Matys
    • 3
  • Vitaly A. Nemashkalov
    • 3
  • Vadim D. Telitsin
    • 2
  • Aleksandra M. Rozhkova
    • 1
    • 2
  • Arkady P. Sinitsyn
    • 1
    • 2
  1. 1.Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of SciencesMoscowRussia
  2. 2.Department of ChemistryM. V. Lomonosov Moscow State UniversityMoscowRussia
  3. 3.G. K. Skryabin Institute of Biochemistry and Physiology of MicroorganismsRussian Academy of SciencesPushchinoRussia

Personalised recommendations