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. 2024 May 21;90(5):e0012224.
doi: 10.1128/aem.00122-24. Epub 2024 Apr 3.

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression

Jesus D Castaño  1 , Irina V El Khoury  2 , Joshua Goering  1 , James E Evans  2   3 , Jiwei Zhang  1
Affiliations

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression

Jesus D Castaño et al. Appl Environ Microbiol. .

Abstract

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Keywords: ROS redox; biocatalyst consortium; brown rot; fungal enzymes; fungal wood degradation; wheat germ cell-free protein expression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
Candidate CAZy enzymes involved in the brown rot fungal oxidative deconstruction of wood cell wall. The fungal enzyme consortium, chelator metabolites, and their mediated redox reactions for generating Fenton reactants, Fe2+ and H2O2, were proposed for the oxidative step of brown rot according to the previous research (A) (6, 16). The putative enzyme catalysts in this model included Fenton-associating oxidoreductases (e.g., benzoquinone reductase and ferric reductase) and their synchronistic enzymes (e.g., heme-thiolate peroxidase and hemicellulose-debranching glycoside hydrolase in the GH51 family). The upregulation or high expression of four corresponding genes at the early oxidation stage of Rhodonia placenta, relative to the advanced decay stages (middle and late decay), suggested that they play a role in the oxidative decomposition of wood cell wall (B). Gene expression data were retrieved from the data set of GSE84529 (6), and the expression of the four candidate genes was presented as FPKM mean values ± SD of three bio-replicates. Gene codes were taken from the JGI database R. placenta MAD 698R v1.0 (https://mycocosm.jgi.doe.gov/Pospl1/Pospl1.home.html). ECM (light blue) represents the fungal extracellular matrix mediating the diffusion of enzymes and metabolites. Significant differences of gene expressions between the oxidation and advanced decay stages were indicated by the FDR values of RNA-seq DEG analysis (*, FDR < 0.05; **, FDR < 0.01; ***, FDR < 0.001).
Fig 2
Fig 2
Cell-free synthesis and preliminary functional characterization of brown rot fungal enzymes. A pipeline from CDS annotation to cell-free protein expression and characterization was used for the study of brown rot fungal enzymes (A). DNA constructs containing the CDS of interest were transcribed and translated using wheat germ-based cell-free expression in a screening mode with the use of a FluoroTect GreenLys reagent—a method described previously (36). Successful expression and solubility of target proteins were then visualized on SDS-PAGE using a laser-based scanner (B). Symbols “c” and “s” refer to the crude and soluble fractions of each cell-free protein sample, respectively. The FluoroTect GreenLys reagent itself manifests as two dark bottom bands in every lane of the gel. The subsequent enzyme assays showed that the synthesized fungal proteins Pospl1|61079, Pospl1|124517, Pospl1|130030, and Pospl1|100251 had the expected heme-thiolate peroxidase, benzoquinone reductase, α-L-arabinofuranosidase, and ferric reductase activities, respectively (C). Mean values ± SD of three bio-replicates were shown for the enzyme assays, and two-tailed paired t tests were used to establish significant differences between the sample and its corresponding control (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The list of protein sample names and their relevant information can be seen in Table 1.
Fig 3
Fig 3
Protein structures, catalytic features, and responses to environmental factors of the synthesized brown rot fungal enzymes. The 3D protein structures and their possible cellular loci were predicted for four active brown rot enzymes—ABF, HTP, BQR, and FRD (A). Kinetic parameters, including the maximum velocity (Vmax) and Km values, were evaluated for each enzyme (B). Catalytic features, including the optimal pH, pH stability, optimal temperature, and temperature stability of four enzymes, were determined and presented in the format of heatmap, with a higher color intensity indicating a higher activity in each case (C). The numerical data set of these measurements is available in Fig. S2 to S5. Potential environmental inhibitors (D) and the effect of oxidative stress (E) on enzyme activities were also evaluated. The effects of oxidative stress were measured by monitoring the enzyme tolerance to the ROS radicals (•OH) generated by the Fenton reaction (Fenton) relative to that of control (i.e., without both H2O2 and Fe2+) and Fe2+- or H2O2-only treatments (1 mM Fe2+ and 5 mM H2O2 were used for these experiments, and see more details in the methods for the Fenton treatment). All enzyme activities were shown as the mean values ± SD of three replicates, and different letters represent significant differences within a series of treatment for each enzyme (P < 0.05).
Fig 4
Fig 4
The influences of phenol-oxidizing enzymes on quinone redox cycle that drives Fenton chemistry in brown rot. (A) The proposed quinone redox cycle driven by a BQR of R. placenta and its partner oxidases: laccase (LCC; Sigma, SIAL-SAE0050) and heme-thiolate peroxidase (HTP). In line with the proposed reactions, the reduction of 2,6-DMBQ by BQR allowed the detection of Fe2+. Additionally, adding LCC to the cycle caused a protein dosage-dependent interference with the generation of both H2O2 (B) and Fe2+ (C). Similarly, adding HTP also lowered the production of Fe2+ (D). A higher amount of Fe3+ was used for the HTP supplemental experiments to increase the detection limits of Fe2+, given that adding exogenous H2O2, required by HTP activity, may accelerate the oxidative consumption of generated Fe2+. Different amounts of LCC or HTP proteins were supplemented into the reactions, including 40 µg (+P40), 80 µg (+P80), 200 µg (+P200), and 600 µg (+P600). Bovine albumin serum (BSA) was used as a control in both cases to account for any unspecific scavenging effects. Mean values ± SD of three bio-replicates were shown for the production of H2O2 and Fe2+. Different letters represent significant differences within a series of treatment (ANOVA, P < 0.05).
Fig 5
Fig 5
Schematic model depicting the functions of fungal enzymes during the oxidative phase of brown rot wood decomposition. Four characterized enzymes by this work, as well as their microenvironments (e.g., oxalate and pH) between the fungal cell and wood substrate, were included to build this model. Briefly, the benzoquinone reductase (BQR) located inside the fungal cell drives the quinone redox cycle, thus translocating intracellular reducing equivalents to drive the extracellular reduction of Fe3+ and O2 for Fenton reaction on wood substrate. A quinone transporter (QT), despite not yet being characterized, is expected in this process to facilitate the diffusions of the quinone metabolites. Microenvironments composed of decreasing concentrations of protons and oxalate, formed while they are diffused from fungal cell to wood, are required to control the localized Fenton reactions adjacent to the wood substrate. Marginal production of ligninolytic oxidases [e.g., laccase (LCC) and heme-thiolate peroxidase (HTP)] by brown rot fungi may slow down the generation of Fenton radicals produced by quinone redox molecules. Bi-functionility is proposed for ferric reductase (FRD) in the model, which includes maintaining iron homeostasis and production of reduced iron to facilitate wood decay processes. Synergy between GH catalysts and Fenton mechanism is evident, but they may have evolved distinct structural or catalytic features to resist the ROS damage. For instance, in this study a GH51 family α-L-arabinofuranosidase (ABF) displayed outstanding ROS-tolerance, and it may be involved in debranching hemicellulose immediately after ROS depolymerization, thus exposing the cellulose chains.
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