Constitutive expression of Botrytis aclada laccase in Pichia pastoris

Kittl, Roman; Gonaus, Christoph; Pillei, Christian; Haltrich, Dietmar; Ludwig, Roland

doi:10.4161/bbug.20037

Cited by 2 publications

(2 citation statements)

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“…That is the case of fed-batch fermentation of P. pastoris producing POXA1b laccase (from the basidiomycete Pleurotus ostreatus), where constitutive production under GAPDH outperformed the enzyme levels obtained in AOX-induced cultures (Pezzella et al, 2017). This is in agreement with results obtained with the ascomycete Botrytis aclada laccase (Kittl et al, 2012), where the enzyme levels were about 40-60 mg l -1 .…”

Section: Heterologous Expression Of Fungal Laccasessupporting

confidence: 85%

Fungal Laccases as Biocatalysts for Wide Range Applications

Salas¹,

Camarero²

2021

Encyclopedia of Mycology

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Section: Heterologous Expression Of Fungal Laccasessupporting

confidence: 85%

Fungal Laccases as Biocatalysts for Wide Range Applications

Salas¹,

Camarero²

2021

Encyclopedia of Mycology

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“…Although the methylotrophic yeast P. pastoris is not the favorite host for directed evolution experiments (the lack of episomal vectors together with low transformation efficiencies constrain the building of mutant libraries) [17], it does show some attractive features which may complement S. cerevisiae in the synthetic evolutionary scenario: specifically, the ability to grow at very high cell densities under the control of strong promoters and secrete high amounts of protein [18]. 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 [19-25], than in S. cerevisiae , where they vary from 2 to 18 mg/L [26-30]. …”

Section: Introductionmentioning

confidence: 99%

Functional expression of a blood tolerant laccase in Pichia pastoris

et al. 2013

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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.

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Constitutive expression of Botrytis aclada laccase in Pichia pastoris

Abstract: ). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h).

Cited by 2 publications

References 6 publications

Fungal Laccases as Biocatalysts for Wide Range Applications

Fungal Laccases as Biocatalysts for Wide Range Applications

Functional expression of a blood tolerant laccase in Pichia pastoris

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