In:
Microbial Biotechnology, Wiley, Vol. 10, No. 6 ( 2017-11), p. 1830-1836
Abstract:
Although the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution experiments. In this study, twelve constructs were prepared by fusing native and evolved α‐factor prepro‐leaders from S. cerevisiae to four different laccases with low‐, medium‐ and high‐redox potential ( PM 1L from basidiomycete PM 1; PcL from Pycnoporus cinnabarinus ; TspC30L from Trametes sp. strain C30; and MtL from Myceliophthora thermophila ). Microcultures of the prepro‐leader:laccase fusions were grown in selective expression medium that used galactose as both the sole carbon source and as the inducer of expression so that the secretion and activity were assessed with low‐ and high‐redox potential mediators in a high‐throughput screening context. With total activity improvements as high as sevenfold over those obtained with the native α‐factor prepro‐leader, the evolved prepro‐leader from PcL (α PcL ) most strongly enhanced secretion of the high‐ and medium‐redox potential laccases PcL, PM 1L and TspC30L in the microtiter format with an expression pattern driven by prepro‐leaders in the order α PcL 〉 α PM 1L ~ α native . By contrast, the pattern of the low‐redox potential MtL was α native 〉 α PcL 〉 α PM 1L . When produced in flask with rich medium, the evolved prepro‐leaders outperformed the α native signal peptide irrespective of the laccase attached, enhancing secretion over 50‐fold. Together, these results highlight the importance of using evolved α‐factor prepro‐leaders for functional expression of fungal laccases in directed evolution campaigns.
Type of Medium:
Online Resource
ISSN:
1751-7915
,
1751-7915
DOI:
10.1111/mbt2.2017.10.issue-6
DOI:
10.1111/1751-7915.12838
Language:
English
Publisher:
Wiley
Publication Date:
2017
detail.hit.zdb_id:
2406063-X
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