title: Functional expression of a blood tolerant laccase in Pichia pastoris.
abstract: 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 (P(AOX1) and P(GAP)) and expressed in Pichia pastoris. The most active construct, which contained the P(AOX1) 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.
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid 28049, Spain.
authors.forename: Diana M
authors.initials: DM
authors.lastname: Mate
authors.forename: David
authors.initials: D
authors.lastname: Gonzalez-Perez
authors.forename: Roman
authors.initials: R
authors.lastname: Kittl
authors.forename: Roland
authors.initials: R
authors.lastname: Ludwig
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
mesh_terms: D001790:Blood Physiological Phenomena; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D042845:Laccase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D012969:Sodium Fluoride / Q000737:chemistry
chemical_list: D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D012969:Sodium Fluoride; D042845:Laccase