Abstract
Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1–8) from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.
Highlights
Laccase is a family of copper-containing oxidases which can oxidize a wide variety of organic and inorganic compounds
HYB07 laccase family contained at least eight members sharing moderate identities to each other as well as other published laccases. These laccases belonged to classes 2 and 3, depending on the amino acid located 10 amino acids downstream of the conserved Cys in the L4 domain [29]
Even though laccases harboring Phe and Leu had been correlated with high and medium redox potentials (E0 ), respectively [29], it was later shown that Phe at this position may not be a prerequisite for a high E0 [30]
Summary
Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a family of copper-containing oxidases which can oxidize a wide variety of organic and inorganic compounds. Fungal laccases usually exist in gene families, and the laccase isozymes display diverse expression patterns and physicochemical characteristics [3,4]. Among the 12 P. ostreatus laccase isozymes, six are biochemically characterized, and five are heterologously expressed in yeast [9,10,11]. In-depth transcriptional analysis showed expression patterns of P. ostreatus laccase genes in various strains responded to growth conditions, and Lacc and 10 are responsible for the majority of laccase activity [9,10]. Heterologously expressed in P. pastoris (Lac1) and characterized [26,27]. We isolated six new members of the laccase gene family in Cerrena sp. These eight laccase genes showed differential expression patterns in submerged and solid state fermentation. The work will provide a theoretical foundation for production and commercialization of Cerrena laccases
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