High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii

Abstract

Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.

Keywords: Archaea; Halophilic; Laccase; Metalloenzyme; Organic solvent-tolerant; Secretion.

Fig. 1

General strategy used to construct an H. volcanii strain for high-level synthesis and secretion of the metalloenzyme biocatalyst LccA, a laccase of the multicopper oxidase family.

Fig. 2

Example of an expression plasmid used for high-level secretion of laccase encoded by lccA, using H. volcanii as the platform organism. Plasmid pJAM824 includes a colE1 origin for replication and an ampicillin resistance (Ap^R) marker for selection in E. coli. The pHV2 origin and novobiocin resistance marker (Nv^R) are used for replication and selection in H. volcanii strains cured of pHV2. The rRNA P2 promoter drives transcription and the T7 terminator is included to enhance expression.

Fig. 3

Gel image illustrating a metalloenzyme (LccA) overexpressed and secreted by H. volcanii. The LccA laccase fractions separated by 10% SDS-PAGE and stained with Coomassie brilliant blue R-250 include: ethanol precipitate of culture broth (lane 1) and further purification of these precipitates by anion exchange chromatography (MonoQ 10/10) (lane 2). Image is modified from Uthandi, S., Saad, B., Humbard, M.A., Maupin-Furlow, J.A. (2010). LccA, an archaeal laccase secreted as a highly stable glycoprotein into the extracellular medium by Haloferax volcanii, Applied and Environmental Microbiology, 76(3) 733–43.