Deletion of two genes improves heterologous secretion of fungal lignin-degrading heme peroxidases in S. cerevisiae

Efficient, large-scale heterologous production of enzymes is a crucial component of the biomass valorization industry. Whereas cellulose utilization has been successful in applications such as bioethanol, its counterpart lignin remains significantly underutilized despite being an abundant potential source of aromatic commodity chemicals. Fungal lignin-degrading heme peroxidases are thought to be the major agents responsible for lignin depolymerization in nature, but their large-scale production remains inaccessible due to the genetic intractability of basidiomycete fungi and the challenges in the heterologous production of these enzymes. In this study, we employ a strain engineering approach based on functional genomics to identify mutants of the model yeast Saccharomyces cerevisiae with enhanced heterologous production of lignin-degrading heme peroxidases. We show that our screening method coupling an activity-based readout with fluorescence-assisted cell sorting enables identification of two single null mutants of S. cerevisiae, pmt2 and cyt2, with up to 11-fold improved secretion of a versatile peroxidase from the lignin-degrading fungus Pleurotus eryngii. We demonstrate that the double deletion strain pmt2cyt2 displays positive epistasis, improving and even enabling production of members from all three classes of lignin-degrading fungal peroxidases. We anticipate that these mutant strains will be broadly applicable for improved heterologous production of this biotechnologically important class of enzymes.

Heme Peroxidases at Unperturbed and Inflamed Mucous Surfaces

In our organism, mucous surfaces are important boundaries against the environmental milieu with defined fluxes of metabolites through these surfaces and specific rules for defense reactions. Major mucous surfaces are formed by epithelia of the respiratory system and the digestive tract. The heme peroxidases lactoperoxidase (LPO), myeloperoxidase (MPO), and eosinophil peroxidase (EPO) contribute to immune protection at epithelial surfaces and in secretions. Whereas LPO is secreted from epithelial cells and maintains microbes in surface linings on low level, MPO and EPO are released from recruited neutrophils and eosinophils, respectively, at inflamed mucous surfaces. Activated heme peroxidases are able to oxidize (pseudo)halides to hypohalous acids and hypothiocyanite. These products are involved in the defense against pathogens, but can also contribute to cell and tissue damage under pathological conditions. This review highlights the beneficial and harmful functions of LPO, MPO, and EPO at unperturbed and inflamed mucous surfaces. Among the disorders, special attention is directed to cystic fibrosis and allergic reactions.

Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases

Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis. Graphic abstract

Characterization of Class V DyP-Type Peroxidase SaDyP1 from Streptomyces avermitilis and Evaluation of SaDyPs Expression in Mycelium

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.

Peroxidase, an Example of Enzymes with Numerous Applications

The enzyme peroxidase is a heme or iron-porphyrin protein that belongs to a large family of enzymes called the oxidoreductases. Their function mainly is to oxidize molecules at the expense of hydrogen peroxide. They are widely distributed in living organisms, and usually show dramatic colour-product formation as a result of their catalytic effect. They generally catalyse many oxygen transfer reactions involving hydrogen peroxide or anyone of the many other peroxides as electron acceptors and substrates. This ability of reducing peroxides at the expense of electron donating substrates is what marks peroxidases as ubiquitous and very important enzymes with many biotechnological applications. Not surprisingly therefore peroxidases play many important roles in different areas of biotechnology. Among others, these include such diverse areas as bioenergy, bioremediation, dye decolorization, humic acid degradation, paper and pulp and textile industries among many others. An important reason for this ability is the different areas from which peroxidases could be sourced as the function of many peroxidases show variations according to its source. This is a character that differentiates peroxidases from many other biological catalysts. Among the many different types of peroxidases are the heme peroxidases which mainly come from plants and fungi and include among others lignin peroxidases, manganese peroxidases and versatile peroxidases. Some important types of peroxidases from humans and animals are glutathione peroxidase, thyroid peroxidase, lactoperoxidase, salivary peroxidase and thyroid peroxidase.

Revealing two important tryptophan residues with completely different roles in a dye-decolorizing peroxidase from Irpex lacteus F17

Background Dye-decolorizing peroxidases (DyPs) represent a novel family of heme peroxidases that use H2O2 as the final electron acceptor to catalyze the oxidation of various organic compounds. A DyP from Irpex lacteus F17 (Il-DyP4, corresponding to GenBank MG209114), obtained by heterologous expression, exhibits a high catalytic efficiency for phenolic compounds and a strong decolorizing ability toward various synthetic dyes. However, the enzyme structure and the catalytic residues involved in substrate oxidation remain poorly understood. Results Here, we obtained a high-resolution structure (2.0 Å, PDB: 7D8M) of Il‑DyP4 with α-helices, anti-parallel β-sheets and one ferric heme cofactor sandwiched between two domains. The crystal structure of Il‑DyP4 revealed two heme access channels leading from the enzyme molecular surface to its heme region, and also showed four conserved amino acid residues forming the pocket for the conversion of hydrogen peroxide into the water molecule. In addition, we found that Trp264 and Trp380, were two important residues with different roles in Il‑DyP4, by using site-directed mutagenesis and an electron paramagnetic resonance (EPR) study. Trp264 is a noncatalytic residue that mainly is used for maintaining the normal spatial conformation of the heme region and the high-spin state of heme Fe3+ of Il‑DyP4, while Trp380 serves as the surface-exposed radical-forming residue that is closely related to the oxidation of substrates including not only bulky dyes, but also simple phenols. Conclusions This study is important for better understanding the catalytic properties of fungal DyPs and their structure–function relationships.

DyP-Type Peroxidases: Recent Advances and Perspectives

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli. We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.

Histidine orientation in artificial peroxidase regioisomers as determined by paramagnetic NMR shifts

A semi-empirical approach allows determining the His axial-ligand orientation with respect to the porphyrin plane in synthetic heme-peroxidases, for structure/function analysis.