2024-03-28T11:28:24Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1739132019-08-20T09:03:01Zcom_10261_101com_10261_5col_10261_354
Tandem-yeast expression system for engineering and producing unspecific peroxygenase
Molina-Espeja, Patricia
Ma, S
Maté, Diana M.
Ludwig, Roland
Alcalde Galeote, Miguel
Ministerio de Economía y Competitividad (España)
European Commission
Alcalde, Miguel [0000-0001-6780-7616]
Molina-Espeja, Patricia [0000-0002-2590-0932]
Saccharomyces cerevisiae
Pichia pastoris
Functional expression
Directed evolution
Unspecific peroxygenase (UPO)
Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (~8. mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217. mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.
EU (FP7-KBBE-2013-7-613549-INDOX, FP7-People-2013-ITN-607793, COST-Action CM1303 Systems Biocatalysis) and the Spanish Government (BIO2010-19697-EVOFACEL, BIO2013-43407-R-DEWRY and CAMBIOS-RTC-2014-1777-3 projects).
Peer Reviewed
2019-01-10T12:32:55Z
2019-01-10T12:32:55Z
2015
2019-01-10T12:32:55Z
artículo
http://purl.org/coar/resource_type/c_6501
Enzyme and Microbial Technology 73-74: 29- 33 (2015)
0141-0229
http://hdl.handle.net/10261/173913
10.1016/j.enzmictec.2015.03.004
1879-0909
http://dx.doi.org/10.13039/501100000780
http://dx.doi.org/10.13039/501100003329
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info:eu-repo/grantAgreement/EC/FP7/613549
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2013-43407
info:eu-repo/grantAgreement/EC/FP7/607793
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2010-19697
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/RTC2014-1777-3
Postprint
http://dx.doi.org/10.1016/j.enzmictec.2015.03.004
open
Elsevier