A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus
            <i>Gloeophyllum trabeum</i>

Li, Weiran; Ayers, Charles R.; Huang, Wei‐Ping; Schilling, Jonathan S.; Cullen, Daniel; Zhang, Jiwei

doi:10.1128/spectrum.04246-22

Cited by 5 publications

(3 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the other hand, during brown rot evolution the loss of these “brake” genes allowed fungi to accelerate Fenton cycles for oxidative attacks. To validate this, we have built a genetic manipulation system in brown rot fungus G. trabeum ( 77 ), and recently we have tested the in vivo functions of laccase and ligninolytic peroxidases and their influences on Fenton cycles.…”

Section: Resultsmentioning

confidence: 99%

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

Castaño,

El Khoury,

Goering

et al. 2024

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

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. IMPORTANCE Brown 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.

show abstract

Section: Resultsmentioning

confidence: 99%

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

Castaño,

El Khoury,

Goering

et al. 2024

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The hygromycin phosphotransferase gene is widely used as an antibiotic transformation marker in fungi, including Eutypella sp. ( Ning et al, 2022 ), Poria cocos ( Xie et al, 2022 ) and Gloeophyllum trabeum ( Li et al, 2023 ). Here, we determined that the lowest hygromycin B inhibition concentration in C. chanhua 022017–3 was 300 μg/mL.…”

Section: Discussionmentioning

confidence: 99%

“…The hygromycin B is widely used as an antibiotic screening agent in fungal genetic transformation ( Gritz and Davies, 1983 ; Ning et al, 2022 ; Li et al, 2023 ). The antibiotic sensitivity of C. chanhua strain 022017-3 was determined in PDA medium with different concentrations of hygromycin B (200, 250, 300, 350, 400, 450 μg/mL).…”

Section: Methodsmentioning

confidence: 99%

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua

Cai,

Xiao,

Xing

et al. 2024

Front. Microbiol.

View full text Add to dashboard Cite

Cordyceps chanhua is a well-known edible and medicinal mushroom with a long history of use in China, and it contains a variety of secondary metabolites with interesting bioactive ingredients. However, recent researches have mainly focused on cultivation conditions, secondary metabolite compositions and pharmacological activities of C. chanhua, the lack of an efficient and stable genetic transformation system has largely limited further research on the relationship between secondary metabolites and biosynthetic gene clusters in C. chanhua. In this study, single-factor experiments were used to compare the effects of different osmotic stabilizers, enzyme concentrations and enzyme digestion times on protoplast yield, and we found that the highest yield of 5.53 × 108 protoplasts/mL was obtained with 0.7 M mannitol, 6 mg/mL snail enzyme and 4 h of enzyme digestion time, and the regeneration rate of protoplasts was up to approximately 30% using 0.7 M mannitol as an osmotic stabilizer. On this basis, a PEG-mediated genetic transformation system of C. chanhua was successfully established for the first time, which lays the foundation for further genetic transformation of C. chanhua.

show abstract

Role of Bacterial Degradation in Lignocellulosic Biomass for Biofuel Production

Kumari,

Kumar,

Bhoi

2024

Valorization of Biomass Wastes for Environmental Sustainability

View full text Add to dashboard Cite

A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum

Abstract: One of the most ubiquitous types of decomposers in nature, brown rot fungi, has lacked robust genetic tools by which to manipulate genes and understand its biology. Brown rot fungi are primary decomposers in northern forests helping recycle the encased carbons in trees back to ecosystem.

Cited by 5 publications

References 43 publications

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

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

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua

Role of Bacterial Degradation in Lignocellulosic Biomass for Biofuel Production

Contact Info

Product

Resources

About