Abstract
BackgroundBasidiomycete 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.ResultsA 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.ConclusionsThe 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.
Highlights
Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids arise great interest in the engineering of 3D-nanobiodevices for biomedical uses
The methylotrophic yeast P. pastoris is not the favorite host for directed evolution experiments [17], it does show some attractive features which may complement S. cerevisiae in the synthetic evolutionary scenario: the ability to grow at very high cell densities under the control of strong promoters and secrete high amounts of protein [18]
Heterologous functional expression of blood tolerant laccases in P. pastoris The departure point of the present study is a thermostable laccase from basidiomycete PM1, which was first subjected to 8 generations of in vitro evolution for functional expression in S. cerevisiae [28] and thereafter to 4 further cycles of evolution to become active in human blood [31]
Summary
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. Over 20 fungal laccases have been heterologously expressed in the yeasts Pichia pastoris and Saccharomyces cerevisiae for different purposes [11,12] In general terms, both organisms are suitable for the expression of eukaryotic genes. The battery of reliable in vivo recombination methods based on S. cerevisiae physiology make this budding yeast a powerful cell factory for plenty of potential applications [15]. Despite these advantages, the practical use of S. cerevisiae in different industrial settings is limited by its rather low secretion levels [11]. Even though the expression levels reported for recombinant fungal laccases in these yeasts are diverse (Table 1), overall they are much higher in P. pastoris, ranging from 4.9 to 517 mg/L [19,20,21,22,23,24,25], than in S. cerevisiae, where they vary from 2 to 18 mg/L [26,27,28,29,30]
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