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The structure and antidepressant like activity of MEPS2 extracted from Marasmius androsaceus subjected to submerged fermentation was systematically studied. MEPS2 is a pyranoid polysaccharide composed of glucose and arabinose, which have a molar ratio of 0.56:0.08. The molecular weight was 85,944 Da. The NMR spectrum suggested the extracted MEPS2 contained uronic acid, and the glucosyl linkage was in α form, in accordance with the analysis of FT-IR spectrum. MEPS2 can considerably enhance the levels of noradrenalin (NE) and dopamine (DA) by ELISA. In addition, western blotting results indicated that MEPS2 can enhance the expression levels of TH, D2DR, and CAMKII. Furthermore, we found that AMPT, raclopride, and prazosin blocked the immobility and time-reducing effect of MEPS2. Overall, the antidepressant-like effect of MEPS2 may be involved in catecholamine synthesis and release, and TH, D2DR and CAMKII play an important role in this process.

Oxidative stress, inflammation and gut microbiota disorders can be induced by long-term high-fat diets (HFD). In order to confirm that polyphenols can improve these symptoms, polyphenols from Shanxi-aged vinegar (SAVEP) were extracted, and the components were detected by Comprehensive two-dimensional gas chromatography mass spectrometry (GC × GC-MS). 41 polyphenols include 18 phenolic acids and 17 polyphenols, which have not been reported. The mechanism of SAVEP on oxidative stress and inflammatory stress induced by HFD in rats and its regulating effect on intestinal flora disorder were studied. The results showed that SAVEP could significantly improve the lipid, inflammatory stress and oxidative stress related indicators compared with the Model group ("Model" refers to the group that successfully constructed a hyperlipidemia model by feeding HFD without any drugs or SAVEP in subsequent experiments.). In addition, SAVEP decreased the Firmicutes/Bacteroidetes ratio compared with the Model group, and elevated the relative abundance of beneficial bacteria. Conclusively, SAVEP can alleviate the oxidative stress and inflammatory stress caused by HFD, improving intestinal microbial disorders. The Spearman's correlation analysis revealed that Desulfovibrio, Lactobacillus and Akkermansia were correlated negatively with all of the inflammatory indicators, whereas Ruminococcus was the opposite. These results suggest that SAVEP may be a novel strategy against oxidative stress and inflammation, restoring the normal microbial community ecology of the gut and the treatment of metabolic syndromes.

Steroidal 17-carbonyl reduction is crucial to the production of natural bioactive steroid medicines, and boldenone (BD) is one of the important C-17-hydroxylated steroids. Although efforts have been made to produce BD through biotransformation, the challenges of the complex transformation process, high substrate costs, and low catalytic efficiencies have yet to be mastered. Phytosterol (PS) is the most widely accepted substrate for the production of steroid medicines due to its similar foundational structure and ubiquitous sources. 17β-Hydroxysteroid dehydrogenase (17βHSD) and its native electron donor play significant roles in the 17β-carbonyl reduction reaction of steroids. In this study, we bridged 17βHSD with a cofactor regeneration strategy in Mycobacterium neoaurum to establish a one-step biocatalytic carbonyl reduction strategy for the efficient biosynthesis of BD from PS for the first time. After investigating different intracellular electron transfer strategies, we rationally designed the engineered strain with the coexpression of 17βhsd and the glucose-6-phosphate dehydrogenase (G6PDH) gene in M. neoaurum. With the establishment of an intracellular cofactor regeneration strategy, the ratio of [NADPH]/[NADP(+)] was maintained at a relatively high level, the yield of BD increased from 17% (in MNR M3M-ayr1(S.c)) to 78% (in MNR M3M-ayr1&g6p with glucose supplementation), and the productivity was increased by 6.5-fold. Furthermore, under optimal glucose supplementation conditions, the yield of BD reached 82%, which is the highest yield reported for transformation from PS in one step. This study demonstrated an excellent strategy for the production of many other valuable carbonyl reduction steroidal products from natural inexpensive raw materials. IMPORTANCE Steroid C-17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for the further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residues of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicine production by Mycobacterium. This study explored a green and efficient one-step biocatalytic carbonyl reduction strategy for the direct conversion of phytosterol to C-17-hydroxylated steroids by bridging 17β-hydroxysteroid dehydrogenase with a cofactor regeneration strategy in Mycobacterium neoaurum. This work has practical value for the production of many valuable hydroxylated steroids from natural inexpensive raw materials.

Shanxi aged vinegar (SAV), a traditional Chinese cereal vinegar, is produced using solid-state fermentation (SSF) technology. Organic acids are the key flavor compounds of vinegar. However, the metabolic mechanism of organic acids during SSF process is still unclear. In this study, metatranscriptomics was used to explore the metabolic profile of main organic acids in SSF. The results show that carbon metabolism is the dominant pathway during fermentation, among which pyruvate metabolism, glycolysis and starch and sucrose metabolism associated with organic acids were the most abundant. The metabolic pathways of acetic acid and lactic acid shift from acetyl-P and pyruvate pathways at early and middle-early stages of fermentation to acetaldehyde and L-lactaldehyde pathways at later stages, respectively, and Lactobacillus and Acetobacter are the predominant microorganisms contributed to them. Temperature and acetic acid are proven to be the environmental factors that regulate the metabolic activity during SSF. This study sheds new lights on metabolism of flavor substances in the spontaneous ecosystems of traditional fermented food.

Chitin deacetylase (CDA) can hydrolyze the acetamido group of chitin polymers and its deacetylated derivatives to produce chitosan, an industrially important biopolymer. Compared with traditional chemical methods, biocatalysis by CDA is more environment-friendly and easy to control. However, most reported CDA-producing microbial strains show low CDA producing capabilities. Thus, the enhancement of CDA production has always been a challenge. In this study, we report co-culture fermentation to significantly promote the CDA production of Rhodococcus equi CGMCC14861 chitin deacetylase (ReCDA). Due to co-culture fermentation with Staphylococcus sp. MC7, ReCDA yield increased to 21.74 times that of pure culture of R. equi. Additionally, the enhancement was demonstrated to be cell-independent by adding cell-free extracts and the filtrate obtained by 10 kDa ultrafiltration of Staphylococcus sp. MC7. By preliminary characterization, we found extracellular, thermosensitive signal substances produced by Staphylococcus that were less than 10 kDa. We investigated the mechanism of promotion of ReCDA production by transcriptomic analysis. The data showed that 328 genes were upregulated and 1,258 genes were downregulated. The transcription level of the gene encoding ReCDA increased 2.3-fold. These findings provide new insights into the research of co-culture fermentation for the production of CDA and quorum sensing regulation., Graphical Abstract GRAPHICAL ABSTRACT

Newly synthesized glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive remodeling prior to transport to the plasma membrane. GPI-AP remodeling events serve as quality assurance signatures, and complete remodeling of the anchor functions as a transport warrant. Using a genetic approach in yeast cells, we establish that one remodeling event, the removal of ethanolamine-phosphate from mannose 2 via Ted1p (yPGAP5), is essential for cell viability in the absence of the Golgi-localized putative phosphodiesterase Dcr2p. While GPI-APs in which mannose 2 has not been remodeled in dcr2 ted1-deficient cells can still be delivered to the plasma membrane, their presence elicits a unique stress response. Stress is sensed by Mid2p, a constituent of the cell wall integrity pathway, whereupon signal promulgation culminates in activation of the spindle assembly checkpoint. Our results are consistent with a model in which cellular stress response and chromosome segregation checkpoint pathways are functionally interconnected.

Chitin deacetylase (CDA) can hydrolyse the acetamido group of chitin polymers to produce chitosans, which are used in various fields including the biomedical and pharmaceutical industries, food production, agriculture, and water treatment. CDA represents a more environmentally-friendly and easier to control alternative to the chemical methods currently utilised to produce chitosans from chitin; however, the majority of identified CDAs display activity toward low-molecular-weight oligomers and are essentially inactive toward polymeric chitin or chitosans. Therefore, it is important to identify novel CDAs with activity toward polymeric chitin and chitosans. In this study, we isolated the bacterium Rhodococcus equi F6 from a soil sample and showed that it expresses a novel CDA (ReCDA), whose activity toward 4-nitroacetanilide reached 19.20 U/mL/h during fermentation and was able to deacetylate polymeric chitin, colloidal chitin, glycol-chitin, and chitosan. Whole genome sequencing revealed that ReCDA is unique to the R. equi F6 genome, while phylogenetic analysis indicated that ReCDA is evolutionarily distant from other CDAs. In conclusion, ReCDA isolated from the R. equi F6 strain expands the known repertoire of CDAs and could be used to deacetylate polymeric chitosans and chitin in industrial applications.

Scleroglucan is widely used in the food and chemical industries because of its good rheological property, stability, and emulsification activity. To investigate the influence of different carbon sources on the properties and synthesis of exopolysaccharides (EPS), the three EPSs (GEPS, glucose was used as the carbon source, LEPS, lactose was used as the carbon source, and SEPS, sucrose was used as the carbon source) were determined, respectively. It was found that the yield and viscosity of exopolysaccharides were different. When sucrose and glucose were used as the carbon sources, the viscosity and yield of EPS were both higher than lactose. The scanning electron microscopy (SEM) images showed that the three EPSs had different morphologies, but the monosaccharide analysis showed that they were all composed of glucose units. Fourier transform infrared spectroscopy (FT-IR) proved that there were no additional substituents for the three EPSs. Furthermore, the high performance liquid chromatography (HPLC) results showed that SEPS and LEPS had two fractions. Through the analysis of proteomics data, there were few differences in the metabolic pathways between GEPS and SEPS, but a significant difference between LEPS and SEPS. Our study provides a theoretical basis and reference for understanding the biosynthesis of exopolysaccharides and the development of different types of EPS products.

Background Aspergillus niger mycelium waste, a by-product from citric acid fermentation is considered a good raw material to produce fungal chitosan and glucosamine (GlcN). However, its successful application has always been a challenge.Results In this work, the effects of combined pretreatment involving steam explosion and ionic liquid (IL) on mycelium waste were investigated. Results show that pretreatment of mycelium waste at 2.5 MPa for 1 min improved the GlcN hydrochloride yield by 65.49%, the release of soluble sugar reached 0.46 g/g of sample, and the deacelation degree of fungal chitosan by hydrolysis of chitin deacetylase from Rhodococcus equi CGMCC14861 (ReCDA) was from scratch and a 1.3-fold increase by combined with IL pretreatment. In addition, a recycling strategy for by-products, which promoted the fermentation of ReCDA used for fungal chitosan, was efficiently developed.Conclusion This is the first report of comprehensive utilization of Aspergillus niger mycelium waste from citric acid fermentation on the basis of steam explosion and ionic liquid pretreatment. This study demonstrates a green-like technology for the comprehensive utilization of A. niger mycelium waste from citric acid fermentation.

Collaborative microbial communities are ubiquitous in nature and exhibit appealing functions for enhanced production of natural products, which provides new possibility for biotechnology development. In this study, we bridged Mycobacterium neoaurum with Pichia pastoris to establish a step-wise biotransformation strategy for efficient biosynthesis of boldenone (BD) from phytosterol (PS). Firstly, the producing strains were rationally designed with overexpression of 3-ketosteroid-Δ1-dehydrogenase (KsdD) and 17β-hydroxysteroid dehydrogenase (17βHSD) in M. neoaurum and P. pastoris, respectively. Then, to shorten the total biotransformation process and provide reducing power, semi-batch fermentation strategy and glucose supplementation strategy were introduced at side-chain degradation stage and carbonyl reduction stage, respectively. Under the optimal transformation conditions, the productivity of BD was increased from 10% to 76% and the total biotransformation process was shortened by 41.7%, which is the shortest among the ever reported. Our results demonstrated an excellent biological strategy for production of many other valuable microbial products from bioresources.

Aspergillus niger mycelium residues, a citric acid fermentation by-product, is a raw material for fungal chitosan and glucosamine (GlcN). However, traditional chemical extraction of fungal chitosan and GlcN uses strong acid at high temperatures for long periods and has several drawbacks, including high cost and environmental pollution. In this work, the residues were pretreated by the combination of steam explosion (SE) and ionic liquid (IL) to produce fungal chitosan and GlcN hydrochloride. Following chemical extraction, GlcN hydrochloride yield increased by 65.49 ± 8.14%, and soluble sugar release reached 0.36–0.46 g/g via SE pretreatment at 2.5 MPa for 1 min. Fungal chitosan was produced via enzymatic hydrolysis. Combined SE and IL pretreatment increased deacetylation degree (1.29 ± 0.06-fold) of fungal chitosan by chitin deacetylase hydrolysis using Rhodococcus equi CGMCC14861 (ReCDA) at 37 °C for 6 h. Besides, fungal chitosan was produced using a by-product recycling strategy, promoting ReCDA fermentation, using supernatant with soluble sugars and proteins. After mixing 0.2 g by-product supernatant with 100 mL medium, ReCDA production was 1.5-fold higher than that in the control. This study demonstrates a green technology for efficient utilization of fungal chitin from A. niger mycelium residues of citric acid fermentation.

Monascus vinegar (MV) exerts beneficial effects on cardiovasular diseases. The present work explored the lipid-lowering mechanism of MV and the correlation among gut microbiota, lipid metabolism and inflammation. The results illustrated that the administration of MV ameliorated hyperlipidemia by regulating Peroxisome proliferator-activated α (PPARα)-, Nuclear factor-E2-related factor 2 (Nrf2)- and Nuclear factor κB (NF-κB)-mediated signals and modulating the gut microbiota composition in hyperlipidemia rats. Moreover, Lactobacillus, Roseburia and Lachnostridium showed positive correlation with antioxidative parameters and negative correlation with lipid metabolism and inflammation parameters. Whereas Clostridium, Desulfovibrio and Streptococcus exhibited the opposite effect. Importantly, the differences in lipid-lowering effect between high-dose of MV (HD) group and low acetic acid and lovastatin (LLA) group were evaluated, which indicated that the other components in MV could improve the lipid-lowering effect of acetic acid and lovastatin. Our findings provided a novel insight into the lipid-lowering effect of MV.

Chitin is the second most abundant renewable polymer on earth, and its deacetylated derivative, chitosan, is a highly useful biopolymer. This work studied for the first time the application of ionic liquid (IL) tetrabutylammonium hydroxide ([TBA][OH]) and chitin deacetylase from Rhodococcus equi CGMCC14861 (ReCDA) for the efficient conversion of chitin into chitosan at room temperature. Results confirmed that chitin had good solubility in 18 wt% aqueous [TBA][OH] solution at 80 ℃. In addition, efficient chitin deactylation was observed with high concentrations of [TBA][OH] exceeding 12 wt% and showing potential application in chitin conversion. ReCDA activity on chitin was activated by [TBA][OH] pretreatment. Sequential and simultaneous strategies were also compared, and the results showed that the simultaneous one-pot deacetylation provided the highest acetic acid yield of 3.78 mg/g chitin powder after 24 h. This study serves as a guide for the dissolution and deacetylation of chitin to produce high value-added chitosan products.

The COPI-interacting KXD/E motif in the C-terminal cytosolic tail of endomembrane proteins (EMPs) is a crucial Golgi retention mechanism for EMPs. KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes., We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.

Vps74p, a member of the GOLPH3 protein family, binds directly to coatomer and the cytoplasmic tails of a subset of Golgi-resident glycosyltransferases to mediate their Golgi retention. We identify a cluster of arginine residues at the N-terminal end of GOLPH3 proteins that are necessary and sufficient to mediate coatomer binding. While loss of coatomer binding renders Vps74p non-functional for glycosyltransferase retention, the Golgi membrane-binding capabilities of the mutant protein are not significantly reduced. We establish that the oligomerization status and phosphatidylinositol-4-phosphate-binding properties of Vps74p largely account for the membrane-binding capacity of the protein and identify an Arf1p–Vps74p interaction as a potential contributing factor in Vps74p Golgi membrane association.

For many glycosyltransferases, the information that instructs Golgi localization is located within a relatively short sequence of amino acids in the N-termini of these proteins comprising: the cytoplasmic tail, the transmembrane spanning region, and the stem region (CTS). Also, one enzyme may be more reliant on a particular region in the CTS for its localization than another. The predominance of these integral membrane proteins in the Golgi has seen these enzymes become central players in the development of membrane trafficking models of transport within this organelle. It is now understood that the means by which the characteristic distributions of glycosyltransferases arise within the subcompartments of the Golgi is inextricably linked to the mechanisms that cells employ to direct the flow of proteins and lipids within this organelle.

Golgi-resident glycosyltransferases are a family of enzymes that sequentially modify glycoproteins in a subcompartment-specific manner. These type II integral membrane proteins are characterized by a short cytoplasmically exposed amino-terminal tail and a luminal enzymatic domain. The cytoplasmic tails play a role in the localization of glycosyltransferases, and coat protein complex I (COPI) vesicle–mediated retrograde transport is also involved in their Golgi localization. However, the tails of these enzymes lack known COPI-binding motifs. Here, we found that Vps74p bound to a pentameric motif present in the cytoplasmic tails of the majority of yeast Golgi-localized glycosyltransferases, as well as to COPI. We propose that Vps74p maintains the steady-state localization of Golgi glycosyltransferases dynamically, by promoting their incorporation into COPI-coated vesicles.