Transcriptomics and Metabolomics Analysis of Sclerotium rolfsii Fermented with Differential Carbon Sources.

Scleroglucan is obtained from Sclerotium rolfsii and is widely used in many fields. In this study, transcriptomics combined with metabolomics were used to study the global metabolites and gene changes. The results of the joint analysis showed that the DEGs (differentially expressed genes) and DEMs (differentially expressed metabolites) of SEPS_48 (fermented with sucrose as a carbon source for 48 h) and GEPS_48 (fermented with glucose as a carbon source for 48 h) comparison groups were mainly related to cell metabolism, focusing on carbohydrate metabolism, amino acid metabolism, and amino sugar and nucleoside sugar metabolism. We therefore hypothesized that the significant differences in these metabolic processes were responsible for the differences in properties. Moreover, the joint analysis provides a scientific theoretical basis for fungal polysaccharides biosynthesis and provides new insights into the effects of carbon sources on the production. As an excellent bioenergy and biological product, scleroglucan can be better applied in different fields, such as the food industry.

Interaction of acetic acid bacteria and lactic acid bacteria in multispecies solid-state fermentation of traditional Chinese cereal vinegar.

The microbial community plays an important role on the solid-state fermentation (SSF) of Chinese cereal vinegar, where acetic acid bacteria (AAB) and lactic acid bacteria (LAB) are the dominant bacteria. In this study, the top-down (in situ) and bottom-up (in vitro) approaches were employed to reveal the interaction of AAB and LAB in SSF of Shanxi aged vinegar (SAV). The results of high-throughput sequencing indicates that Acetobacter pasteurianus and Lactobacillus helveticus are the predominant species of AAB and LAB, respectively, and they showed negative interrelationship during the fermentation. A. pasteurianus CGMCC 3089 and L. helveticus CGMCC 12062, both of which were isolated from fermentation of SAV, showed no nutritional competition when they were co-cultured in vitro. However, the growth and metabolism of L. helveticus CGMCC 12062 were inhibited during SSF due to the presence of A. pasteurianus CGMCC 3089, indicating an amensalism phenomenon between these two species. The transcriptomic results shows that there are 831 differentially expressed genes (|log2 (Fold Change)| > 1 and, p ≤ 0.05) in L. helveticus CGMCC 12062 under co-culture condition comparing to its mono-culture, which are mainly classified into Gene Ontology classification of molecular function, biological process, and cell composition. Of those 831 differentially expressed genes, 202 genes are up-regulated and 629 genes are down-regulated. The down-regulated genes were enriched in KEGG pathways of sugar, amino acid, purine, and pyrimidine metabolism. The transcriptomic results for A. pasteurianus CGMCC 3089 under co-culture condition reveals 529 differentially expressed genes with 393 up-regulated and 136 down-regulated, and the genes within KEGG pathways of sugar, amino acid, purine, and pyrimidine metabolism are up-regulated. Results indicate an amensalism relationship in co-culture of A. pasteurianus and L. helveticus. Therefore, this work gives a whole insight on the interaction between the predominant species in SSF of cereal vinegar from nutrient utilization, endogenous factors inhibition and the regulation of gene transcription.

Remodeling-defective GPI-anchored proteins on the plasma membrane activate the spindle assembly checkpoint.

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.

Efficient One-Step Biocatalytic Multienzyme Cascade Strategy for Direct Conversion of Phytosterol to C-17-Hydroxylated Steroids.

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. 17ß-Hydroxysteroid dehydrogenase (17ßHSD) and its native electron donor play significant roles in the 17ß-carbonyl reduction reaction of steroids. In this study, we bridged 17ßHSD 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 17ßhsd 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-ayr1S.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 17ß-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.

Metabolic profile of main organic acids and its regulatory mechanism in solid-state fermentation of Chinese cereal vinegar.

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.

Enhanced Chitin Deacetylase Production Ability of Rhodococcus equi CGMCC14861 by Co-culture Fermentation With Staphylococcus sp. MC7.

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.

Structure feature and antidepressant-like activity of a novel exopolysaccharide isolated from Marasmius androsaceus fermentation broth.

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.

GC × GC-MS analysis and hypolipidemic effects of polyphenol extracts from Shanxi-aged vinegar in rats under a high fat diet.

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.

Isolation, characterisation, and genome sequencing of Rhodococcus equi: a novel strain producing chitin deacetylase.

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.

Initial Analysis on the Characteristics and Synthesis of Exopolysaccharides from Sclerotium rolfsii with Different Sugars as Carbon Sources.

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.

Dissolution and deacetylation of chitin in ionic liquid tetrabutylammonium hydroxide and its cascade reaction in enzyme treatment for chitin recycling.

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.

A highly efficient step-wise biotransformation strategy for direct conversion of phytosterol to boldenone.

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 17ß-hydroxysteroid dehydrogenase (17ßHSD) 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.

Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms.

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.

A conserved N-terminal arginine-motif in GOLPH3-family proteins mediates binding to coatomer.

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.

Localization of Golgi-resident glycosyltransferases.

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.

Signal-mediated dynamic retention of glycosyltransferases in the Golgi.

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.