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Chromosomal integration of exogenous genes is preferred for industrially related fermentation, as plasmid-mediated fermentation leads to extra metabolic burden and genetic instability. Moreover, with the development and advancement of genome engineering and gene editing technologies, inserting genes into chromosomes has become more convenient; integration expression is extensively utilized in microorganisms for industrial bioproduction and expected to become the trend of recombinant protein expression. However, in actual research and application, it is important to enhance the expression of heterologous genes at the host genome level. Herein, we summarized the basic principles and characteristics of genomic integration; furthermore, we highlighted strategies to improve the expression of chromosomal integration of genes and pathways in host strains from three aspects, including chassis cell optimization, regulation of expression elements in gene expression cassettes, optimization of gene dose level and integration sites on chromosomes. Moreover, we reviewed and summarized the relevant studies on the application of integrated expression in the exploration of gene function and the various types of industrial microorganism production. Consequently, this review would serve as a reference for the better application of integrated expression.

The dietary supplementation of prebiotics is considered a promising strategy for the modulation of gut microbiota. Due to the wide variety of animal models and tremendous inter-individual variability from human investigations, the prebiotic effect of fibers is often difficult to compare between studies. Here, the effects of 11 dietary fibers on human fecal microbiota were studied using an in vitro human fecal fermentation model under well-controlled conditions. All fibers showed positive regulatory effects on short chain fatty acids (SCFAs) and several beneficial bacteria, including Parabacteroides distasonis and Bifidobacterium spp. Cultures supplemented with xylo-oligosaccharide and konjac flour showed the highest SCFAs. According to regulatory effects, fibers were divided into three groups, with 13 indicator OTUs (operational taxonomic units) identified. Fecal microbiota regulated by isomalto-oligosaccharide and chitosan-oligosaccharide were similar to fructo-oligosaccharide and inulin outputs. As a supplement to in vivo studies, our results comprehensively summarized the similarities and distinctiveness of fibers in regulating fecal microbiota structures. KEY POINTS: • Fibers were divided into three groups based on the regulatory effects in microbiota. • Thirteen indicator OTUs were identified using pairwise comparisons. • Fiber similarities and distinctive traits in regulating microbiota effect were identified.

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates l-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL l-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application.

The mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent research and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB’s characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice; however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating the pit mud microbiome. Esters (50.3% to 64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate, and butyrate) increased with cellar age. Bacteria (9.5 to 10.4 log10 [lg] copies/g) and archaea (8.3 to 9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7% to 81.2%) and Methanomicrobia (32.8% to 92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial and archaeal genera, Caproiciproducens and Methanosarcina. Correlation analysis revealed significantly (P

Hericium erinaceus (H. erinaceus) is widely studied as a medicinal and edible fungus. Recent studies have shown that H. erinaceus has protective effects for diseases, such as inflammatory bowel disease and cancer, which are related to gut microbiota. To investigate the benefits of H. erinaceus intake on gut microbiota and blood indices in adulthood, we recruited 13 healthy adults to consume H. erinaceus powder as a dietary supplement. Blood changes due to H. erinaceus consumption were determined by routine hematological examination and characterized by serum biochemical markers. Microbiota composition was profiled by 16S ribosomal RNA gene sequencing. Results showed that daily H. erinaceus supplementation increased the alpha diversity within the gut microbiota community, upregulated the relative abundance of some short-chain fatty acid (SCFA) producing bacteria (Kineothrix alysoides, Gemmiger formicilis, Fusicatenibacter saccharivorans, Eubacterium rectale, Faecalibacterium prausnitzii), and downregulated some pathobionts (Streptococcus thermophilus, Bacteroides caccae, Romboutsia timonensis). Changes within the gut microbiota were correlated with blood chemical indices including alkaline phosphatase (ALP), low-density lipoprotein (LDL), uric acid (UA), and creatinine (CREA). Thus, we found that the gut microbiota alterations may be part of physiological adaptations to a seven-day H. erinaceus supplementation, potentially influencing beneficial health effects.

High-temperature Daqu, usually used as a fermentation starter for sauce-flavor Baijiu production, plays an essential role in the yield and flavor quality of Baijiu. The environmental heterogeneity of different locations in the workshop during fermentation led to the final production of Daqu with three different types (i.e., white, yellow, and black Daqu). How to use these three types of Daqu in Baijiu production mainly depends on the workers' experience so far. Here, we aimed to reveal the potential functions of different types of Daqu by comparing enzyme activity, volatile metabolites, and microbiota characteristics. White_Qu exhibited the highest liquefaction and saccharification enzyme activities, while the highest neutral proteinase and cellulase enzyme activities were detected in black_Qu. The total volatile content of yellow_Qu and black_Qu was roughly double that of white_Qu, and multivariate analysis revealed distinct volatile dissimilarities across different types of Daqu. Significant differences in bacterial and fungal community structures, assembly patterns, and potential functional profiles were discovered among different types of Daqu. At the genus level, Oceanobacillus and Thermomyces dominated the white_Qu microbiota, and the abundant microbes in yellow_Qu and black_Qu were scattered in Kroppenstedtia and Thermoascus. Bacterial and fungal communities were dominated by deterministic and stochastic assembly processes, respectively, suggesting that bacteria may be more affected by abiotic environmental factors and species interaction than fungi. Co-occurrence network analysis showed positive correlations characterized Daqu microbial networks, and network topological features indicated stronger interactions between bacterial taxa compared with fungal community. The Spearman correlation analysis revealed that four bacterial genera (Kroppenstedtia, Virgibacillus, Scopulibacillus, and Staphylococcus) and two fungal genera (Thermoascus and Aspergillus) exhibited positive correlations with almost all of the abundant volatiles. This work reveals that spatially varying environments lead to the microbiome and metabolome heterogeneity of high-temperature Daqu in the same workshop.

Acetoin, giving a creamy yogurt aroma and buttery taste, exists in cereal vinegar as an important flavor substance and is mainly produced by the metabolism of Lactobacillus and Acetobacter during multispecies solid-state acetic acid fermentation. However, the impacts of Lactobacillus-Acetobacter interactions on acetoin accumulation and the microbial metabolism during acetic acid fermentation are not completely clear. Here, six strains isolated from vinegar fermentation culture and associated with acetoin metabolism, namely, Lactobacillus reuteri L-0, L. buchneri F2-6, L. brevis 4–20, L. fermentum M10-7, L. casei M1-6 and Acetobacter pasteurianus G3-2, were selected for microbial growth and metabolism analysis in monoculture and coculture fermentations. Lactobacillus sp. and A. pasteurianus G3-2 respectively utilized glucose and ethanol preferentially. In monocultures, L. casei M1-6 (183.7 mg/L) and A. pasteurianus G3-2 (121.0 mg/L) showed better acetoin-producing capacity than the others. In the bicultures with Lactobacillus sp. and A. pasteurianus G3-2, biomass analysis in the stationary phase demonstrated that significant growth depressions of Lactobacillus sp. occurred compared with monocultures, possibly due to intolerance to acetic acid produced by A. pasteurianus G3-2. Synergistic effect between Lactobacillus sp. and A. pasteurianus G3-2 on enhanced acetoin accumulation was identified, however, cocultures of two Lactobacillus strains could not apparently facilitate acetoin accumulation. Coculture of L. casei M1-6 and A. pasteurianus G3-2 showed the best performance in acetoin production amongst all mono-, bi- and triculture combinations, and the yield of acetoin increased from 1827.7 to 7529.8 mg/L following optimization of culture conditions. Moreover, the interactions of L. casei M1-6 and A. pasteurianus G3-2 regulated the global metabolism of vinegar microbiota during fermentation through performing in situ bioaugmentation, which could accelerate the production of acetic acid, lactic acid, acetoin, ethyl acetate, ethyl lactate, ligustrazine and other important flavoring substances. This work provides a promising strategy for the production of acetoin-rich vinegar through Lactobacillus sp.-A. pasteurianus joint bioaugmentation.

Keratinases are becoming biotechnologically important since they have shown potential in hydrolysis of recalcitrant keratins with highly rigid and strongly cross-linked structures. However, the large-scale application of keratinases has been limited by the inefficient expression level and low enzyme activity. In this work, we employed pro-peptide engineering and saturation mutagenesis to construct excellent keratinase variants with improved activities. It turned out that amino acid substitutions at the pro-peptide cleavage site (P1) could accelerate the release of active mature enzymes, resulting in a 3-fold activity increase. Eighteen sites of the pro-peptide area were targeted for codon mutagenesis, and a multisite saturation mutagenesis library of the six potential sites was generated, achieving a significant improvement of keratinase activity from 179 to 1114 units/mL. Also, the mutants exhibited alterant catalytic properties. Finally, fermentation for keratinase production in a 15 L fermenter was carried out, and the enzyme activity reached up to over 3000 units/mL. Our results demonstrated that pro-peptide engineering played a crucial role in high expression and engineering of proteases. This study provides a universal route toward improvement of industrial enzymes that were first synthesized as precursors in the form of pre-pro-protein.

Microbial ecosystems of fermented foods are largely interfered by human activities in myriad ways. The aim of this study was to illuminate the impacts of various starters and environmental variables on the fermentation process of Zhenjiang aromatic vinegar (ZAV), one of the four representative cereal vinegars in China. The effects of environmental variables (e.g., ethanol, total acidity, temperature) and starters (e.g., jiuqu, maiqu, seed pei) on the profiles of microbiome and metabolome (e.g., organic acids, amino acids and volatiles) during fermentation process of ZAV were analyzed. Amongst the four fermentation stages, acetic acid fermentation was the main stage for the accumulation of flavor substances, and subsequently, the contents of acids (mainly acetic, lactic and citric acids) and volatile metabolites (e.g., 2,3-butanedione, acetoin, etc.) continued to enrich in sealed fermentation stage. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) showed that the fungal and bacterial community structures of four fermentation stages were significantly different. As for bacterial community, the dominant OTUs with average relative abundance over 10% in at least one fermentation stage were assigned to the genera Acetilactobacillus, Acetobacter, Acinetobacter, Aeromonas, Lactobacillus, and Pseudomonas. The dominant fungal populations in each fermentation stage were obviously divergent, including Wickerhamomyces, Saccharomyces, Alternaria, Fusarium, etc. SourceTracker analysis demonstrated that jiuqu and seed pei provided microorganisms to initiate starch saccharification and acetic acid fermentation stages, respectively, and maiqu was mainly the donor of enzymes in alcohol fermentation. Spearman correlation coefficients revealed positive relationships between fungal community and various flavor metabolites, indicating the essential role of fungi in the flavor formation of ZAV. This study systematically reveals the effects of fermentation starters and environmental variables on vinegar production and deepens the understanding of the traditional production craft.

Over thousands of years, humans have mastered the natural vinegar fermentation technique of cultivating functional microbiota on different raw materials. Functional microbial communities that form reproducibly on non-autoclaved raw materials through repeated batch acetic acid fermentation underpin the flavour development of traditional cereal vinegars. However, how to modulate rationally and optimise the metabolic function of these naturally engineered acidic ecosystems remains unclear. Exploring two key minorities in a vinegar ecosystem, including microbial functions (e.g., flavour and aroma synthesis) and microbial strains, is a crucial step for the vinegar industry to modulate the metabolic function of vinegar microbiota, to monitor the fermentation process, and to maintain the flavour quality of final product.

Solid-state fermentation of Chinese traditional cereal vinegar is a complex and retractable ecosystem with multi-species involved, including few abundant and many non-abundant species. However, the roles of non-abundant species in vinegar fermentation remain unknown. Here, we studied the assembly and co-occurrence patterns for abundant and non-abundant bacterial sub-communities using Zhenjiang aromatic vinegar fermentation as a model system. Our results showed that the change of reducing sugar and total titratable acid were the main driving forces for the assembly of abundant and non-abundant sub-communities, respectively. The non-abundant sub-community was more sensitive to the environmental variation of acetic acid fermentation (AAF) process. Integrated co-occurrence network revealed that non-abundant sub-communities occupied most of the nodes in the network, which play fundamental roles in network stability. Importantly, non-abundant species-Komagataeibacter europaeus, showed the highest value of degree in the co-occurrence network, implying its importance for the metabolic function and resilience of the microbial community. Bioaugmentation of K. europaeus JNP1 verified that it can effectively modulate bacterial composition and improve the robustness of co-occurrence network in situ, accompanied by (i) increased acetic acid content (14.78%) and decreased reducing sugar content (40.38%); and (ii) increased the gene numbers of phosphogluconate dehydratase (212.24%) and aldehyde dehydrogenase (192.31%). Overall, the results showed that non-abundant bacteria could be used to regulate the desired metabolic function of the community, and might play an important ecological significance in traditional fermented foods.

The nitrilase-mediated green synthesis of nicotinic acid, an important reaction in synthetic organic chemistry, has attracted considerable attention in recent years. However, the application potential of nitrilase is hindered by several limitations, such as low catalytic efficiency and byproduct formation. In this study, the site saturation mutagenesis of asparagine 40, phenylalanine 50, and glutamine 207 in recombinant nitrilase from Pseudomonas putida CGMCC3830 was conducted to improve the specificity of nitrilase for nicotinic acid. The resulting mutants, which contain the mutations N40G (asparagine → glycine), F50W (phenylalanine → tryptophan), and Q207E (glutamine → glutamic acid) produced higher nicotinic acid yields than the wild type. Furthermore, double and triple mutations were introduced, and four mutants containing N40G/F50W, N40G/Q207E, F50W/Q207E, and N40G/F50W/Q207E were obtained and evaluated for their capacity to produce nicotinic acid. The double mutant F50W/Q207E and triple mutant N40G/F50W/Q207E displayed the highest activity, which was nearly two-fold higher than that of the wild type. The kinetics analysis of nicotinic acid synthesis with the mutant nitrilase revealed the higher catalytic capability of all the mutants compared with the wild type. These results provide new insights into the catalytic performance of P. putida nitrilase toward 3-cyanopyridine to promote nicotinic acid production.

A novel keratinase was mined and expressed in Escherichia coli BL21 (DE3) via function-driven screening with fosmid library. The catalytic properties of purified keratinase were investigated in detail following enzyme purification. The recombinant keratinase was purified to homogeneity with an estimated molecular weight of 26 kDa using nickel affinity chromatography, of which the optimal reaction pH and temperature were 10.0 and 55 °C, respectively. It could remain stable at pH 5.0–12.0 and 40–60 °C. Metal ions such as Ca2+, Mn2+, Ag+, Na+, Mg2+, Li+, Sn2+ (1 mM) displayed positive influence on keratinase, and particularly, Ca2+ exhibited remarkable improvement effect by 2.6 folds. It was strongly inhibited by PMSF as a protease inhibitor. On the contrary, it could be obviously activated by various surfactants, such as Tween 40 and Triton X-114. The recombinant keratinase showed high specificity towards casein, soluble keratin, BSA, and wool. The keratinase could efficiently degrade the feathers, which demonstrated its applicable potential for biodegradation of keratin wastes and regeneration of soluble protein.

An alginate lyase producing bacterial strain, Cobetia sp. WG-007, was isolated and identified from rotting seaweed. The alginate lyase, Aly-W02, was purified by procedures of ultrafiltration, Q-Sepharose Fast Flow, Phenyl Sepharose 6 Fast Flow, and Superdex-G100 with specific activity of 21,285.5 U/mg. Aly-W02 had an apparent molecular mass of 35 kDa. It exhibited maximum activity at 45 °C in 50 mM sodium phosphate buffer (pH 8.5). This alginate lyase was stable in the pH range of 6.0-8.5. Among the tested metal ions, the addition of K+ , Na+ , and Mg2+ ions can enhance the enzyme activities, while Ba2+ , Ni+ , Cu2+ , Mn2+ , Zn2+ , Ag+ , and ethylenediaminetetraacetic acid decreased the activities. It displayed high salt-tolerant ability; 0.8 M NaCl or 1.5 M KCl significantly enhanced the enzyme activity. Furthermore, Aly-W02 mainly released disaccharide, trisaccharide, and tetrasaccharid from alginate. It showed potential in producing low molecular weight alginate oligosaccharides.

Acetoin (3-hydroxy-2-butanone) formation in vinegar microbiota is crucial for the flavor quality of Zhenjiang aromatic vinegar, a traditional vinegar produced from cereals. However, the specific microorganisms responsible for acetoin formation in this centuries-long repeated batch fermentation have not yet been clearly identified. Here, the microbial distribution discrepancy in the diacetyl/acetoin metabolic pathway of vinegar microbiota was revealed at the species level by a combination of metagenomic sequencing and clone library analysis. The results showed that Acetobacter pasteurianus and 4 Lactobacillus species ( Lactobacillus buchneri , Lactobacillus reuteri , Lactobacillus fermentum , and Lactobacillus brevis ) might be functional producers of acetoin from 2-acetolactate in vinegar microbiota. Furthermore, A. pasteurianus G3-2, L. brevis 4-22, L. fermentum M10-3, and L. buchneri F2-5 were isolated from vinegar microbiota by a culture-dependent method. The acetoin concentrations in two cocultures ( L. brevis 4-22 plus A. pasteurianus G3-2 and L. fermentum M10-3 plus A. pasteurianus G3-2) were obviously higher than those in monocultures of lactic acid bacteria (LAB), while L. buchneri F2-5 did not produce more acetoin when coinoculated with A. pasteurianus G3-2. Last, the acetoin-producing function of vinegar microbiota was regulated in situ via augmentation with functional species in vinegar Pei . After 72 h of fermentation, augmentation with A. pasteurianus G3-2 plus L. brevis 4-22, L. fermentum M10-3, or L. buchneri F2-5 significantly increased the acetoin content in vinegar Pei compared with the control group. This study provides a perspective on elucidating and manipulating different metabolic roles of microbes during flavor formation in vinegar microbiota. IMPORTANCE Acetoin (3-hydroxy-2-butanone) formation in vinegar microbiota is crucial for the flavor quality of Zhenjiang aromatic vinegar, a traditional vinegar produced from cereals. Thus, it is of interest to understand which microbes are driving the formation of acetoin to elucidate the microbial distribution discrepancy in the acetoin metabolic pathway and to regulate the metabolic function of functional microbial groups in vinegar microbiota. Our study provides a perspective on elucidating and manipulating different metabolic roles of microbes during flavor formation in vinegar microbiota.

Most available methods for modifying the catalytic properties of enzymes are costly and time-consuming, as they rely on the information of enzyme crystal structure or require handling large amounts of mutants. This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermedia was performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Large-scale submerged fermentation (SmF) of Antrodia camphorata (A. camphorata) usually encounters challenges including tedious preparation of mycelial inoculum, long fermentation period (10-14 d), and poor repeatability. Here we developed an asexual reproduction-based repeated batch fermentation (RBF) process for bioactive metabolites production by A. camphorata ATCC 200183. Compared with traditional batch fermentation, production time was shortened to 58 d from 80 d (overall time for eight cycles) using the RBF process established in this study, and accordingly, the productivities of bioactive metabolites (including antrodins) were improved by 40-60%. Kinetic parameters (α is 2.1-18.7 times as β) indicated that the cell growth was the major contribution for bioactive metabolites production. The RBF shows excellent batch-repeatability (Pearson correlation coefficient of 0.998±0.001), together with advantages of energy-efficient, low cost, and labor-saving, RBF process can be implemented to SmF by other filamentous fungi.

Solid-state fermentation of traditional Chinese vinegar is a mixed-culture refreshment process that proceeds for many centuries without spoilage. Here, we investigated bacterial community succession and flavor formation in three batches of Zhenjiang aromatic vinegar using pyrosequencing and metabolomics approaches. Temporal patterns of bacterial succession in the Pei (solid-state vinegar culture) showed no significant difference (P > 0.05) among three batches of fermentation. In all the batches investigated, the average number of community operational taxonomic units (OTUs) decreased dramatically from 119 ± 11 on day 1 to 48 ± 16 on day 3, and then maintained in the range of 61 ± 9 from day 5 to the end of fermentation. We confirmed that, within a batch of fermentation process, the patterns of bacterial diversity between the starter (took from the last batch of vinegar culture on day 7) and the Pei on day 7 were similar (90%). The relative abundance dynamics of two dominant members, Lactobacillus and Acetobacter, showed high correlation (coefficient as 0.90 and 0.98 respectively) among different batches. Furthermore, statistical analysis revealed dynamics of 16 main flavor metabolites were stable among different batches. The findings validate the batch-to-batch uniformity of bacterial community succession and flavor formation accounts for the quality of Zhenjiang aromatic vinegar. Based on our understanding, this is the first study helps to explain the rationality of age-old artistry from a scientific perspective.

β-Mannanases can efficiently hydrolyze both the polysaccharide mannan and manno-oligosaccharides. In order to characterize a novel mannanase for potential industrial application, an endo-1,4-β-mannanase from Bacillus subtilis YH12 was purified to homogeneity and its biochemical characterization was performed. The optimal conditions for the purified enzyme were pH 6.5 and 55 °C, and it remained stable at temperature up to 60 °C and pH of 4.5–7.5. The enzyme had specific activity on complex structure polysaccharide. And it showed high activity on locust bean gum, konjac powder, guar and fenugreek gums. Furthermore, it efficiently hydrolyzed xanthan, and carrageenan gums, and it exhibited no activity on starch, and xylan. The mannanase exhibited higher activity on galactomannan branched with (1→6)-linked α- d -galactose than glucomannan. The predominant products resulting from mannanase hydrolysis were 1–7 units of manno-oligosaccharides from locust bean gum, 2–7 units of manno-oligosaccharides from konjac powder, and 1–2 units of manno-oligosaccharides from xanthan gum. The B. subtilis YH12 mannanase encoding gene was successfully cloned. This study provided a novel mannanase that demonstrates potential application in food and animal feeds technology.

An α-keratinase producing strain was isolated with wool as the sole carbon and nitrogen source and identified as Bacillus pumilus K9. The major amino acids liberated from the keratin degradation of wool by B. pumilus K9 were glutamic acid and leucine. The α-keratinase was purified to electrophoretic homogeneity with a molecular weight of 32000. The purified enzyme exhibits an optimum activity at 60 °C and pH=9.0. It was stable at pH values between 8 and 11. Bacillas pumilus keratinase displays a high activity towards casein, keratin, wool and feather, which indicates its wide application range. The keratinase was completely inhibited by phenylmethyl-sulfonyl fluoride(PMSF) and β-mercaptoethanol, and moderately inhibited by ethylemediamine-tetraacetic acid(EDTA), suggesting it is a metallo-cysteine keratinase. This enzyme could remain stable that could even be promoted in the presence of surfactants, including sodium dodecyl sulfate(SDS), Tween and Triton. And Tween 40 and Triton X-100 could substantially enhance the activity of the enzyme by 54% and 35%, respectively. It may indicate the prominent feature of the keratinase to tolerate surfactants. The enzymatic properties distinguish this keratinase from others in the literature. Furthermore, this enzyme is extremely stable in the presence of a commercially available detergent with 1% concentration. Detergents ARIEL, Bluemoon and WhiteCat can enhance the activity of the keratinase by 43.56%, 15.22%, and 22.48%, respectively.

Medicinal mushroom Antrodia camphorata sporulate large numbers of arthroconidia in submerged fermentation, which is rarely reported in basidiomycetous fungi. Nevertheless, the molecular mechanisms underlying this asexual sporulation (conidiation) remain unclear. Here, we used comparative transcriptomic and proteomic approaches to elucidate possible signaling pathway relating to the asexual sporulation of A. camphorata. First, 104 differentially expressed proteins and 2586 differential cDNA sequences during the culture process of A. camphorata were identified by 2DE and RNA-seq, respectively. By applying bioinformatics analysis, a total of 67 genes which might play roles in the sporulation were obtained, and 18 of these genes, including fluG, sfgA, SfaD, flbA, flbB, flbC, flbD, nsdD, brlA, abaA, wetA, ganB, fadA, PkaA, veA, velB, vosA, and stuA might be involved in a potential FluG-mediated signaling pathway. Furthermore, the mRNA expression levels of the 18 genes in the proposed FluG-mediated signaling pathway were analyzed by quantitative real-time PCR. In summary, our study helps elucidate the molecular mechanisms underlying the asexual sporulation of A. camphorata, and provides also useful transcripts and proteome for further bioinformatics study of this valuable medicinal mushroom.

Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.

The I-V and C-V characteristics of Au/n-GaN Schottky diodes irradiated with 290-MeV U-238(32+) ions are presented. The U ions can penetrate the n-type GaN epi-layer with a thickness about 3 mu m grown on the c-plane of a sapphire substrate using the MOCVD technique, leaving a purely electronic energy deposition. The Au/n-GaN Schottky diodes were irradiated to successively increasing fluences from 1 x 10(9) to 5 x 10(11) ions cm(-2). The measured I-V curves show that the height of the Schottky barrier decreases after irradiation and that the Schottky barrier almost disappears when the ion fluence reaches 5 x 10(9) ions cm(-2). Meanwhile, the irradiation increases the series resistance. The C-V curves show that the capacitance drops sharply when the ion fluence reaches 5 x 10(10) ions cm(-2). The dielectric constant also decreases following the irradiation. The changes of the electrical properties are ascribed to the neutralization of the donor-like surface state and the acceptor-like surface state due to the migration of Au atoms at the interface of Au/n-GaN under energetic U ions irradiations. (C) 2014 Elsevier B.V. All rights reserved.

Large-scale submerged fermentation (SmF) of Antrodia camphorata usually encounters engineering problems, such as long period and complex fermentation. In this study, a modified inoculation method using arthroconidia sporulated in liquid medium was developed for an efficient large-scale fermentation of A. camphorata ATCC 200183. The optimum environmental and nutritional conditions for arthroconidial germination in the dark control were estimated as follows: temperature, 26 °C; glucose, 20 g/L; soybean peptone, 10 g/L; Mg2+ concentration, 2 mmol/L; and initial pH, 3.5. All the arthroconidia rapidly germinated within 32 h after inoculation under optimal conditions. In the shaking flask, prepared according to the arthroconidial inoculation method, the fermentation period, maximal cell growth rate, and triterpenoid productivity of SmF were 0.6-, 1.7-, and 1.6-fold of those obtained from the culture prepared according to the conventional mycelial inoculation method. Furthermore, efficient fermentation was obtained in a 4000 L fermentor, with a biomass of 7.84 ± 0.20 g/L and a triterpenoid production of 61.21 ± 2.12 mg/L. Therefore, the modified inoculation method can be used in engineering applications with several advantages, including simplicity, good controllability, and high efficiency (shorten the operation time of mycelium-based inoculation from ≥240 h to 168 h).

Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH2 terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters Km and Vmax for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.

Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only

Inonotus obliquus is a medicinal mushroom used in Russian and Eastern European folk medicine for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes. Previous studies in our laboratory have demonstrated that the mycelium powders of I. obliquus possess significant antihyperglycemic effects in a mouse model of diabetic disease induced by alloxan. However, the active ingredients of mycelium powders responsible for the diabetes activity have not been identified. This study aims to identify the active ingredients of I. obliquus mycelium powders by a bioassay-guided fractionation approach and explore the mechanism of action of these active ingredients by using a well-established DPP-4 (an important enzyme as a new therapeutic target for diabetes) inhibitory assay model. The results showed the chloroform extract of mycelium was potential inhibitory against DPP-4. Bioactivity guided fractionation led to the identification of 19 compounds using UPLC-Q-TOF-MS. Molecular docking between the compounds and DPP-4 revealed that compounds 5, 8, 9, 14, 15 may be the active components responsible for the DPP-4 inhibitory activity.

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.

The main purpose of this study was to determine the volatile composition of Zhenjiang aromatic vinegar, one of the four famous China-style cereal vinegars, by using headspace solid-phase microextraction (HS-SPME)/gas chromatography-mass spectrum (GC-MS) and chemometrics. For this purpose, the HS-SPME sampling method for the volatile compounds of Zhenjiang aromatic vinegar was optimized by a second-order rotatable central composite experimental design (CCD). A HS extraction of the volatile compounds by incubation on a 65 µm thickness polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre during 44.2 min at 69.5°C with 1.9 g NaCl add gave the most effective and accurate extraction. By the optimized method, a total of 58 volatile compounds, including 9 alcohols, 13 acids, 16 esters, 5 aldehydes, 4 ketones and 8 heterocycle compounds, were identified from 13 aromatic vinegar samples manufactured in Zhenjiang region. By principal components analysis (PCA), the thirteen vinegar samples were classified into 3 groups, and 10 volatile compounds were chosen as characteristic compounds of Zhenjiang aromatic vinegars. Copyright © 2012 The Institute of Brewing & Distilling

Zhenjiang aromatic vinegar is one of the most famous Chinese traditional vinegars. In this study, change of the microbial community during its fermentation process was investigated. DGGE results showed that microbial community was comparatively stable, and the diversity has a disciplinary series of changes during the fermentation process. It was suggested that domestication of microbes and unique cycle-inoculation style used in the fermentation of Zhenjiang aromatic vinegar were responsible for comparatively stable of the microbial community. Furthermore, two clone libraries were constructed. The results showed that bacteria presented in the fermentation belonged to genus Lactobacillus, Acetobacter, Gluconacetobacter, Staphylococcus, Enterobacter, Pseudomonas, Flavobacterium and Sinorhizobium, while the fungi were genus Saccharomyces. DGGE combined with clone library analysis was an effective and credible technique for analyzing the microbial community during the fermentation process of Zhenjiang aromatic vinegar. Real-time PCR results suggested that the biomass showed a "system microbes self-domestication" process in the first 5 days, then reached a higher level at the 7th day before gradually decreasing until the fermentation ended at the 20th day. This is the first report to study the changes of microbial community during fermentation process of Chinese traditional solid-state fermentation of vinegar.