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This study was performed to investigate the fermentation efficiency of Lactobacillus acidophilus and compare the effects of dietary soybean meal (SM) or L. acidophilus-fermented SM (LASM) on turbot. Two hundred and seventy juvenile turbots ( 13.50 ± 0.13 g ) were randomly divided into three treatments fed with a fishmeal-based diet (CNT) and two experimental diets with 45% fishmeal protein replaced by SM or LASM for 8 weeks. The results showed that fermentation significantly improved the essential amino acid profile, increased the lactic acid content, and reduced the antinutritional factor level in SM. Inferior growth of fish was observed in the SM group after the feeding trial, while the LASM group exhibited comparable performance to the CNT group. Activities of intestinal digestive enzymes (trypsin and diastase) and immune-related enzymes (alkaline phosphatase, acid phosphatase, and lysozyme) decreased significantly in the SM group, while no significant reduction was found in the LASM group compared to the CNT group. Dietary LASM alleviated SM-induced intestinal pathological disruption with higher villus and normal lamina propria width. Dietary LASM positively regulated the intestinal microbiota, making the overall profile more coherent with that in the CNT group. Spearman’s correlation analysis revealed that the altered intestinal microbiota was closely linked to the digestive enzyme activities. Collectively, this study indicated that L. acidophilus fermentation significantly improved the nutritional quality of SM and relieved SM-induced adverse effects on turbot, in terms of growth, intestinal digestive and immune-related enzyme activities, morphology, and microbiota.

Over-substitution of fishmeal with soybean meal (SBM) commonly induces inferior growth and intestinal dysfunction in fish. This study aims to evaluate whether dietary gamma-aminobutyric acid (GABA) could ameliorate the adverse effects in turbot fed a high-SBM diet (HSD). Two hundred and seventy turbots were randomly divided into three treatment groups including turbots fed on a control diet (CNT, containing 60% fishmeal), an HSD (with 45% fishmeal protein replaced by SBM), and an HSD supplemented with GABA (160 mg kg

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

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.

Itaconic acid is a value-added organic acid that is widely applied in industrial production. It can be converted from citric acid by some microorganisms including Aspergillus terreus and Aspergillus niger. Because of high citric acid production (more than 200 g/L), A. niger strains may be developed into powerful itaconic acid-producing microbial cell factories. In this study, industrial citric acid-producing strain A. niger YX-1217, capable of producing 180.0–200.0 g/L, was modified to produce itaconic acid by metabolic engineering. A key gene cadA encoding aconitase was expressed in A. niger YX-1217 under the control of three different promoters. Analyses showed that the PglaA promoter resulted in higher levels of gene expression than the PpkiA and PgpdA promoters. Moreover, the synthesis pathway of itaconic acid was extended by introducing the acoA gene, and the cadA gene, encoding aconitate decarboxylase, into A. niger YX-1217 under the function of the two rigid short-peptide linkers L1 or L2. The resulting recombinant strains L-1 and L-2 were induced to produce itaconic acid in fed-batch fermentations under three-stage control of agitation speed. After fermentation for 104 h, itaconic acid concentrations in the recombinant strain L-2 culture reached 7.2 g/L, which represented a 71.4% increase in itaconic acid concentration compared with strain Z-17 that only expresses cadA. Therefore, co-expression of acoA and cadA resulted in an extension of the citric acid metabolic pathway to the itaconic acid metabolic pathway, thereby increasing the production of itaconic acid by A. niger.

Background Aspergillus niger is a highly important industrial microorganism because of its amazing capacity to produce citric acid (CA). To explore the metabolic mechanism and physiological phenotype associated with high CA productivity, the transcriptomes of high CA-producing A. niger YX-1217 and degenerative strain YX-1217G were investigated using A. niger ATCC1015 as a control. Results These strains showed distinct transcriptional differences in CA production. By contrast, the genes encoding glycoside hydrolases, aspartyl endoproteases, and carboxypeptidases were unusually upregulated in CA-producing strain YX-1217, which involved the carbohydrate hydrolysis and polypeptide degradation pathways, and should be related to its powerful capacity to utilize cornmeal fluidified liquid as raw material for the production of CA. In central metabolism of YX-1217, gene 9.735.1, which encodes glyceraldehyde 3-phosphate dehydrogenase, and two transcriptionally outstanding genes, 6000119 (An15g01920) and 3.2152.1 (An08g10920) that encode citrate synthase, were upregulated, thereby ensuring CA accumulation. In addition, a relatively strong electron transport chain, a regeneration system for NAD+/NADP+, and an efficient resistance mechanism may have contributed to the high CA production rate of YX-1217. Conclusions These comparisons have shed light on the mechanism underlying high CA yield in A. niger YX-1217 as well as provide insights into the development of novel strains that produce other organic acids.

meso--Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible oxidative deamination of the d-configuration of meso-2,6-diaminopimelate (meso-DAP) and is thought to have substrate specificity toward meso-DAP. The discovery of the meso-DAPDH from Symbiobacterium thermophilum IAM14863 (StDAPDH) revealed meso-DAPDH members with broad substrate specificity. In order to elucidate the substrate-preference mechanism of StDAPDH, it is necessary to identify the key residues related to this mechanism. Our previous work suggested that the non-active-site R71 of StDAPDH was related to substrate preference. Here, we report the key roles of the non-active site on the catalysis of StDAPDH. In order to explore the mechanism through which non-active-site R71 only affected the amination activity of StDAPDH, we performed molecular dynamic simulations and investigated the functional role of R71 in the type II meso-DAPDH StDAPDH. Site-directed mutagenesis with the allelic site A69 of CgDAPDH as a target proved that when replaced by Arg at position 71 of StDAPDH, the CgA69R mutant showed higher catalytic efficiencies toward a series of 2-keto acids, ranging from 1.2- to 1.5-fold. These findings provide some guidelines for improving our understanding of the broad substrate specificity of StDAPDH.

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.

Itaconic acid is a value-added organic acid that is widely applied in industrial production. It can be converted from citric acid by some microorganisms including Aspergillus terreus and Aspergillus niger. Because of high citric acid production (more than 200 g/L), A. niger strains may be developed into powerful itaconic acid-producing microbial cell factories. In this study, industrial citric acid-producing strain A. niger YX-1217, capable of producing 180.0–200.0 g/L, was modified to produce itaconic acid by metabolic engineering. A key gene cadA encoding aconitase was expressed in A. niger YX-1217 under the control of three different promoters. Analyses showed that the PglaA promoter resulted in higher levels of gene expression than the PpkiA and PgpdA promoters. Moreover, the synthesis pathway of itaconic acid was extended by introducing the acoA gene, and the cadA gene, encoding aconitate decarboxylase, into A. niger YX-1217 under the function of the two rigid short-peptide linkers L(1) or L(2). The resulting recombinant strains L-1 and L-2 were induced to produce itaconic acid in fed-batch fermentations under three-stage control of agitation speed. After fermentation for 104 h, itaconic acid concentrations in the recombinant strain L-2 culture reached 7.2 g/L, which represented a 71.4% increase in itaconic acid concentration compared with strain Z-17 that only expresses cadA. Therefore, co-expression of acoA and cadA resulted in an extension of the citric acid metabolic pathway to the itaconic acid metabolic pathway, thereby increasing the production of itaconic acid by A. niger.

Glucosamine (GlcN) is an amine sugar, in which a hydroxyl group of glucose is replaced with an amino group. It is an important part of the polysaccharides chitin and chitosan and is highly hydrophilic. It is also an important compound required for the formation of cartilage cells and represents one of the elementary units of the cartilage matrix and joint fluid. GlcN has been widely used in food, cosmetics, health care, and pharmaceutical industries. This paper fully addresses the categories and biomanufacturing methods of GlcN, including its production by fermentation with wild-type as well as engineered microorganisms and enzymatic catalysis with a series of chitinolytic enzymes. However, GlcN is usually produced from glucose by fermentation in a coupled manner with N-acetylglucosamine (GlcNAc). Enzymatic catalysis is thus a specific pathway for production of GlcN where chitin can be directly hydrolyzed to GlcN. In industry, GlcN produced with fungal mycelium as raw materials (plant GlcN) is thought as a high-end product because of vegetarian and non-transgenosis. In our opinion, more studies should be performed in order to develop a competitive enzymatic pathway using Aspergillus niger mycelium for the preparation of high-end GlcN.

meso-Diaminopimelate dehydrogenase ( meso-DAPDH) is a good candidate for one-step synthesis of d-amino acid from 2-keto acids. Our previous research revealed the classification of meso-DAPDH family and showed that type II meso-DAPDH, such as the meso-DAPDH from Symbiobacterium thermophilum (StDAPDH), could catalyze reductive amination. In this article, seven residues of StDAPDH, which are highly conserved in each subfamily but are different between two subfamilies, were targeted to explore the relationships between structure and function. Determination of kinetic parameters showed that the amino acid residues, including P69, K159, V68, S90, V14, and V156, played very important roles in the catalytic function of StDAPDH. Molecular dynamics simulation revealed that these point mutations reduced the productive conformations by the newly formed or eliminated interactions between the residues and ligands. These results strengthen our understanding of the catalytic mechanism and evolution of meso-DAPDH and can aid future endeavors in enzyme engineering.

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.

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.

Additional file 3: Table S3. Number of clean reads and mapping ratio in three A. niger strains.

d-Amino acids exist widely in microbes, plants, animals, and food and can be applied in pharmaceutical, food, and cosmetics. Because of their widespread applications in industry, d-amino acids have recently received more and more attention. Enzymes including d-hydantoinase, N-acyl-d-amino acid amidohydrolase, d-amino acid amidase, d-aminopeptidase, d-peptidase, l-amino acid oxidase, d-amino acid aminotransferase, and d-amino acid dehydrogenase can be used for d-amino acids synthesis by kinetic resolution or asymmetric amination. In this review, the distribution, industrial applications, and enzymatic synthesis methods are summarized. And, among all the current enzymatic methods, d-amino acid dehydrogenase method not only produces d-amino acid by a one-step reaction but also takes environment and atom economics into consideration; therefore, it is deserved to be paid more attention.