Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent—A comparative study

Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:69–80, 2018     

Towards industrially-feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator

The ability of two natural phenols to act as mediators of the recombinant Myceliophthora thermophila laccase (MtL) in eucalypt-pulp delignification was investigated. After alkaline peroxide extraction, the properties of the enzymatically-treated pulps improved with respect to the control. The pulp brightness increased (3.1 points) after the enzymatic treatment with MtL alone, but the highest improvements were obtained after the MtL treatment using syringaldehyde (4.7 points) and especially methyl syringate (8.3 points) as mediators. Likewise, a decrease in kappa number up to 2.7 points was obtained after the MtL-methyl syringate treatment, followed by decreases of 1.4 and 0.9 points after the treatments with MtL-syringaldehyde and MtL alone, respectively. On the other hand, removal of the main lipophilic extractives present in eucalypt pulp was observed after the above laccase-mediator treatments. Finally, the doses of both MtL and methyl syringate were reduced, and results compatible with industrial implementation were obtained.     

Thermostability of native and pegylated Myceliophthora thermophila laccase in aqueous and mixed solvents

A commercial preparation of laccase (EC 1.10.3.2), cloned from Myceliophthora thermophila and expressed in Aspergillus oryzae (MtL), was purified and modified by conjugation with poly(ethylene glycol) (M(r) = 5000) and is labeled PEG-MtL. Native enzyme was found to have a molecular mass of 80 kDa, as determined by gel filtration, and 110 kDa, by SDS-PAGE. The oxidative dimerization of 2,6-dimethoxyphenol (DMP) to produce the corresponding dibenzoquinone was catalyzed by MtL in a manner comparable to that for a diffusion-controlled reaction (k(cat)/K(M) approximately = 10(8) M(-)(1) s(-)(1) and E(a) approximately = 18 kJ M(-)(1)). PEG-MtL was found, by TNBS titration, to have blocked 54% of lysine groups; its hydrodynamic and charge properties were different from those of MtL. Catalytic efficiency (k(cat)/K(M)) of PEG-MtL was similar to that of MtL with DMP as substrate; however, k(cat)/K(M) was 2-fold reduced for the reaction in which 2',2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is oxidized to form a radical cation. E(a) values were similar in both enzyme preparations when assayed in buffered solutions. Far-UV CD spectra were similar for MtL and PEG-MtL and consistent with a protein rich in beta-sheet structure with negligible content of alpha-helices. A blue shift of near-UV CD spectrum for PEG-MtL as compared to MtL was consistent with the decreased polarity of the tyrosyl side chains upon PEG conjugation. Also the blue band of the copper active site was shifted from lambda approximately 610 nm (MtL) to lambda approximately 575 nm (PEG-MtL). Scanning microcalorimetry showed small denaturation enthalpies (6.3 and 7.5 J g(-)(1) for MtL and PEG-MtL, respectively), indicating the high stability of the beta-sheet folding pattern of laccases. However, PEG-MtL proved to be more stable, its half-denaturation temperature being 2 degrees C higher than that of MtL. In 30% alcohol, pegylated laccase showed slower enzyme-activity decay rates than the unmodified enzyme; this behavior was caused by a decrease in the activation entropy of the denaturation reaction. Results can be explained by entropic stabilization by PEG conjugation because of the restricted motion of some surface amino acid side chains, which results in a more stable active site.     

Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V_HH antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.     

Molecular Cloning and Expression in Saccharomyces cerevisiae of a Laccase Gene from the Ascomycete Melanocarpus albomyces

The lac1 gene encoding an extracellular laccase was isolated from the thermophilic fungus Melanocarpus albomyces. This gene has five introns, and it encodes a protein consisting of 623 amino acids. The deduced amino acid sequence of the laccase was shown to have high homology with laccases from other ascomycetes. In addition to removal of a putative 22-amino-acid signal sequence and a 28-residue propeptide, maturation of the translation product of lac1 was shown to involve cleavage of a C-terminal 14-amino-acid extension. M. albomyces lac1 cDNA was expressed in Saccharomyces cerevisiae under the inducible GAL1 promoter. Extremely low production was obtained with the expression construct containing laccase cDNA with its own signal and propeptide sequences. The activity levels were significantly improved by replacing these sequences with the prepro sequence of the S. cerevisiae α-factor gene. The role of the C-terminal extension in laccase production in S. cerevisiae was also studied. Laccase production was increased sixfold with the modified cDNA that had a stop codon after the native processing site at the C terminus.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

Enzymatic degradation of lignocellulosic biomass by continuous process using laccase and cellulases with the aid of scaffoldin for ethanol production

Biological processes for the degradation of intractable materials are still not considered to be practical due to the slow rates of enzymatic degradation. Cellulosomes are complexed enzyme systems with great degradative potential and one of the strategies for overcoming this problem. In this study, the laccase CueO from Escherichia coli was fused to the dockerin domain of a cellulosome system and further assembled with the scaffoldin miniCbpA, forming a laccase–miniCbpA complex. Compared to the individual subunits, laccase–miniCbpA complex caused a noticeable 2.1-fold increase in enzyme activity levels and enhanced degradation of various synthetic dyes, showing an increase of approximately 1.6-fold. Also, pretreated barley straw by laccase complexes was efficiently converted to bioethanol using a cellulase producing Saccharomyces cerevisiae strain. The laccase complexes caused a 2.6-fold increase in the amount of reduced sugar with an insoluble substrate in conditions with an identical amount of enzymes. The cellulolytic yeast with the aid of laccase complexes produced 2.34 g/L ethanol after 72 h, indicating an increase of approximately 2.1-fold compared to fermentation without the laccase complexes. This demonstrates the feasibility of developing an efficient laccase complex based on the cellulosome and this strategy may be used to degrade recalcitrant materials.     

Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, a pI of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl activity is positively affected by calcium. A 10-fold decrease in the K_m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.     

Enhancement of copper content and specific activity of CotA laccase from Bacillus licheniformis by coexpression with CopZ copper chaperone in E. coli

Copper depletion of bacterial laccases obtained by heterologous expression in Escherichia coli is a common problem in production of these versatile biocatalysts. We demonstrate that coexpression of small soluble copper chaperones can mitigate this problem. The laccase CotA and the copper chaperone CopZ both from Bacillus licheniformis were used as model system. The use of the E. coli BL21(DE3) strain expressing CopZ and CotA simultaneously from two plasmids resulted in an 20% increase in copper occupancy and in 26% higher specific activity. We conclude that not only intracellular copper ion concentration, but also presence of an appropriate copper chaperone influences copper ion insertion into CotA laccase. Moreover, E. coli BL21(DE3) seems to lack such a copper chaperone which can be partially complemented by heterologous expression thereof. The presented system is simple and can routinely be used for improved heterologous production of bacterial laccase in E. coli.     

Development of a Saccharomyces cerevisiae Strain with Enhanced Resistance to Phenolic Fermentation Inhibitors in Lignocellulose Hydrolysates by Heterologous Expression of Laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.     

Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor

A cDNA encoding LccIV, a previously uncharacterized laccase isozyme of the white-rot basidiomycete Trametes versicolor, was expressed in the methylotrophic yeast Pichia pastoris. The LccIV isozyme is not expressed by T. versicolor under normal culture conditions and the enzyme was, therefore, investigated to determine whether it had any unusual properties. The native signal peptide of LccIV directed efficient secretion and correct proteolytic processing of LccIV to the mature form, whereas, substitution with the Saccharomyces cerevisiae α-mating factor signal peptide led to retention of an additional tetrapeptide at the amino-terminus of the secreted enzyme and ∼25% lower specific activity in fermentor medium. Active LccIV was purified to homogeneity by sequential steps of ion-exchange, size-exclusion and hydrophobic interaction chromatography. The enzyme contains ∼25% N-linked glycans (∼40% total carbohydrate) and has an apparent molecular mass of ∼85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ∼100 kDa by size-exclusion chromatography, indicating a monomeric structure. A pH of 5.5 was optimal for oxidation of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Thus, the LccIV isozyme appears to be similar in these respects to the laccase isozymes constitutively expressed by T. versicolor.     

Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature

Laccase can be used for enzymatic detoxification of lignocellulosic hydrolysates. A Saccharomyces cerevisiae strain with enhanced resistance to phenolic inhibitors and thereby improved ability to ferment lignocellulosic hydrolysates would presumably be obtained by heterologous expression of laccase. Sequencing of the cDNA for the novel laccase gene lcc2 from the lignin-degrading basidiomycete Trametes versicolor showed that it encodes an isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide. By comparison with Edman degradation data, it was concluded that lcc2 encodes an isoenzyme corresponding to laccase A. The gene product of lcc2 displays 71% identity with the previously characterized T. versicolor lcc1 gene product. An alignment of laccase sequences revealed that the T. versicolor isoenzymes in general are more closely related to corresponding isoenzymes from other white-rot fungi than to the other T. versicolor isoenzymes. The multiplicity of laccase is thus a conserved feature of T. versicolor and related species of white-rot fungi. When the T. versicolor lcc2 cDNA was expressed in S. cerevisiae, the production of active enzyme was strongly dependent on the temperature. After 3 days of incubation, a 16-fold higher laccase activity was found when a positive transformant was kept at 19 °C instead of 28 °C. Similar experiments with Pichia pastoris expressing the T. versicolor laccase gene lcc1 also showed that the expression level was favoured considerably by lower cultivation temperature, indicating that the observation made for the S. cerevisiae expression system is of general significance. Received: 8 December 1998 / Received revision: 9 April 1999 / Accepted: 16 April 1999     

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETαA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae α-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the α-factor signal peptide was 9.79 U ml⁻¹. The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.     

Cytochrome b5 Coexpression Increases Tetrahymena thermophila Δ6 Fatty Acid Desaturase Activity in Saccharomyces cerevisiae

Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2^Δ9,12 substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b₅ protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b₅ domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b₅s were coexpressed with the desaturase. Coexpression of a cytochrome b₅ from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b₅ proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.     

Computational analysis of phenotypic space in heterologous polyketide biosynthesis—Applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.     

Switching from blue to yellow: altering the spectral properties of a high redox potential laccase by directed evolution

Abstract

During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs₆₁₀T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs₆₁₀T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs₆₁₀T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.