Versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems. With a broad substrate scope and minor requirements, VP is an extremely attractive blueprint to be designed by the directed evolution tool-box. In recent years, improved VP variants have been generated that meet industrial requirements in terms of improved heterologous functional expression and activity, as well as stability at high temperatures or in the presence of strong inhibitors. This review describes the making of VP by directed evolution, addressing the most important findings and challenges that were faced, along with the future prospects for this research field. ; European UnionFP7-KBBE-2013-7-613549-INDOXCOST Action CM1303 Spanish Government BIO2016-79106-R- Lignolution Seventh Framework Programme10.13039/100011102FP7-KBBE-2013-7-613549-INDOXEuropean Cooperation in Science and Technology10.13039/501100000921CM1303 Systems BiocatalysisThis work was supported by grants from the European Union [FP7-KBBE-2013-7-613549-INDOX], the COST Action [CM1303 Systems Biocatalysis] and the Spanish Government [BIO2016-79106-R-Lignolution]. ; Peer reviewed
12 páginas, 6 figuras ; [Background] Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory. ; [Results] A blood-active laccase (ChU-B mutant) fused to the native/evolved α-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the α-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers. ; [Conclusions] The tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications. ; This study is based upon a work funded by European Union Projects (grant numbers NMP4-SL-2009-229255-3D-Nanobiodevice, FP7-KBBE-2010-4-26537-Peroxicats and COST Action CM0701) and a Spanish National Project (Evofacel, BIO2010-19697) and by the Austrian Science Fund (FWF, P25148-B20). We thank Prof. S. Shleev from Malmö University (Sweden) for carrying out the measurements of the laccase activity in human plasma and blood. D.M.M. is grateful to the CSIC for a JAE fellowship. ; Peer reviewed
High-redox potential laccases are powerful biocatalysts with a wide range of applications in biotechnology. We have converted a thermostable laccase from a white-rot fungus into a blood tolerant laccase. Adapting the fitness of this laccase to the specific composition of human blood (above neutral pH, high chloride concentration) required several generations of directed evolution in a surrogate complex blood medium. Our evolved laccase was tested in both human plasma and blood, displaying catalytic activity while retaining a high redox potential at the T1 copper site. Mutations introduced in the second coordination sphere of the T1 site shifted the pH activity profile and drastically reduced the inhibitory effect of chloride. This proof of concept that laccases can be adapted to function in extreme conditions opens an array of opportunities for implantable nanobiodevices, chemical syntheses, and detoxification. ; This study was based upon work funded by EU Projects (NMP4-SL-2009-229255-3D-Nanobiodevice, FP7-KBBE-2010-4-26537-Peroxicats and COST Action CM0701) and a project from the Spanish Government (BIO2010-19697). D.M.M. was supported by a JAE grant and D.G.P. was supported by a Peroxicats contract. M.P. received support from the 2009 Ramón y Cajal programme of the Spanish MINECO. ; Peer reviewed
