A key O-demethylase in the degradation of guaiacol by Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoAR, which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoAR and its partner protein gcoBR resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoAR and gcoBR restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoAR could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl-O-demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O-demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O-demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O-demethylase in vitro. This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization.

Wood structure-inspired injectable lignin-based nanogels as blood-vessel-embolic sustained drug-releasing stent for interventional therapies on liver cancer.

An embolic reagent with easy injection, well-controlled target embolization, and sustained release of chemotherapy drugs is urgently needed for successful trans-arterial chemo-embolization (TACE) treatment. However, the development of a highly effective embolic reagent is still challenged. Here, inspired and guided by the structural supporting properties and defense mechanisms of wood cell walls, an ideal lignin-based embolic nanogel (DOX-pN-KL) was explored. Based on the mechanical support of branched lignin and the π-π stacking force between the lignin aromatic ring with anti-tumor drug doxorubicin (DOX), DOX-pN-KL showed the highest mechanical strength among the reported thermosensitive embolization nanogel and performed high drug-loading and favorable sustained-release. Moreover, further TACE treatment and tumor microenvironment evaluation of VX2 tumor-bearing rabbits showed that this nanogel can completely block all levels of vessels in long term and continuously release DOX, thus having effective inhibition on tumor growth and metastasis. DOX-pN-KL is expected to be a promising alternative reagent for interventional therapy.

Lytic polysaccharide monooxygenase synergized with lignin-degrading enzymes for efficient lignin degradation.

Even though the discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally shifted our understanding of biomass degradation, most of the current studies focused on their roles in carbohydrate oxidation. However, no study demonstrated if LPMO could directly participate to the process of lignin degradation in lignin-degrading microbes. This study showed that LPMO could synergize with lignin-degrading enzymes for efficient lignin degradation in white-rot fungi. The transcriptomics analysis of fungi Irpex lacteus and Dichomitus squalens during their lignocellulosic biomass degradation processes surprisingly highlighted that LPMOs co-regulated with lignin-degrading enzymes, indicating their more versatile roles in the redox network. Biochemical analysis further confirmed that the purified LPMO from I. lacteus CD2 could use diverse electron donors to produce H2O2, drive Fenton reaction, and synergize with manganese peroxidase for lignin oxidation. The results thus indicated that LPMO might uniquely leverage the redox network toward dynamic and efficient degradation of different cell wall components.

Copper Ion Mediates Yeast-to-Hypha Transition in Yarrowia lipolytica.

Copper is an essential element that maintains yeast physiological function at low concentrations, but is toxic in excess. This study reported that Cu(II) significantly promoted the yeast-to-hypha transition of Yarrowia lipolytica in dose-dependent manner. Strikingly, the intracellular Cu(II) accumulation was drastically reduced upon hyphae formation. Moreover, we investigated the effect of Cu(II) on the physiological function of Y. lipolytica during the dimorphic transition and found that cellular viability and thermomyces lanuginosus lipase (TLL) were both influenced by the Cu(II)-induced yeast-to-hypha transition. Overall, hyphal cells survived better than yeast-form cells with copper ions. Furthermore, transcriptional analysis of the Cu(II)-induced Y. lipolytica before and after hyphae formation revealed a transition state between them. The results showed multiple differentially expressed genes (DEGs) were turned over between the yeast-to-transition and the transition-to-hyphae processes. Furthermore, gene set enrichment analysis (GSEA) identified that multiple KEGG pathways, including signaling, ion transport, carbon and lipid metabolism, ribosomal, and other biological processes, were highly involved in the dimorphic transition. Importantly, overexpression screening of more than thirty DEGs further found four novel genes, which are encoded by YALI1_B07500g, YALI1_C12900g, YALI1_E04033g, and YALI1_F29317g, were essential regulators in Cu-induced dimorphic transition. Overexpression of each of them will turn on the yeast-to-hypha transition without Cu(II) induction. Taken together, these results provide new insight to explore further the regulatory mechanism of dimorphic transition in Y. lipolytica.

Fungal-Modified Lignin-Enhanced Physicochemical Properties of Collagen-Based Composite Films.

Renewable and biodegradable materials have attracted broad attention as alternatives to existing conventional plastics, which have caused serious environmental problems. Collagen is a potential material for developing versatile film due to its biosafety, renewability, and biodegradability. However, it is still critical to overcome the low mechanical, antibacterial and antioxidant properties of the collagen film for food packaging applications. To address these limitations, we developed a new technology to prepare composite film by using collagen and fungal-modified APL (alkali pretreatment liquor). In this study, five edible and medical fungi, Cunninghamella echinulata FR3, Pleurotus ostreatus BP3, Ganoderma lucidum EN2, Schizophyllum commune DS1 and Xylariaceae sp. XY were used to modify the APL, and that showed that the modified APL significantly improved the mechanical, antibacterial and antioxidant properties of APL/Collagen composite films. Particularly, the APL modified by BP3, EN2 and XY showed preferable performance in enhancing the properties of the composite films. The tensile strength of the film was increased by 1.5-fold in the presence of the APL modified by EN2. To further understand the effect of fungal-biomodified APL on the properties of the composite films, a correlation analysis between the components of APL and the properties of composite films was conducted and indicated that the content of aromatic functional groups and lignin had a positive correlation with the enhanced mechanical and antioxidant properties of the composite films. In summary, composite films prepared from collagen and fungal biomodified APL showed elevated mechanical, antibacterial and antioxidant properties, and the herein-reported novel technology prospectively possesses great potential application in the food packaging industry.

Phospholipid and antioxidant responses of oleaginous fungus Cunninghamella echinulata against hydrogen peroxide stress.

Hydrogen peroxide (H2O2) is one of the major oxidative stress intracellularly and extracellularly, which may affect lipid membrane or cell membrane. However, the mechanism remains unclear. The present study investigated phospholipid and antioxidant responses of Cunninghamella echinulata under exogenous H2O2 stress by integrating lipidomics and transcriptomics. H2O2 significantly affected phospholipid profile of C. echinulata exposed to exogenous H2O2. The phospholipid content was reduced from 6.41% to 2.47% on the first day, and to 1.03% on the 7th day, which was 5-6 times lower than that in the control. Phosphatidyl choline was reduced significantly from 29.71% to 2.73% on the 7th day. The lipid-related metabolic maps of C. echinulata responding to H2O2 were constructed based on transcriptomics, lipidomics and biochemical analysis. Results showed that H2O2 almost mobilized all the signaling pathways in the cell, especially the AMPK and cAMP signaling pathway, which regulated the metabolism of proteins and fatty acids. H2O2-stress triggered the high expression of heat shock genes. The antioxidant enzymes were activated to supply more NADPH, which contributed to the modulation of intracellular redox balance, and continuously scavenged active substances, thus improving the mycelial resistance to oxidative stress.

Microbial lignin valorization through depolymerization to aromatics conversion.

Lignin is the most abundant source of renewable aromatic biopolymers and its valorization presents significant value for biorefinery sustainability, which promotes the utilization of renewable resources. However, it is challenging to fully convert the structurally complex, heterogeneous, and recalcitrant lignin into high-value products. The in-depth research on the lignin degradation mechanism, microbial metabolic pathways, and rational design of new systems using synthetic biology have significantly accelerated the development of lignin valorization. This review summarizes the key enzymes involved in lignin depolymerization, the mechanisms of microbial lignin conversion, and the lignin valorization application with integrated systems and synthetic biology. Current challenges and future strategies to further study lignin biodegradation and the trends of lignin valorization are also discussed.

Recent Advancements and Challenges in Lignin Valorization: Green Routes towards Sustainable Bioproducts.

The aromatic hetero-polymer lignin is industrially processed in the paper/pulp and lignocellulose biorefinery, acting as a major energy source. It has been proven to be a natural resource for useful bioproducts; however, its depolymerization and conversion into high-value-added chemicals is the major challenge due to the complicated structure and heterogeneity. Conversely, the various pre-treatments techniques and valorization strategies offers a potential solution for developing a biomass-based biorefinery. Thus, the current review focus on the new isolation techniques for lignin, various pre-treatment approaches and biocatalytic methods for the synthesis of sustainable value-added products. Meanwhile, the challenges and prospective for the green synthesis of various biomolecules via utilizing the complicated hetero-polymer lignin are also discussed.

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin-carbohydrate complexes during fungal degradation of lignocellulose.

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin-carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.

Integrative Analysis of Selected Metabolites and the Fungal Transcriptome during the Developmental Cycle of Ganoderma lucidum Strain G0119 Correlates Lignocellulose Degradation with Carbohydrate and Triterpenoid Metabolism.

To systemically understand the biosynthetic pathways of bioactive substances, including triterpenoids and polysaccharides, in Ganoderma lucidum, the correlation between substrate degradation and carbohydrate and triterpenoid metabolism during growth was analyzed by combining changes in metabolite content and changes in related enzyme expression in G. lucidum over 5 growth phases. Changes in low-polarity triterpenoid content were correlated with changes in glucose and mannitol contents in fruiting bodies. Additionally, changes in medium-polarity triterpenoid content were correlated with changes in the lignocellulose content of the substrate and with the glucose, trehalose, and mannitol contents of fruiting bodies. Weighted gene coexpression network analysis (WGCNA) indicated that changes in trehalose and polyol contents were related to carbohydrate catabolism and polysaccharide synthesis. Changes in triterpenoid content were related to expression of the carbohydrate catabolic enzymes laccase, cellulase, hemicellulase, and polysaccharide synthase and to the expression of several cytochrome P450 monooxygenases (CYPs). It was concluded that the products of cellulose and hemicellulose degradation participate in polyol, trehalose, and polysaccharide synthesis during initial fruiting body formation. These carbohydrates accumulate in the early phase of fruiting body formation and are utilized when the fruiting bodies mature and a large number of spores are ejected. An increase in carbohydrate metabolism provides additional precursors for the synthesis of triterpenoids. IMPORTANCE Most studies of G. lucidum have focused on its medicinal function and on the mechanism of its activity, whereas the physiological metabolism and synthesis of bioactive substances during the growth of this species have been less studied. Therefore, theoretical guidance for cultivation methods to increase the production of bioactive compounds remains lacking. This study integrated changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum with enzyme expression from transcriptomics data using WGCNA. The findings helped us better understand the connections between substrate utilization and the synthesis of polysaccharides and triterpenoids during the cultivation cycle of G. lucidum. The results of WGCNA suggest that the synthesis of triterpenoids can be enhanced not only through regulating the expression of enzymes in the triterpenoid pathway, but also through regulating carbohydrate metabolism and substrate degradation. This study provides a potential approach and identifies enzymes that can be targeted to regulate lignocellulose degradation and accelerate the accumulation of bioactive substances by regulating substrate degradation in G. lucidum.

Recycling of Lignin and Si Waste for Advanced Si/C Battery Anodes.

The ever-increasing silicon photovoltaics industry produces a huge annual production of silicon waste (2.03 × 105 tons in 2019), while lignin is one of the main waste materials in the traditional paper industry (7.0 × 107 tons annually), which lead to not only enormous wastage of resources but also serious environment pollution. Lithium-ion batteries (LIBs) are the dominating power sources for portable electronics and electric vehicles. Silicon (Si)-based material is the most promising anode choice for the next-generation high-energy-density LIBs due to its much higher capacity than the commercial graphite anode. Here, we proposed the use of these silicon and lignin waste as sustainable raw materials to fabricate high-capacity silicon/carbon (Si/C) anode materials for LIBs via a facile coprecipitation method utilizing electrostatic attracting force, followed by a thermal annealing process. The as-achieved Si/C composite featured an advanced material structure with micrometer-sized secondary particles and Si nanoparticles embedded in the carbon matrix, which could tackle the inherent challenges of Si materials, including low conductivity and large volume change during the lithiation/delithiation processes. As expected, the obtained Si/C composite displayed an initial charge capacity of 1016.8 mAh g-1, which was 3 times that of a commercial graphite anode in the state-of-the-art LIBs, as well as a high capacity retention of 74.5% at 0.2 A g-1 after 100 cycles. In addition, this Si/C composite delivered superior rate capability with a high capacity of 575.9 mAh g-1 at 2 A g-1, 63.4% of the capacity at 0.2 A g-1. The utilization of industrial Si and lignin waste provides a sustainable route for the fabrication of advanced high-capacity anode materials for the next-generation LIBs with high economic and environmental feasibility.

CRISPR/Cas9-based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi.

Filamentous fungi have several industrial, environmental, and medical applications. However, they are rarely utilized owing to the limited availability of full-genome sequences and genetic manipulation tools. Since the recent discovery of the full-genome sequences for certain industrially important filamentous fungi, CRISPR/Cas9 technology has drawn attention for the efficient development of engineered strains of filamentous fungi. CRISPR/Cas9 genome editing has been successfully applied to diverse filamentous fungi. In this review, we briefly discuss the use of common genetic transformation techniques as well as CRISPR/Cas9-based systems in filamentous fungi. Furthermore, we describe potential limitations and challenges in the practical application of genome engineering of filamentous fungi. Finally, we provide suggestions and highlight future research prospects in the area.

Biodegradable and Electroactive Regenerated Bacterial Cellulose/MXene (Ti3 C2 Tx ) Composite Hydrogel as Wound Dressing for Accelerating Skin Wound Healing under Electrical Stimulation.

Traditional wound dressings mainly participate in the passive healing processes and are rarely engaged in active wound healing by stimulating skin cell behaviors. Electrical stimulation (ES) has been known to regulate skin cell behaviors. Herein, a series of multifunctional hydrogels based on regenerated bacterial cellulose (rBC) and MXene (Ti3 C2 Tx ) are first developed that can electrically modulate cell behaviors for active skin wound healing under external ES. The composite hydrogel with 2 wt% MXene (rBC/MXene-2%) exhibits the highest electrical conductivity and the best biocompatibility. Meanwhile, the rBC/MXene-2% hydrogel presents desired mechanical properties, favorable flexibility, good biodegradability, and high water-uptake capacity. An in vivo study using a rat full-thickness defect model reveals that this rBC/MXene hydrogel exhibits a better therapeutic effect than the commercial Tegaderm film. More importantly, in vitro and in vivo data demonstrate that coupling with ES, the hydrogel can significantly enhance the proliferation activity of NIH3T3 cells and accelerate the wound healing process, as compared to non-ES controls. This study suggests that the biodegradable and electroactive rBC/MXene hydrogel is an appealing candidate as a wound dressing for skin wound healing, while also providing an effective synergistic therapeutic strategy for accelerating wound repair process through coupling ES with the hydrogel dressing.

In-situ lignin drives lytic polysaccharide monooxygenases to enhance enzymatic saccharification.

Recently low-molecular lignin was reported to activate lytic polysaccharide monooxygenases (LPMOs) to oxidize cellulose. However, whether lignin formed in cell wall can play the role as electron donor for LPMOs is still largely unknown due to the complex ultrastructure of lignocellulosic biomass. In this study, we presented a new strategy to elucidate in-situ lignin function in LPMOs reaction. A lignocellulosic mimicking model was used as substrate, which was equipped with a polysaccharide template of self-assembled bacterial cellulose film and synthesized lignin. Remarkably, it has been demonstrated that lignin polymer deposited on cellulose can reduce LPMOs in-situ for cellulose oxidation and then boost cellulose hydrolysis, and the cellulose conversion ratio of the mimicked lignocellulosic film was increased by 26.0%. More importantly, lignin in-situ might exceed the well-known reductant of ascorbic acid to drive LPMOs for cellulase enzymatic hydrolysis with equivalent cellulose oxidation efficiency and extremely lower H2O2 generation, avoiding the inactivation of enzymes. The maximum H2O2 yield from lignin-driven LPMO reaction was 75.8% lower than that from ascorbic acid-driven reaction. Therefore, by using the lignocellulosic mimicking model, we have elucidated the function of in-situ lignin in boosting enzymatic hydrolysis. Such understanding could significantly promote current utilization of LPMOs in lignocellulosic biorefinery.

Mechanism-Guided Design of Highly Efficient Protein Secretion and Lipid Conversion for Biomanufacturing and Biorefining.

Bacterial protein secretion represents a significant challenge in biotechnology, which is essential for the cost-effective production of therapeutics, enzymes, and other functional proteins. Here, it is demonstrated that proteomics-guided engineering of transcription, translation, secretion, and folding of ligninolytic laccase balances the process, minimizes the toxicity, and enables efficient heterologous secretion with a total protein yield of 13.7 g L-1. The secretory laccase complements the biochemical limits on lignin depolymerization well in Rhodococcus opacus PD630. Further proteomics analysis reveals the mechanisms for the oleaginous phenotype of R. opacus PD630, where a distinct multiunit fatty acid synthase I drives the carbon partition to storage lipid. The discovery guides the design of efficient lipid conversion from lignin and carbohydrate. The proteomics-guided integration of laccase-secretion and lipid production modules enables a high titer in converting lignin-enriched biorefinery waste to lipid. The fundamental mechanisms, engineering components, and design principle can empower transformative platforms for biomanufacturing and biorefining.

Combinatorial pretreatment and fermentation optimization enabled a record yield on lignin bioconversion.

BACKGROUND: Lignin valorization has recently been considered to be an essential process for sustainable and cost-effective biorefineries. Lignin represents a potential new feedstock for value-added products. Oleaginous bacteria such as Rhodococcus opacus can produce intracellular lipids from biodegradation of aromatic substrates. These lipids can be used for biofuel production, which can potentially replace petroleum-derived chemicals. However, the low reactivity of lignin produced from pretreatment and the underdeveloped fermentation technology hindered lignin bioconversion to lipids. In this study, combinatorial pretreatment with an optimized fermentation strategy was evaluated to improve lignin valorization into lipids using R. opacus PD630. RESULTS: As opposed to single pretreatment, combinatorial pretreatment produced a 12.8-75.6% higher lipid concentration in fermentation using lignin as the carbon source. Gas chromatography-mass spectrometry analysis showed that combinatorial pretreatment released more aromatic monomers, which could be more readily utilized by lignin-degrading strains. Three detoxification strategies were used to remove potential inhibitors produced from pretreatment. After heating detoxification of the lignin stream, the lipid concentration further increased by 2.9-9.7%. Different fermentation strategies were evaluated in scale-up lipid fermentation using a 2.0-l fermenter. With laccase treatment of the lignin stream produced from combinatorial pretreatment, the highest cell dry weight and lipid concentration were 10.1 and 1.83 g/l, respectively, in fed-batch fermentation, with a total soluble substrate concentration of 40 g/l. The improvement of the lipid fermentation performance may have resulted from lignin depolymerization by the combinatorial pretreatment and laccase treatment, reduced inhibition effects by fed-batch fermentation, adequate oxygen supply, and an accurate pH control in the fermenter. CONCLUSIONS: Overall, these results demonstrate that combinatorial pretreatment, together with fermentation optimization, favorably improves lipid production using lignin as the carbon source. Combinatorial pretreatment integrated with fed-batch fermentation was an effective strategy to improve the bioconversion of lignin into lipids, thus facilitating lignin valorization in biorefineries.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study.

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass, is discovered to promote the degradation of Azo dye by white-rot fungus Irpex lacteus CD2 in the lignin/dye/fungus system. Shotgun proteomics technique was used to understand degradation mechanism at the protein level for the lignin/dye/fungus system. Our proteomics study can identify about two thousand proteins (one third of the predicted white-rot fungal proteome) in a single experiment, as one of the most powerful proteomics platforms to study the fungal system to date. The study shows a significant enrichment of oxidoreduction functional category under the dye/lignin combined treatment. An in vitro validation is performed and supports our hypothesis that the synergy of Fenton reaction and manganese peroxidase might play an important role in DR5B dye degradation. The results could guide the development of effective bioremediation strategies and efficient lignocellulosic biomass conversion.

Genomic and molecular mechanisms for efficient biodegradation of aromatic dye.

Understanding the molecular mechanisms for aromatic compound degradation is crucial for the development of effective bioremediation strategies. We report the discovery of a novel phenomenon for improved degradation of Direct Red 5B azo dye by Irpex lacteus CD2 with lignin as a co-substrate. Transcriptomics analysis was performed to elucidate the molecular mechanisms of aromatic degradation in white rot fungus by comparing dye, lignin, and dye/lignin combined treatments. A full spectrum of lignin degradation peroxidases, oxidases, radical producing enzymes, and other relevant components were up-regulated under DR5B and lignin treatments. Lignin induced genes complemented the DR5B induced genes to provide essential enzymes and redox conditions for aromatic compound degradation. The transcriptomics analysis was further verified by manganese peroxidase (MnP) protein over-expression, as revealed by proteomics, dye decolorization assay by purified MnP and increased hydroxyl radical levels, as indicated by an iron reducing activity assay. Overall, the molecular and genomic mechanisms indicated that effective aromatic polymer degradation requires synergistic enzymes and radical-mediated oxidative reactions to form an effective network of chemical processes. This study will help to guide the development of effective bioremediation and biomass degradation strategies.

Exploration of Natural Biomass Utilization Systems (NBUS) for advanced biofuel--from systems biology to synthetic design.

Efficient degradation and utilization of lignocellulosic biomass remains a challenge for sustainable and affordable biofuels. Various natural biomass utilization systems (NBUS) evolved the capacity to combat the recalcitrance of plant cell walls. The study of these NBUS could enable the development of efficient and cost-effective biocatalysts, microorganisms, and bioprocesses for biofuels and bioproducts. Here, we reviewed the recent research progresses for several NBUS, ranging from single cell microorganisms to consortiums such as cattle rumen and insect guts. These studies aided the discovery of biomass-degrading enzymes and the elucidation of the evolutionary and functional relevance in these systems. In particular, advances in the next generation 'omics' technologies offered new opportunities to explore NBUS in a high-throughput manner. Systems biology helped to facilitate the rapid biocatalyst discovery and detailed mechanism analysis, which could in turn guide the reverse design of engineered microorganisms and bioprocesses for cost-effective and efficient biomass conversion.