Transcriptome analysis of genes associated with autolysis of Coprinus comatus.

Coprinus comatus, widely known as "Jituigu", is an important commodity and food in China. The yield of C. comatus, however, is substantially reduced by the autolysis of the fruiting bodies after harvest. To gain insight into the molecular mechanism underlying this autolysis, we divided the growth of C. comatus fruiting bodies into four stages: infant stage (I), mature stage (M), discolored stage (D), and autolysis stage (A). We then subjected these stages to de novo transcriptomic analysis using high-throughput Illumina sequencing. A total of 12,946 unigenes were annotated and analyzed with the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). We analyzed the differentially expressed genes (DEGs) between stages I and M, M and D, and D and A. Because the changes from M to D are thought to be related to autolysis, we focused on the DEGs between these two stages. We found that the pathways related to metabolic activity began to vary in the transition from M to D, including pathways named as autophagy-yeast, peroxisome, and starch and sucrose metabolism. This study also speculates the possible process of the autolysis of Coprinus comatus. In addition, 20 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study, which is the first to describe the transcriptome of C. comatus, provides a foundation for future studies concerning the molecular basis of the autolysis of its fruiting bodies.

Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

Discovery of microRNA-like RNAs during early fruiting body development in the model mushroom Coprinopsis cinerea.

Coprinopsis cinerea is a model mushroom particularly suited for the study of fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) have been extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), one for Argonaute (AGO), one for AGO-like and one for quelling deficient-2 (QDE-2) proteins, were also identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to those in animals and plants. Taken together, our findings provided the first evidence for milRNAs in the model mushroom and their potential roles in regulating fruiting body development. New information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights for guiding further functional and evolutionary studies of miRNAs.

Effects of pex1 disruption on wood lignin biodegradation, fruiting development and the utilization of carbon sources in the white-rot Agaricomycete Pleurotus ostreatus and non-wood decaying Coprinopsis cinerea.

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

Accumulation and tolerance to cadmium heavy metal ions and induction of 14-3-3 gene expression in response to cadmium exposure in Coprinus atramentarius.

Cadmium (Cd), one of the most toxic heavy-metal pollutants, has a strong and irreversible tendency to accumulate. Bioremediation is a promising technology to remedy and control heavy metal pollutants because of its low cost and ability to recycle heavy metals. Coprinus atramentarius is recognized as being able to accumulate heavy metal ions. In this work, C. atramentarius is cultivated on a solid medium containing Cd2+ ions to analyze its ability to tolerate different concentrations of the heavy metal ion. It is found that the growth of C. atramentarius is not significantly inhibited when the concentration of Cd2+ is less than 0.6mgL-1. The accumulation capacity of C. atramentarius at different Cd2+ concentrations also was determined. The results show that 76% of the Cd2+ present can be accumulated even when the concentration of the Cd2+ is 1mgL-1. The different proteins of C. atramentarius exposed to Cd2+ were further analyzed using gel electrophoresis. A 14-3-3 protein was identified and shown to be significantly up-regulated. In a further study, a full-length 14-3-3 gene was cloned containing a 759bp open reading frame encoding a polypeptide consisting of 252 amino acids and 3 introns. The gene expression work also showed that the 14-3-3 was significantly induced, and showed coordinated patterns of expression, with Cd2+ exposure.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.

A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/ß)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.

Coprinopsis cinerea intracellular lactonases hydrolyze quorum sensing molecules of Gram-negative bacteria.

Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-ß-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle.

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala.

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.

Determination of biodiversity of Coprinus comatus using genotyping and metabolic profiling tools.

Coprinus comatus strains (CCMs) originating from Poland were identified using ITS region sequencing. Based on the sequences obtained, the genetic relationship between the CCM strains was determined and a clear separation of all strains into two main clusters was obtained. The Coprinus strains were also genetically characterized for the first time by the AFLP technique. The analysis showed that the CCMs separated into four main clusters and a high complication of a UPGMA-based dendrogram was achieved. C. comatus strains included in the analysis displayed an AFLP profile similarity level in the range from 44 to 66%. The highest similarity coefficient, 0.490, was found between CCM12 and CCM13, and the lowest (0.202) between the CCM2 and CCM5 isolates. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mycelial growth. The analysis allowed comparison of the functional diversity of the CCM strains and revealed a broad variability within the analyzed Coprinus species based on substrate utilization profiles. Significant differences (2-48) have been shown in the substrate richness values. The Biolog experiments proved to be a good profiling technology for studying the diversity in shaggy manes due to metabolic differences and demonstrated that all the strains might be considered individually. It is evident that the strain metabolic grouping does not correlate with the grouping based on the ITS sequences and AFLP profiles, however, some similarities may be observed.

[Expression and characterization of Coprinus cinereus peroxidase].

OBJECTIVE: The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently. METHODS: We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115. RESULTS: We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol. CONCLUSION: The highly secretory expression of CIP and high special activity lay the good foundation for it' s industrial applications in waste water treatment, decolouration of dyestuffs.

Production of antibacterial peptide from bee venom via a new strategy for heterologous expression.

Honey bee is important economic insect that not only pollinates fruits and crops but also provides products with various physiological activities. Bee venom is a functional agent that is widely applied in clinical treatment and pharmacy. Secapin is one of these agents that have a significant role in therapy. The functions of secapin from the bee venom have been documented, but little information is known about its heterologous expression under natural condition. Moreover, few scholars verified experimentally the functions of secapin from bee venom in vitro. In this study, we successfully constructed a heterologous expression vector, which is different from conventional expression system. A transgenic approach was established for transformation of secapin gene from the venom of Apis mellifera carnica (Ac-sec) into the edible fungi, Coprinus cinereus. Ac-sec was encoded by a 234 bp nucleotide that contained a signal peptide domain and two potential phosphorylation sites. The sequence exhibited highly homology with various secapins characterized from honey bee and related species. Southern blot data indicated that Ac-sec was present as single or multiple copy loci in the C. cinereus genome. By co-transformation and double-layer active assay, Ac-sec was expressed successfully in C. cinereus and the antibacterial activity of the recombinants was identified, showing notable antibacterial activities on different bacteria. Although Ac-sec is from the venom of Apidae, phylogenetic analysis demonstrated that Ac-sec was more closely related to that of Vespid than to bee species from Apidae. The molecular characteristics of Ac-sec and the potential roles of small peptides in biology were discussed.

A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cell elongation.

We have identified and characterized a Coprinopsis cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cortex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically.

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea.

BACKGROUND: The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. RESULTS: We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. CONCLUSIONS: We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.

Global gene expression in Coprinopsis cinerea meiotic mutants reflects checkpoint arrest.

The basidiomycete Coprinopsis cinerea is well-suited to studies of meiosis because meiosis progresses synchronously in 10 million cells within each mushroom cap. Approximately 20% of C. cinerea genes exhibit changing expression during meiosis, but meiosis and mushroom development happen concurrently and therefore differentially expressed genes might not be directly involved in meiotic processes. By using microarrays, we examined global gene expression across a meiotic time course in two mutants in which meiosis arrests but mushrooms develop normally. Genes differentially expressed in the mutants compared with the wild type are likely to be involved in meiosis and sporulation as opposed to mushroom development. In rad50-1, which arrests in late prophase, RNA abundance for a group of early meiotic genes remains high, whereas the expression of a group of late meiotic genes is never induced. In contrast, in msh5-22 (which fails to undergo premeiotic DNA replication), both early and late meiotic genes are underexpressed relative to wild type at late meiotic time points as the cells die. Genes that are differentially expressed relative to wild type in both mutants are particularly strong candidates for playing roles in meiosis and sporulation.

Efficient gene targeting in ΔCc.ku70 or ΔCc.lig4 mutants of the agaricomycete Coprinopsis cinerea.

Coprinopsis cinerea is a model for studies of sexual development in agaricomycetes (homobasidiomycetes). Efficient gene targeting should facilitate such studies, especially because increasing genome and transcriptome information is now available in C. cinerea. To estimate the frequency of gene disruption by homologous integration in this fungus, we tried to disrupt Cc.wc-2, which encodes a WC-2 homolog, a partner of the fungal blue-light photoreceptor, WC-1. Disruption of Cc.wc-2 did not occur when recipients (protoplasts) of the disrupting construct were prepared from asexual spores, oidia, from the wild type, 326, while it occurred when protoplasts were prepared from mycelial cells from the same strain, albeit at a low frequency (3%). Double-stranded RNA-mediated silencing of a ku70 homolog, named Cc.ku70, or the lig4 homolog Cc.lig4 more or less increased the frequency of Cc.wc-2 targeting. On the basis of these results, we disrupted Cc.ku70 using a Cc.lig4-silenced strain. We then disrupted Cc.lig4 using the Cc.ku70 disruptant. We found that the disruption of Cc.ku70 or Cc.lig4 greatly enhanced gene targeting. In addition, this study demonstrates that Cc.wc-2 is involved in blue light perception in this fungus.

A mutation in the Cc.ubc2 gene affects clamp cell morphogenesis as well as nuclear migration for dikaryosis in Coprinopsis cinerea.

The formation and proliferation of the dikaryon in the agaricomycete Coprinopsis cinerea is controlled by the mating type genes, A and B. The B genes, which encode pheromones and pheromone receptors, control nuclear migration for dikaryosis as well as the fusion of the clamp cell with the subterminal cell while the A genes, which encode two classes of homeodomain proteins, control conjugate nuclear division associated with clamp connection development. We characterized the mutant, B28, which was newly isolated as a strain that fails to form a primary hyphal knot, the first visible sign toward fruiting, from a homokaryotic fruiting strain after REMI mutagenesis. Detailed phenotypic analysis revealed that strain B28 exhibits, in addition to the fruiting defect, a defect in A-regulated clamp cell morphogenesis as well as a defect in B-regulated nuclear migration for dikaryosis. The mutant clamp cells are unique in that they continue growing like branches without fusing with the subterminal cells, in contrast to the unfused pseudoclamp which are normally formed in A-on B-off strains, providing evidence for the existence of an as yet unidentified mechanism for the growth suppression of the clamp cell. Molecular analysis revealed that the gene responsible for the phenotypes, designated Cc.ubc2, encodes a protein similar to Ubc2, an adaptor protein for filamentous growth, pheromone response and virulence in the smut fungus Ustilago maydis. In addition, western blot analysis demonstrated that the Cc.ubc2-1 mutation blocks phosphorylation of a presumptive MAP kinase.

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design.

Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild-type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins.