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Strain improvement of Aspergillus sojae for increased l-leucine aminopeptidase and protease production

  • Jaeho Lim
  • Yong-Ho Choi
  • Byung-Serk Hurh
  • Inhyung Lee
Article
  • 29 Downloads

Abstract

Conventional random mutagenesis was implemented to improve l-leucine aminopeptidase (LAP) and protease production in Aspergillus sojae. Through successive mutagenesis by ethyl methanesulfonate (EMS), UV, and 1-methyl-2-nitro-1-nitrosoguanidine (NTG), EMS25, EU36, and EUN13 mutants from each mutagenesis process were screened using a newly developed quick and easy screening method. The mutant EUN13 exhibited a 9.6-fold increase in LAP [50.61 ± 4.36 U/g-initial dried substrate (IDS)] and a 3.8-fold increase in protease production (13.36 ± 0.31 U/g-IDS) on solid-state fermentation. This mutant showed more frequent branching and higher lap1 mRNA expression as compared to the parent strain SMF 131, which at least in part contributed to the increased LAP and protease production. The mutant EUN13 can be used as a starter organism for diverse industrial soybean fermentation processes for the production of conventional products such as meju, doenjang, and ganjang as well as for the production of new fermented soybean-based sauces.

Keywords

l-leucine aminopeptidase (LAP) Protease Aspergillus sojae Random mutagenesis Soybean fermentation 

Notes

Acknowledgements

This study was supported by the World-Class 300 project, and by the Partnership programs of Strengthening technological competency for Small and Medium Enterprise (S2483241) funded by the Ministry of SMEs and Startups (MSS, Korea).

References

  1. Abdullah R, Ikram-Ul-Haq I, Iftikhar T, Butt Z, Khattak M. Random mutagenesis for enhanced production of alpha-amylase by Aspergillus oryzae IIB-30. Pak. J. Bot. 45: 269–274 (2013)Google Scholar
  2. Adrio LA, Demain AL. Genetic improvement of processes yielding microbial products. FEMS Microbiol. Rev. 30: 187–214 (2006)CrossRefGoogle Scholar
  3. Al-Jailawi MH, Al-Shekdhaher A, Al-Zaiadi R. Genetic improvement of Saccharomyces boulardii R7 and generate suitable strains for synthesis and expression of recombinant products. Brit. Biotechnol. J. 11: 1–9 (2016)CrossRefGoogle Scholar
  4. Chand P, Aruna A, Maqsood A, Rao L. Novel mutation method for increased cellulase production. J. Appl. Microbiol. 98: 318–323 (2005)CrossRefGoogle Scholar
  5. Chien H-CR, Lin L-L, Chao S-H, Chen C-C, Wang W-C, Shaw C-Y, Tsai Y-C, Hu H-Y, Hsu W-H. Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae. Biochim. Biophys. Acta Gene Struct. Expr. 1576: 119–126 (2002)Google Scholar
  6. Finlay M-F, McCloud L. Intestinal leucine aminopeptidase and alkaline phosphatase: Genetic regulation and development in mice. Biochem. Genet. 28: 267–281 (1990)CrossRefGoogle Scholar
  7. Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS. Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J. Biol. Chem. 278: 31988–31997 (2003)CrossRefGoogle Scholar
  8. Heerd D, Tari C, Fernández-Lahore M. Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae. Appl. Microbiol. Biotechnol. 98: 7471–7481 (2014)CrossRefGoogle Scholar
  9. Hong EJ, Kim NK, Lee D, Kim WG, Lee I. Overexpression of the laeA gene leads to increased production of cyclopiazonic acid in Aspergillus fumisynnematus. Fungal Biol. 119: 973–983 (2015)CrossRefGoogle Scholar
  10. Javed S, Asgher M, Sheikh MA, Nawaz H. Strain improvement through UV and chemical mutagenesis for enhanced citric acid production in molasses-based solid state fermentation. Food Biotechnol. 24: 165–179 (2010)CrossRefGoogle Scholar
  11. Kim KM, Lim J, Lee JJ, Hurh B-S, Lee I. Characterization of Aspergillus sojae isolated from meju, Korean traditional fermented soybean brick. J. Microbiol. Biotechnol. 27: 251–261 (2017)CrossRefGoogle Scholar
  12. Le Crom S, Schackwitz W, Pennacchio L, Magnuson JK, Culley DE, Collett JR, Martin J, Druzhinina IS, Mathis H, Monot F. Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing. Proc. Natl. Acad. Sci. 106: 16151–16156 (2009)CrossRefGoogle Scholar
  13. Lee I, Oh JH, Shwab EK, Dagenais TR, Andes D, Keller NP. HdaA, a class 2 histone deacetylase of Aspergillus fumigatus, affects germination and secondary metabolite production. Fungal Genet. Biol. 46: 782–790 (2009)CrossRefGoogle Scholar
  14. Liu G, Zhang L, Qin Y, Zou G, Li Z, Yan X, Wei X, Chen M, Chen L, Zheng K, Zhang J, Ma L, Li J, Liu R, Xu H, Bao X, Fang X, Wang L, Zhong Y, Liu W, Zheng H, Wang S, Wang C, Xun L, Zhao G-P, Wang T, Zhou Z, Qu Y. Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes. Sci. Rep. 3: 1–7 (2013)Google Scholar
  15. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402–408 (2001)CrossRefGoogle Scholar
  16. Müller C, McIntyre M, Hansen K, Nielsen J. Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis. Appl. Environ. Microbiol. 68: 1827–1836 (2002)CrossRefGoogle Scholar
  17. Manildi ER. Isoenzymes of leucine aminopeptidase (LAP)—Values in normal human serum. Lab. Med. 3: 19–21 (1972)CrossRefGoogle Scholar
  18. Molzahn S. A new approach to the application of genetics to brewing yeast. J. Am. Soc. Brew. Chem. 35: 54 (1977)CrossRefGoogle Scholar
  19. Nampoothiri K, Nagy V, Kovacs K, Szakacs G, Pandey A. l-leucine aminopeptidase production by filamentous Aspergillus fungi. Lett. Appl. Microbiol. 41: 498–504 (2005)CrossRefGoogle Scholar
  20. Sato A, Oshima K, Noguchi H, Ogawa M, Takahashi T, Oguma T, Koyama Y, Itoh T, Hattori M, Hanya Y. Draft genome sequencing and comparative analysis of Aspergillus sojae NBRC4239. DNA Res. 18: 165–176 (2011)CrossRefGoogle Scholar
  21. Seitz LM. Ergosterol as a measure of fungal growth. Phytopathology 69: 1202–1203 (1979)CrossRefGoogle Scholar
  22. Sempio FC. Yondu. Korea Trademark 4,012,227,080,000 (2016)Google Scholar
  23. Simpson I, Caten C. Recurrent mutation and selection for increased penicillin titre in Aspergillus nidulans. Microbiology 113: 209–217 (1979)Google Scholar
  24. Te Biesebeke R, Record E, Van Biezen N, Heerikhuisen M, Franken A, Punt P, Van Den Hondel C. Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates. Appl. Microbiol. Biotechnol. 69: 44 (2005)CrossRefGoogle Scholar
  25. Toldrá F, Aristoy M-C, Flores M. Contribution of muscle aminopeptidases to flavor development in dry-cured ham. Food Res. Int. 33: 181–185 (2000)CrossRefGoogle Scholar
  26. Vitikainen M, Arvas M, Pakula T, Oja M, Penttilä M, Saloheimo M. Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties. BMC Genom. 11: 441 (2010)CrossRefGoogle Scholar
  27. Wösten HA, Moukha SM, Sietsma JH, Wessels JG. Localization of growth and secretion of proteins in Aspergillus niger. Microbiology 137: 2017–2023 (1991)Google Scholar
  28. Ward OP. Production of recombinant proteins by filamentous fungi. Biotechnol. Adv. 30: 1119–1139 (2012)CrossRefGoogle Scholar
  29. Xu D, Pan L, Zhao H, Zhao M, Sun J, Liu D. Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger. J. Ind. Microbiol. Biotechnol. 38: 1255–1265 (2011)CrossRefGoogle Scholar

Copyright information

© The Korean Society of Food Science and Technology and Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Bio and Fermentation Convergence Technology, BK21 PLUS ProjectKookmin UniversitySeoulKorea
  2. 2.Sempio Fermentation Research CenterSempio Foods CompanyOsongKorea

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