The filamentous fungus Trichoderma reesei serves as an industrial workhorse for production of cellulolytic enzymes. However, the regulatory network governing cellulase biosynthesis in T. reesei remains incompletely understood, which limits rational engineering towards obtaining of hyper-producing strains. Herein, a previously uncharacterized NDT80/PhoG family transcription factor (TrNf1) was identified and characterized as a negative regulator of cellulase expression. Deletion of TrNf1 led to increases in the concentration of total secreted protein and overall cellulase activity by up to 4.2-fold and 3.1-fold, respectively. Deletion of TrNf1 induced global transcriptional reprogramming by activating a large array of carbohydrate-active enzyme (CAZyme) genes, remodeling the transcriptional regulatory network, and upregulating key activators such as xyr1 and ace3. Overexpressing xyr1 in the TrNf1-deleting strain substantially improved overall cellulase production by achieving a 3.2-fold of increase in the extracellular cellulase activity. Importantly, the repressive function is evolutionarily conserved in Aspergillus nidulans, where the homolog of TrNf1, i.e. AnNf1, similarly suppresses cellulase production. Collectively, our findings uncover a novel negative regulatory role for an NDT80/PhoG protein in fungal cellulase production and provide a conserved target for engineering the fungi into cellulase hyperproducers.

Enzymes can modulate the gut microbiota, whose integrity significantly impacts human health. Herein, CbXyn10C, a xylanase from Caldicellulosiruptor bescii, was investigated for its health-promoting effect on high-fat diet-induced obesity in mice. CbXyn10C-catalyzed hydrolysate from wheat bran promoted proliferation of 10 gut probiotics among 13 tested in vitro. In obese mice, while feeding 5% wheat bran alone was ineffective, oral administration of CbXyn10C reduced body weight and lipid accumulation, improved glycolipid metabolic disorders, reduced systemic inflammation, and fortified intestinal barrier function. Furthermore, CbXyn10C with wheat bran alleviated HFD-induced gut microbiota dysbiosis and significantly increased the abundance of probiotics, such as Lactobacillus, Bifidobacterium, and Faecalibaculum. CbXyn10C with wheat bran greatly affected lipid metabolism, particularly steroid hormone biosynthesis and sphingolipids. Spearman analysis revealed close association between the gut microbes, their metabolites, and obesity-related indicators. In conclusion, oral co-administration of a xylanase with wheat bran represents a valuable strategy for alleviating HFD-induced obesity.

As a facultative chemolithoautotrophic bacterium, Cupriavidus necator H16 uses the Entner-Doudoroff (ED) pathway for heterotrophic growth on carbohydrates such as fructose and the Calvin cycle for lithoautotrophic carbon dioxide fixation. In a previous study, we found that an ED pathway-deficient C. necator strain can survive on fructose, but the underlying metabolic pathway remained unclear. This study aimed to elucidate the metabolic mechanism of fructose metabolism in this ED pathway-deficient C. necator strain. First, the metabolic characteristics of fructose catabolism in the deficient strain were examined. Then, the roles of glycolysis/gluconeogenesis, the Calvin shunt, and the non-oxidative pentose phosphate pathway (non-OxPPP) in the metabolism of fructose were identified through comparative transcriptomic analysis combined with 13C tracer experiments. Further growth experiments using knockout strains of key genes involved in these pathways confirmed that the non-OxPPP compensates for the blocked ED pathway to metabolize fructose and provide a precursor for the Calvin shunt, thereby driving subsequent carbon fluxes. Additionally, phosphoglycolate salvage pathways, particularly the malate cycle, are crucial for recycling glycolate-2-phosphate produced during RuBisCO-catalyzed oxidation. This study revealed a novel fructose metabolism mechanism in C. necator and highlighted its metabolic flexibility, thereby deepening our understanding of its carbon utilization strategies and providing a theoretical basis for further metabolic engineering research.

The thermophilic fungus Myceliophthora thermophila serves as a vital platform for producing cellulolytic complex enzymes. However, their efficiency still requires enhancement to meet the cost-effective demands of lignocellulosic biomass conversion. Herein, secretome analysis revealed that the cellulolytic enzyme system of M. thermophila comprises the oxidative system consisting of lytic polysaccharide monooxygenases (LPMOs) and the hydrolytic system that includes endoglucanase, cellobiohydrolase, and β-glucosidase. Both in vitro supplementation and in vivo overexpression of MtLPMOs with C1 or C1/C4 oxidizing activity enhanced the enzymatic saccharification of Avicel using M. thermophila fermentation broth, resulting in a maximum increase of 485

Detoxification of aflatoxin B-1 (AFB(1)) and zearalenone (ZEN), which pose substantial threats to both agriculture and food safety, using fungal laccases is an efficient and environmentally friendly technology. However, the difficulty in heterologous expression of fungal laccases has hindered their practical application. In this study, the expression of a Steccherinum ochraceum-originated laccase in Pichia pastoris X33 was achieved through fusion with an Aspergillus niger xylanase (AnXynB). The volumetric activity of laccase was enhanced from an undetectable level to 9,102.0 U/L in a 15-L fermenter. Molecular docking of fusion proteins with mycotoxins indicated that SoLac potentially interacts with AFB(1) and ZEN directly. Optimized conditions were established for the mediator-free catalytic transformation of AFB(1) and ZEN using the AnXynB-SoLac fusion protein, achieving transformation efficiencies of 71.0 % and 72.9 %, respectively, under 50 degrees C and pH 7.0 for 20 h. The main AFB(1) and ZEN transformation products were AFQ(1), 13-OH-ZEN-quinone, 15-OH-ZEN, and ZOM-1, respectively. Evaluation of toxicity revealed that the transformed products exhibited markedly reduced toxicity. Furthermore, AnXynB-SoLac fusion protein effectively transformed AFB(1) and ZEN in raw feed materials, including soybean meal and maize flour. This study describes a novel and feasible strategy for the production of fungal laccases that are difficult to express in P. pastoris in a food-grade manner and illustrated its promise in addressing the detoxification of AFB(1) and ZEN residues in food and feed-related matrices.

Understanding the molecular mechanisms of cellobiohydrolase I (CBHI), a key enzyme in cellulase complexes, is crucial for developing efficient enzymes for the degradation of lignocellulosic biomasses (LCB). Building on our previous discovery that Chaetomium thermophilum CBHI (C-CBH) exhibits significantly higher specific activity than Trichoderma reesei CBHI (T-CBH), systematic domain-swapping experiments were conducted to elucidate the structural determinants of catalytic efficiency in CBHI. Herein, the carbohydrate-binding modules (CBM) of the CBHIs from Trichoderma reesei (T-CBH) and Chaetomium thermophilum (C-CBH) were interchanged and to obtain two chimeric mutants TC-CBH and CT-CBH. These four CBHs were expressed in T. reesei, and the enzyme properties were analyzed. Comparative characterization revealed that while module exchange preserved native temperature/pH adaptability, it significantly altered substrate specificity and catalytic performance. The CT-CBH variant was identified as the most efficient biocatalyst, exhibiting four key advantages over T-CBH: (1) protein expression levels that far exceed those of T-CBH, (2) specific activity enhanced by 2.6-fold (734.5 U/μM vs. 282.5 U/μM on MU-cellobiose), (3) superior degradation capacities for filter paper (1.6-fold) and xylan, and (4) improved binding affinity for crystalline cellulose. These findings establish cross-species domain engineering as a viable strategy for creating high-performance cellulases, providing both mechanistic insights and practical solutions for lignocellulose degradation.

l-isoleucine, a value-added branched-chain amino acid, has been widely used in the food, feed, and pharmaceutical industries. However, the production efficiency of l-isoleucine is relatively low due to the complex and inefficient biosynthetic network of the canonical threonine pathway. Here, we report the exploitation of a concise citramalate pathway for the development of an efficient l-isoleucine producer. First, chassis strain and key genes were screened for establishment of the citramalate pathway. Subsequently, a citramalate importer was identified and applied to enhance citramalate's utilization efficiency. Finally, a plasmid-free high-l-isoleucine producer was developed by enhancement of l-isoleucine efflux, optimization of rate-limiting gene expression, and introduction of a nonoxidative glycolysis pathway. Fed-batch fermentation of the final strain in a 10-L bioreactor produced 56.6 g/L l-isoleucine with a productivity of 1.66 g/L/h, which is the highest l-isoleucine titer and productivity reported. This study paves the way for construction of efficient microbial cell factories for production of l-isoleucine and related derivatives based on the citramalate pathway.

Coccidiosis is a parasitic disease caused by Eimeria species in chickens, leading to substantial threats to the poultry industry worldwide. Vaccination has emerged as a pivotal preventive strategy, with subunit vaccines gaining significant attention due to their safety. Microneme protein 2 (MIC2) of Eimeria tenella (E. tenella) is a promising candidate antigen for coccidiosis vaccination. However, its immune protection efficiency still needs to be further improved. Here, we report that MIC2, cholera toxin B subunit (CTB) and their fusion protein CTB-MIC2 are successfully recombinant expressed and produced in Pichia pastoris (P. pastoris). Notably, CTB and CTB-MIC2 exhibit high affinity for GM1 ganglioside, whereas MIC2 does not. Furthermore, upon administration to mice, the fusion protein CTB-MIC2 triggered a more robust cellular and humoral immune response than MIC2 alone, as evidenced by inducing significantly higher levels of cytokines IFN-γ and IL-4, along with an elevated production of antigen-specific IgG antibodies. More importantly, CTB-MIC2 provided higher immune protection against E. tenella infections in chickens than MIC2. Taken together, these findings reveal that CTB fusion enhances the immunogenicity of MIC2, highlighting a promising strategy for developing effective anti-coccidial subunit vaccines.

Clostridium perfringens infection can induce necrotic enteritis and lead to significant economic loss to the chicken industry. In this study, a xylanase (CbXyn10C), which effectively promotes the growth of probiotics, and a protease, which degrades the biofilm of C. perfringens, were analyzed for their ability to alleviate C. perfringens-induced necrotic enteritis in broiler chickens. A total of 300 male AA chickens were divided into five treatment groups (control, no enzyme and no C. perfringens challenge; Cp, no enzyme, C. perfringens challenge; Xyn, CbXyn10C plus C. perfringens challenge; Xyn+Am, CbXyn10C+Amylase plus C. perfringens challenge; Xyn+Ap, CbXyn10C+Alkaline protease plus C. perfringens challenge). The C. perfringens CVCC 60102 was administered orally on a daily basis to the chickens from 14 to 20 days. In comparison with Cp, Xyn+Ap significantly reduced intestinal damage in the duodenum, jejunum, and ileum of chickens challenged with C. perfringens (p < 0.05). The enzymes, and particularly Xyn+Ap, notably enhanced the expression of key intestinal barrier genes, reduced the IL-6 level, and decreased the DAO (diamine oxidase) level. Not unexpectedly, feeding enzymes influenced the abundance of Lactobacillus and Butyricicoccus bacteria in the intestine. These results indicated that CbXyn10C and protease can be used to alleviate intestinal damage caused by C. perfringens infection.

Intestinal alkaline phosphatase (AP) detoxifies lipopolysaccharides, which has been exploited in treating infection of Gram-negative bacterial pathogens such as Escherichia coli. Orally administered AP has the potential to modulate the gut microbiota, which is in part ascribed to its ability to degrade ATP, a well-known inhibitor of gut commensal bacteria. In addition, AP can fortify the gut barrier. Therefore, we hypothesized that the enzyme might also be used to control intestinal Gram-positive pathogens such as Clostridium perfringens. Herein, broiler chickens were challenged with 3 × 108 colony-forming units of C. perfringens daily from 14 to 20 days. Low (1000 U/kg of feed) and high (5000 U/kg of feed) doses of a recombinant bovine intestinal AP (bIAP) were orally administered to the chickens throughout the study. bIAP could indeedalleviate the gut lesion and diarrhea symptom in chickens challenged with C. perfringens and reversed the decline in their growth performance. 16S rRNA gene sequencing and non-targeted metabolomics analyses revealed that bIAP could modulate the gut microbiota, which was accompanied with a change of the gut metabolites profile, the improved intestinal integrity and immunity, and an ultimate protection of the animals from C. perfingens infection. Strikingly, the RT-qPCR assay showed that the transcript levels of key tight junction proteins zonula occludens-1, Mucin 2, claudin-1, and occludin in the duodenum, jejunum, and ileum were even superior in the high dose group than those in the unchallenged group, suggestive of enhanced integrity of the intestinal barrier. Enzymes such as bIAP are, therefore, a powerful tool in modulating the gut microbiota for better health of the host animals.

Plant cell wall polysaccharides (PCWPs) serve as an abundant but recalcitrant carbon source for many microbes living in the gut of humans and animals. An adhesion to PCWPs is common in gut bacteria and can even be observed in the lactobacilli, which are supposed to promote the growth competence of these non-PCWP degraders because of the facilitated acquisition of newly released oligosaccharides. Nevertheless, the binding of molecules of lactobacilli to PCWPs and the underlying mechanisms remain largely unknown. By analyzing the transcriptome of Lactobacillus brevis grown in xylan supplemented with a xylanase, a gene was identified to encode a putative S-layer PCWP-binding protein (Lb1145). Lb1145 was predicted to have four domains, among which domains 1 and 2 were responsible for binding PCWPs. The binding was nonspecific, since structurally distinct PCWPs, e.g., cellulose, xylan, mannan, and chitin, and even lignin, were all bound by Lb1145. Both of the two N-terminal domains have a high pI, and we demonstrated that a non-enzymatic glycosylation-like process plays an important role in binding. Compared with another L. brevis surface protein, i.e., the WxL protein Lb630, Lb1145 displayed a binding preference for the phloem sieve tube in the wheat stem section. Moreover, Lb1145 could bind ten strains within the Lactobacillus, Enterococcus, Pediococcus, and Bacillus genera among the seventeen selected gut bacterial species. An analysis of the reported S-layer proteins from the Gram-positive bacteria (lactobacilli and bifidobacteria) and outer membrane proteins from the Gram-negative (Bacteroides fragilis and Prevotella intermedia) indicated that bacterial cell surface proteins with high pI values are not rare. The high pI-based and non-enzymatic glycosylation-like process-mediated binding represents a new paradigm and may be popular in gut bacterial surface proteins binding to PCWPs, with important physiological implications in growth competition in the gut microbiota.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

Mycotoxins are widely present from cereal crops to food products, posing a significant risk to food safety and causing serious health and economic losses. Therefore, it is imperative to develop a sustainable and efficient detoxification strategy to address mycotoxin contamination. Herein, a dye-decolorizing peroxidase, BaDyP, from the white-rot fungus Bjerkandera adusta was successfully expressed as a soluble form in Escherichia coli with the help of molecular chaperones. Purified recombinant BaDyP could efficiently degrade multiple mycotoxins including aflatoxin B1, zearalenone, and deoxynivalenol in the presence of mediators such as Mn2+ or 1-HBT. Meanwhile, their degradation products were identified using UPLC-MS/MS, including AFB1-diol, AFQ1, 15-OH-ZEN, HZEN, and C15H18O8. Furthermore, cell survival tests exhibited a significant reduction in the biological toxicity of these degradation products. These findings indicate that the use of dye-decolorizing peroxidase could be a promising approach for the effective removal of multiple mycotoxins in cereal crops and food products.

ABSTRACT Genetic engineering at the genomic scale provides a rapid means to evolve microbes for desirable traits. However, in many filamentous fungi, such trials are daunted by low transformation efficiency. Differentially expressed genes under certain conditions may contain important regulatory factors. Accordingly, although manipulating these subsets of genes only can largely reduce the time and labor, engineering at such a sub-genomic level may also be able to improve the microbial performance. Herein, first using the industrially important cellulase-producing filamentous fungus Trichoderma reesei as a model organism, we constructed suppression subtractive hybridization (SSH) libraries enriched with differentially expressed genes under cellulase induction (MM-Avicel) and cellulase repression conditions (MM-Glucose). The libraries, in combination with RNA interference, enabled sub-genomic engineering of T. reesei for enhanced cellulase production. The ability of T. reesei to produce endoglucanase was improved by 2.8~3.3-fold. In addition, novel regulatory genes ( tre49304 , tre120391 , and tre123541 ) were identified to affect cellulase expression in T. reesei . Iterative manipulation using the same strategy further increased the yield of endoglucanase activity to 75.6 U/mL, which was seven times as high as that of the wild type (10.8 U/mL). Moreover, using Humicola insolens as an example, such a sub-genomic RNAi-assisted strain evolution proved to be also useful in other industrially important filamentous fungi. H. insolens is a filamentous fungus commonly used to produce catalase, albeit with similarly low transformation efficiency and scarce knowledge underlying the regulation of catalase expression. By combining SSH and RNAi, a strain of H. insolens producing 28,500 ± 288 U/mL of catalase was obtained, which was 1.9 times as high as that of the parent strain. IMPORTANCE Genetic engineering at the genomic scale provides an unparalleled advantage in microbial strain improvement, which has previously been limited only to the organisms with high transformation efficiency such as Saccharomyces cerevisiae and Escherichia coli . Herein, using the filamentous fungus Trichoderma reesei as a model organism, we demonstrated that the advantage of suppression subtractive hybridization (SSH) to enrich differentially expressed genes and the convenience of RNA interference to manipulate a multitude of genes could be combined to overcome the inadequate transformation efficiency. With this sub-genomic evolution strategy, T. reesei could be iteratively engineered for higher cellulase production. Intriguingly, Humicola insolens , a fungus with even little knowledge in gene expression regulation, was also improved for catalase production. The same strategy may also be expanded to engineering other microorganisms for enhanced production of proteins, organic acids, and secondary metabolites.

AbstractChicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite‐specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well‐known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV‐SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV‐SAG1 is achieved through fermentation in a 15‐L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV‐SAG1 in P. pastoris holds promise for the development of effective recombinant anti‐coccidial subunit vaccine.

Commensal bacteria in the intestine release enzymes to degrade and ferment dietary components, producing beneficial metabolites. However, the regulatory effects of microbial-derived enzymes on the intestinal microbiota composition and the influence on host health remain elusive. Xylanase can degrade xylan into oligosaccharides, showing wide application in feed industry. To validate the immune-protective effects of xylanase, Nile tilapia was used as the model and fed with xylanase. The results showed that dietary xylanase improved the survival rate of Nile tilapia when they were challenged with Aeromonas hydrophila. The transcriptome analysis showed significant enrichment of genes related to interleukin-17d (il-17d) signaling pathway in the xylanase treatment group. High-throughput sequencing revealed that dietary xylanase altered the composition of the intestinal microbiota and directly promoted the proliferation of Allobaculum stercoricanis which could produce butyrate in vitro. Consequently, dietary xylanase supplementation increased the butyrate level in fish gut. Further experiment verified that butyrate supplementation enhanced the expression of il-17d and regenerating islet-derived 3 gamma (reg3g) in the gut. The knockdown experiment of il-17d confirmed that il-17d is necessary for butyrate to protect Nile tilapia from pathogen resistance. Flow cytometry analysis indicated that butyrate increased the abundance of IL-17D+ intestinal epithelial cells in fish. Mechanistically, butyrate functions as an HDAC3 inhibitor, enhancing il-17d expression and playing a crucial role in pathogen resistance. Dietary xylanase significantly altered the composition of intestinal microbiota and increased the content of butyrate in the intestine. Butyrate activated the transcription of il-17d in intestinal epithelial cells by inhibiting histone deacetylase 3, thereby protecting the Nile tilapia from pathogen infection. This study elucidated how microbial-derived xylanase regulates host immune function, providing a theoretical basis for the development and application of functional enzymes.

Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation. This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively. This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.