Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-ß) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-ß production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Rotavirus Viroplasm Biogenesis Involves Microtubule-Based Dynein Transport Mediated by an Interaction between NSP2 and Dynein Intermediate Chain.

Rotaviruses are the causative agents of severe and dehydrating gastroenteritis in children, piglets, and many other young animals. They replicate their genomes and assemble double-layered particles in cytoplasmic electron-dense inclusion bodies called "viroplasms." The formation of viroplasms is reportedly associated with the stability of microtubules. Although material transport is an important function of microtubules, whether and how microtubule-based transport influences the formation of viroplasms are still unclear. Here, we demonstrate that small viroplasms move and fuse in living cells. We show that microtubule-based dynein transport affects rotavirus infection, viroplasm formation, and the assembly of transient enveloped particles (TEPs) and triple-layered particles (TLPs). The dynein intermediate chain (DIC) is shown to localize in the viroplasm and to interact directly with nonstructural protein 2 (NSP2), indicating that the DIC is responsible for connecting the viroplasm to dynein. The WD40 repeat domain of the DIC regulates the interaction between the DIC and NSP2, and the knockdown of the DIC inhibited rotaviral infection, viroplasm formation, and the assembly of TEPs and TLPs. Our findings show that rotavirus viroplasms hijack dynein transport for fusion events, required for maximal assembly of infectious viral progeny. This study provides novel insights into the intracellular transport of viroplasms, which is involved in their biogenesis. IMPORTANCE Because the viroplasm is the viral factory for rotavirus replication, viroplasm formation undoubtedly determines the effective production of progeny rotavirus. Therefore, an understanding of the virus-host interactions involved in the biogenesis of the viroplasm is critical for the future development of prophylactic and therapeutic strategies. Previous studies have reported that the formation of viroplasms is associated with the stability of microtubules, whereas little is known about its specific mechanism. Here, we demonstrate that rotavirus viroplasm formation takes advantage of microtubule-based dynein transport mediated by an interaction between NSP2 and the DIC. These findings provide new insight into the intracellular transport of viroplasms.

The Emerging Biotherapeutic Agent: Akkermansia.

The human gastrointestinal tract (GIT) is a well-recognized hub of microbial activities. The microbiota harboring the mucus layer of the GIT act as a defense against noxious substances, and pathogens including Clostridium difficile, Enterococcus faecium, Escherichia coli, Salmonella Typhimurium. Toxins, pathogens, and antibiotics perturb the commensal floral composition within the GIT. Imbalanced gut microbiota leads to dysbiosis, manifested as diseases ranging from obesity, diabetes, and cancer to reduced lifespan. Among the bacteria present in the gut microbiome, the most beneficial are those representing Firmicutes and Bacteroidetes. Recent studies have revealed the emergence of a novel biotherapeutic agent, Akkermansia, which is instrumental in regaining eubiosis and conferring various health benefits.

Cloning, Expression and Characterization of Two Beta Carbonic Anhydrases from a Newly Isolated CO2 Fixer, Serratia marcescens Wy064.

Bacterial strains from karst landform soil were enriched via chemostat culture in the presence of sodium bicarbonate. Two chemolithotrophic strains were isolated and identified as Serratia marcescens Wy064 and Bacillus sp. Wy065. Both strains could grow using sodium bicarbonate as the sole carbon source. Furthermore, the supplement of the medium with three electron donors (Na2S, NaNO2, and Na2S2O3) improved the growth of both strains. The activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) could be detected in the crude enzyme of strain Wy064, implying that the strain Wy064 might employ Calvin cycle to fix CO2. S. marcescens genome mining revealed four potential CA genes designated CA1-CA4. The proteins encoded by genes CA1-3 were cloned and expressed in Escherichia coli. The purified recombinant enzymes of CA1 and CA3 exhibited CO2 hydration activities, whereas enzyme CA2 was expressed in inclusion bodies. A CO2 hydration assay demonstrated that the specific activity of CA3 was significantly higher than that of CA1. The maximum CO2 hydration activities for CA1 and CA3 were observed at pH 7.5 and 40 °C. The activities of CA1 and CA3 were significantly enhanced by several metal ions, especially Zn2+, which resulted in 21.1-fold and 26.1-fold increases of CO2 hydration activities, respectively. The apparent K m and V max for CO2 as substrate were 27 mM and 179 WAU/mg for CA1, and 14 mM and 247 WAU/mg for CA3, respectively. Structure modeling combined with sequence analysis indicated that CA1 and CA3 should belong to the Type II ß-CA.

Importance of a Laccase Gene (Lcc1) in the Development of Ganoderma tsugae.

In this study, a novel laccase gene (Lcc1) from Ganoderma tsugae was isolated and its functions were characterized in detail. The results showed that Lcc1 has the highest expression activity during mycelium development and fruit body maturation based on the analysis of Lcc1 RNA transcripts at different developmental stages of G. tsugae. To investigate the exact contribution of Lcc1 to mycelium and fruit body development in G. tsugae, Lcc1 transgenic strains were constructed by targeted gene replacement and over-expression approaches. The results showed that the lignin degradation rate in Lcc1 deletion mutant was much lower than the degradation efficiency of the wild-type (WT), over-expression and rescue strains. The lignin degradation activity of G. tsugae is dependent on Lcc1 and the deletion of Lcc1 exerted detrimental influences on the development of mycelium branch. Furthermore, the study uncovered that Lcc1 deletion mutants generated much shorter pale grey fruit bodies, suggesting that Lcc1 contributes directly to pigmentation and stipe elongation during fruit body development in G. tsugae. The information obtained in this study provides a novel and mechanistic insight into the specific role of Lcc1 during growth and development of G. tsugae.

Efficient (3S)-Acetoin and (2S,3S)-2,3-Butanediol Production from meso-2,3-Butanediol Using Whole-Cell Biocatalysis.

(3S)-Acetoin and (2S,3S)-2,3-butanediol are important platform chemicals widely applied in the asymmetric synthesis of valuable chiral chemicals. However, their production by fermentative methods is difficult to perform. This study aimed to develop a whole-cell biocatalysis strategy for the production of (3S)-acetoin and (2S,3S)-2,3-butanediol from meso-2,3-butanediol. First, E. coli co-expressing (2R,3R)-2,3-butanediol dehydrogenase, NADH oxidase and Vitreoscilla hemoglobin was developed for (3S)-acetoin production from meso-2,3-butanediol. Maximum (3S)-acetoin concentration of 72.38 g/L with the stereoisomeric purity of 94.65% was achieved at 24 h under optimal conditions. Subsequently, we developed another biocatalyst co-expressing (2S,3S)-2,3-butanediol dehydrogenase and formate dehydrogenase for (2S,3S)-2,3-butanediol production from (3S)-acetoin. Synchronous catalysis together with two biocatalysts afforded 38.41 g/L of (2S,3S)-butanediol with stereoisomeric purity of 98.03% from 40 g/L meso-2,3-butanediol. These results exhibited the potential for (3S)-acetoin and (2S,3S)-butanediol production from meso-2,3-butanediol as a substrate via whole-cell biocatalysis.

Characterization of a novel polysaccharide isolated from Phyllanthus emblica L. and analysis of its antioxidant activities.

Phyllanthus emblica L. is a tropical deciduous tree producing edible berries with potential medicinal value. In this study, a novel water-soluble phyllanthus emblica polysaccharide (PEP) from the berries was isolated by precipitation and purification, and analyzed for its structure features. The results showed that PEP was a α-pyran acidic heteropolysaccharide with a molecular weight of 1.31 × 105 Da, which included galacturonic acid, galactose, rhamnose, and arabinose with a molar ratio of 3.21:6.59:1:0.23. Furthermore, the antioxidant activities of PEP were determined and showed remarkable antioxidant capacities in DPPH, superoxide anion- and hydroxyl-radical scavenging, ferric-reducing antioxidant power, and lipid peroxidation inhibition. This work indicated that PEP as a natural antioxidant agent from the berries of Phyllanthus emblica L. had potential application for developing valuable nutraceutical in food industry.

Molecular analysis and biochemical characteristics of degenerated strains of Cordyceps militaris.

Cordyceps militaris has commercially been cultivated, but its degenerated subcultures have gradually resulted in the reduced production. In this study, the biological characteristics and DNA change of degenerated strains of C. militaris were analyzed in detail. The results showed that the degenerated strains exhibited the lower growth rate, and the deficiency in fruit body formation and pigment production. The degradation of strains was not attributable to DNA changes identified by RAPD and SRAP. Compared to normal strains, the biochemical indexes of degradation strains and normal strains showed that the carotenoid content of degradation strains was significantly lower, the activities of cellulase and amylase of degradation strains were slight lower, and the EPS content was lower, but the IPS was higher. All these results suggested that the degradation of C. militaris may be caused by the inhibition or in harmony of metabolite synthesis involved in the metabolic regulation, which should be further verified.

Engineered Serratia marcescens for efficient (3R)-acetoin and (2R,3R)-2,3-butanediol production.

(3R)-Acetoin and (2R,3R)-2,3-butanediol are important pharmaceutical intermediates. However, until now, the quantity of natural microorganisms with the ability to produce single configuration of optically pure (3R)-acetoin and (2R,3R)-2,3-butanediol is rare. In this study, a meso-2,3-butanediol dehydrogenase encoded by the slaC gene from Serratia marcescens MG1 was identified for meso-2,3-butanediol and (2S,3S)-2,3-butanediol biosynthesis. Inactivation of the slaC gene could significantly decrease meso-2,3-butanediol and (2S,3S)-2,3-butanediol and result in a large quantity of (3R)-acetoin accumulation. Furthermore, a (2R,3R)-2,3-butanediol dehydrogenase encoded by the bdhA gene from Bacillus subtilis 168 was introduced into the slaC mutant strain of Serratia marcescens MG1. Excess (2R,3R)-2,3-butanediol dehydrogenase could accelerate the reaction from (3R)-acetoin to (2R,3R)-2,3-butanediol and lead to (2R,3R)-2,3-butanediol accumulation. In fed-batch fermentation, the excess (2R,3R)-2,3-butanediol dehydrogenase expression strain could produce 89.81 g/l (2R,3R)-2,3-butanediol with a productivity of 1.91 g/l/h at 48 h. These results provided potential applications for (3R)-acetoin and (2R,3R)-2,3-butanediol production.

A new NAD(H)-dependent meso-2,3-butanediol dehydrogenase from an industrially potential strain Serratia marcescens H30.

The budC gene coding for a new meso-2,3-butanediol dehydrogenase (BDH) from Serratia marcescens H30 was cloned and expressed in Escherichia coli BL21(DE3), purified, and characterized for its properties. The recombinant BDH with a molecular weight of 27.4 kDa exhibited a reversible transformation between acetoin and 2,3-butanediol. In the presence of NADH, BDH could catalyze the reduction of diacetyl and (3R)-acetoin to (3S)-acetoin and meso-2,3-butanediol, respectively, while (3S)-acetoin as a substrate could be further transformed into (2S, 3S)-2,3-butanediol at pH 9.0. For diol oxidation reactions, (3R)-acetoin and (3S)-acetoin were obtained when meso-2,3-butanediol and (2S,3S)-2,3-butanediol were used as the substrates with BDH and NAD(+). (2R,3R)-2,3-butanediol was not a substrate for the BDH at all. The low K m value (4.1 mM) in meso-2,3-butanediol oxidation reaction and no activity for diacetyl, acetoin, and 2,3-butanediol as the substrates with NADP(+)/NADPH suggested that the budC gene product belongs to a NAD(H)-dependent meso-2,3-BDH. Maximum activities for diacetyl and (3S/3R)-acetoin reduction were observed at pH 8.0 and pH 5.0 while for meso-2,3-butanediol oxidation it was pH 8.0. However, the optimum temperature for oxidation and reduction reactions was about 40 °C. In addition, the BDH activity for meso-2,3-butanediol oxidation was enhanced in the presence of Fe(2+) and for diacetyl and (3S/3R)-acetoin reduction in the presence of Mg(2+) and Mn(2+), while several metal ions inhibited its activity, particularly Fe(3+) for reduction of diacetyl and acetoin. Sequence analysis showed that the BDH from S. marcescens H30 possessed two conserved sequences including the coenzyme binding motif (GxxxGxG) and the active-site motif (YxxxK), which are present in the short-chain dehydrogenase/reductase superfamily.

Cloning, expression and characterization of glycerol dehydrogenase involved in 2,3-butanediol formation in Serratia marcescens H30.

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation-reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa based on ligninolytic enzymes.

A novel breeding strategy for new strains of Hypsizygus marmoreus and Grifola frondosa using ligninolytic enzymes as markers was evaluated with the detection and analysis of activities and composition of 15 edible fungi. The results showed that the activity and composition of ligninolytic enzyme system varied in response to changes of fungal strains. By analyzing the growth rate of mycelia and their ability to produce ligninolytic enzymes, H. marmoreus and P. geesteranus, G. frondosa and P. sajor-caju were screened for further study. Three colonies of 26 regenerated colonies of H. marmoreus and P. geesteranus protoplast fusion and one colony of 48 regenerated colonies of G. frondosa and P. sajor-caju were selected respectively. At the same time, these four strains were identified using RAPD and ISSR molecular markers. The results showed that the strains HM5G1 and PS7F1 are new strains and have low similarity to parental strains H. marmoreus and G. frondosa. These results are supported by the results of antagonism tests. These two fusants were significantly higher in their ligninolytic enzyme activity than H. marmoreus and G. frondosa. The growth rates of strains HM5G1and PS7F1 were also noticeably higher than those of H. marmoreus and G. frondosa, by 1.36 and 1.5 times respectively. The biological efficiency of the strain HM5G1 was 11.5% higher than that of the parental strain H. marmoreus. This work suggests that it is an efficient way of breeding new strains to use the decolorization of ligninolytic enzymes as a preliminary screening marker.

Effects of Geo-Stress and Natural Fracture Strength on Hydraulic Fracture Propagation in a Deep Fractured Tight Sandstone Reservoir.

Hydraulic fracturing technology has been widely used in the tight reservoir reconstruction. Unfortunately, with the deepening of mining depth and the increase of geo-stress, the propagation mechanism of medium-pressure fractures in the reservoir is significantly different from that of conventional shallow reservoirs. Based on the combined finite discrete element method, this paper conducts numerical simulation research on deep tight sandstone reservoirs in the west. The discrete fracture network modeling method is used to establish a tight sandstone reservoir model with natural bedding, and the influence of geo-stress difference and natural fracture strength on hydraulic fracture propagation law in a high geo-stress environment is discussed in detail. The results show that the difference between geo-stress and the strength of natural fractures has a significant effect on the shape and expansion of hydraulic fractures under the high geo-stress conditions. The greater the difference in ground stress, the more obvious the tendency of the main fractures of the reservoir, and the shorter the branch fractures. With the increase of natural fracture strength, the changes in propagation pressure, fracture length, area, and width, which can be fitted with a linear function with a goodness of fit as high as 0.99. In addition, the morphological results of hydraulic fractures in the simulation are not only affected by the constitutive parameters of the model but also may be affected by the randomness of the natural fracture network, thus, showing a certain degree of dispersion. Therefore, it is extremely necessary to build a reservoir fracturing model in a specific area based on more detailed geological monitoring data to guide actual construction. The above achievements have certain reference significance for the field operation of deep tight sandstone reservoirs.

Identification of two novel B-cell epitopes located on the spike protein of swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging alpha-coronavirus that causes diarrhea in piglets and results in serious economic losses. During SADS-CoV infection, the spike protein (S) serves as a crucial structural component of the virion, interacting with receptors and eliciting the production of neutralizing antibodies. Due to the potential risk of zoonotic transmission of SADS-CoV, the identification and screening of epitopes on the S glycoproteins will be crucial for development of sensitive and specific diagnostic tools. In this study, we immunized BALB/c mice with recombinant SADS-CoV S trimer protein and generated two S1-specific monoclonal antibodies (mAbs): 8D6 and 6E9, which recognized different linear B-cell epitopes. The minimal fragment recognized by mAb 8D6 was mapped to 311NPDQRD316, the minimal fragment recognized by mAb 6E9 was mapped to 492ARFVDRL498. Homology analysis of the regions corresponding to 13 typical strains of different SADS-CoV subtypes showed high conservation of these two epitopes. These findings contribute to a deeper understanding of the structure of the SADS-CoV S protein, which is valuable for vaccine design and holds potential for developing diagnostic methods to detect SADS-CoV.

IFITM3 restricts porcine deltacoronavirus infection by targeting its Spike protein.

The discovery of antiviral molecules is crucial for controlling porcine deltacoronavirus (PDCoV). Previous studies have provided evidence that the IFN-inducible transmembrane protein 3 (IFITM3), which is coded by an interferon-stimulated gene, prevents the infections of a number of enveloped viruses. Nevertheless, the involvement of IFITM3 in PDCoV infection remains unexplored. In this study, it was observed that the overexpression of IFITM3 successfully restrictes the infection of PDCoV in cell cultures. Conversely, the suppression of IFITM3 facilitates the infection of PDCoV in IPI-2I and IPEC-J2 cells. Further studies revealed that IFITM3 limits the attachment phase of viral infection by interacting with the S1 subunit of the PDCoV Spike (S) protein. In addition, IFITM3 is verified as a member of the CD225 family, the GxxxG conserved motif of this family is important for it to limit PDCoV infection. In summary, this study reveals the mechanism of IFITM3 as an antiviral molecule to inhibit PDCoV infection, and also provides theoretical supports for screening effective anti-PDCoV drugs.

Correction: Liu et al. Cholesterol 25-Hydroxylase Suppresses Swine Acute Diarrhea Syndrome Coronavirus Infection by Blocking Spike Protein-Mediated Membrane Fusion. Viruses 2023, 15, 2406.

In the original publication [...].

Rotavirus infection inhibits SLA-I expression on the cell surface by degrading ß2 M via ERAD-proteasome pathway.

Group A Rotavirus (RVA) is a major cause of diarrhea in infants and piglets. ß2-microglobulin (ß2 M), encoded by the B2M gene, serves as a crucial subunit of the major histocompatibility complex class I (MHC-I) molecules. ß2 M is indispensable for the transport of MHC-I to the cell membrane. MHC-I, also known as swine leukocyte antigen class I (SLA-I) in pigs, presents viral antigens to the cell surface. In this study, RVA infection down-regulated ß2 M expression in both porcine intestinal epithelial cells-J2 (IPEC-J2) and MA-104 cells. RVA infection did not down-regulate the mRNA level of the B2M gene, indicating that the down-regulation of ß2 M occurred on the protein level. Mechanismly, RVA infection triggered ß2 M aggregation in the endoplasmic reticulum (ER) and enhanced the Lys48 (K48)-linked ubiquitination of ß2 M, leading to the degradation of ß2 M through ERAD-proteasome pathway. Furthermore, we found that RVA infection significantly impeded the level of SLA-I on the surface, and the overexpression of ß2 M could recover its expression. In this study, our study demonstrated that RVA infection degrades ß2 M via ERAD-proteasome pathway, consequently hampering SLA-I expression on the cell surface. This study would enhance the understanding of the mechanism of how RVA infection induces immune escape.

Analysis and modification of central carbon metabolism in Hypsizygus marmoreus for improving mycelial growth performance and fruiting body yield.

Hypsizygus marmoreus is one of the main industrially cultivated varieties of edible fungi, with a delicious taste and high nutritional value. However, the long harvest period of 130-150 days greatly limits its large-scale expansion. This study aimed to investigate the effects of central carbon metabolism (CCM) on the mycelial growth performance and fruiting body formation of H. marmoreus. Nine edible fungi with different harvest periods were collected and used to evaluate their intracellular carbon metabolic differences in the CCM, which revealed that the imbalanced distribution of intracellular carbon metabolic levels in the CCM of H. marmoreus might be one of the key factors resulting in a slow mycelial growth rate and a long harvest period. Further analysis by three strategies, including metabolomics, adaptation of different carbon sources, and chemical interference, confirmed that low carbon flux into the pentose phosphate pathway (PPP) limited the supply of raw materials, reduced power, and thus influenced the mycelial growth of H. marmoreus. Furthermore, four transformants with increased expression levels of glucose-6-phosphate dehydrogenase (G6PDH), a key rate-limiting enzyme in the PPP of H. marmoreus, were developed and showed more extracellular soluble protein secretion and higher sugar assimilation rates, as well as improved mycelial growth rates in bottle substrate mixtures. Finally, cultivation experiments indicated that the maturation periods of the fruiting body with ~4-5 days in advance and the maximum fruiting body yield of 574.8 g per bag with an increase of 7.4% were achieved by improving the G6PDH expression level of the PPP in H. marmoreus. This study showed that CCM played an important role in the mycelial growth and development of H. marmoreus, which provided new insights for future advancements in cultivating and breeding edible fungi.