Identification of the high-yield monacolin K strain from Monascus spp. and its submerged fermentation using different medicinal plants.

BACKGROUND: Medical plants confer various benefits to human health and their bioconversion through microbial fermentation can increase efficacy, reduce toxicity, conserve resources and produce new chemical components. In this study, the cholesterol-lowering monacolin K genes and content produced by Monascus species were identified. The high-yield monacolin K strain further fermented with various medicinal plants. The antioxidant and anti-inflammatory activities, red pigment and monacolin K content, total phenolic content, and metabolites in the fermented products were analyzed. RESULTS: Monacolin K was detected in Monascus pilosus (BCRC 38072), and Monascus ruber (BCRC 31533, 31523, 31534, 31535, and 33323). It responded to the highly homologous mokA and mokE genes encoding polyketide synthase and dehydrogenase. The high-yield monacolin K strain, M. ruber BCRC 31535, was used for fermentation with various medicinal plants. A positive relationship between the antioxidant capacity and total phenol content of the fermented products was observed after 60 days of fermentation, and both declined after 120 days of fermentation. By contrast, red pigment and monacolin K accumulated over time during fermentation, and the highest monacolin K content was observed in the fermentation of Glycyrrhiza uralensis, as confirmed by RT-qPCR. Moreover, Monascus-fermented medicinal plants including Paeonia lactiflora, Alpinia oxyphylla, G. uralensis, and rice were not cytotoxic. Only the product of Monascus-fermented G. uralensis significantly exhibited the anti-inflammatory capacity in a dose-dependent manner in lipopolysaccharide-induced Raw264.7 cells. The metabolites of G. uralensis with and without fermentation (60 days) were compared by LC/MS. 2,3-Dihydroxybenzoic acid, 3,4-dihydroxyphenylglycol, and 3-amino-4-hydroxybenzoate were considered to enhance the antioxidant and anti-inflammatory ability. CONCLUSIONS: Given that highly homologous monacolin K and citrinin genes can be observed in Monascus spp., monacolin K produced by Monascus species without citrinin genes can be detected through the complementary methods of PCR and HPLC. In addition, the optimal fermentation time was important to the acquisition of antioxidants, red pigment and monacolin K. These bioactive substances were significantly affected by medicinal plants over fermentation time. Consequently, Monascus-fermented G. uralensis had a broad spectrum of biological activities.

Agricultural management practices influence the soil enzyme activity and bacterial community structure in tea plantations.

BACKGROUND: The soil quality and health of the tea plantations are dependent on agriculture management practices, and long-term chemical fertilizer use is implicated in soil decline. Hence, several sustainable practices are used to improve and maintain the soil quality. Here, in this study, changes in soil properties, enzymatic activity, and dysbiosis in bacterial community composition were compared using three agricultural management practices, namely conventional (CA), sustainable (SA), and transformational agriculture (TA) in the tea plantation during 2016 and 2017 period. Soil samples at two-months intervals were collected and analyzed. RESULTS: The results of the enzyme activities revealed that acid phosphatase, arylsulfatase, ß-glucosidase, and urease activities differed considerably among the soils representing the three management practices. Combining the redundancy and multiple regression analysis, the change in the arylsulfatase activity was explained by soil pH as a significant predictor in the SA soils. The soil bacterial community was predominated by the phyla Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, and Bacteroidetes in the soil throughout the sampling period. Higher Alpha diversity scores indicated increased bacterial abundance and diversity in the SA soils. A significant relationship between bacterial richness indices (SOBS, Chao and ACE) and soil pH, K and, P was observed in the SA soils. The diversity indices namely Shannon and Simpson also showed variations, suggesting the shift in the diversity of less abundant and more common species. Furthermore, the agricultural management practices, soil pH fluctuation, and the extractable elements had a greater influence on bacterial structure than that of temporal change. CONCLUSIONS: Based on the cross-over analysis of the bacterial composition, enzymatic activity, and soil properties, the relationship between bacterial composition and biologically-driven ecological processes can be identified as indicators of sustainability for the tea plantation.

Evaluation of synthetic gene encoding α-galactosidase through metagenomic sequencing of paddy soil.

Many genes of industrial relevance can be found in soil. In this study, metagenome sequencing of paddy soil was performed with 55.68 Gb sequences and 1,787,113 putative open reading frames (ORFs). The functional profiles and metabolic pathway of soil metagenomes were examined using Gene Ontology, Metagenomics RAST, and Kyoto Encyclopedia of Genes and Genomes. To verify the protein function and assembly of ORFs, a putative gene encoding α-galactosidase, namely GalR, which shares 65% identity with an unpublished glycoside hydrolase (GH) 27 family protein, was synthesized using its optimal codon for overexpression in Escherichia coli. GalR was successfully obtained and characterized. The optimal temperature and pH for GalR activity were 30°C and pH 9, respectively. Enzymatic activity indicated that GalR was alkaliphilic and different from acidophilic α-galactosidase in the GH 27 family. Furthermore, 50% of the relative activity of GalR can be attained for 1.7 and 0.7 h preincubation at 40°C and 50°C, respectively. Significant inhibition of GalR was observed in the presence of ethylenediaminetetraacetic acid (EDTA), MgCl2, sodium dodecyl sulfate (SDS), and H2O2; however, it was resistant to 0.1% methanol and ethanol and was slightly activated with NaCl and KCl. The specific activity of GalR was achieved at 11.6 and 0.59 µmol/min/mg of protein using p-nitrophenyl-α-d-galactopyranoside and raffinose as substrates, respectively. Consequently, the metagenomic sequencing-based strategy can provide information for mining novel genes.

Inspecting the genome sequence and agarases of Microbulbifer pacificus LD25 from a saltwater hot spring.

Neoagaro-oligosaccharides prepared by agar hydrolysis have various application fields, including the pharmaceutical, cosmetic, and food industries. In this study, an agarolytic strain was isolated from a saltwater hot spring and identified as Microbulbifer pacificus LD25 by 16S rRNA. The whole genome sequence of M. pacificus LD25 was obtained. It had a size of 4.27 Mb and comprised 3062 predicted genes in 37 contigs with a G+C content of 58.0%. Six agarases were annotated and classified into three families, namely, GH16 (AgaL1), GH86 (AgaL2, AgaL3), and GH50 (AgaL4, AgaL5, AgaL6), which shared 75-96% identities with unpublished hypothetical proteins and agarases. AgaL1, AgaL4, and AgaL6 can be successfully expressed and purified in Escherichia coli. AgaL1 and AgaL4 displayed a significantly agarolytic capability, whereas AgaL6 exhibited a rarely detectable enzymatic activity. The optimal temperature and pH required for the activity of AgaL1 and AgaL4 was 50°C and 60°C, respectively, at pH 7. The specific activities of AgaL1 and AgaL4 were achieved at 16.8 and 9.6 U per mg of protein. Both agarases were significantly inhibited in the presence of EDTA, MgO, ZnCl2, and H2O2. However, AgaL1 was resistant to 0.1% SDS and AgaL4 was slightly activated by CaCl2. Substrate hydrolysis detected by LC-MS/MS analysis indicated that neoagarobiose was the main product during AgaL1 and AgaL4 catalysis. Furthermore, AgaL4 was thermostable and retained over 93% of its relative activity after pre-incubation at 70°C for 180 min. Consequently, M. pacificus LD25 has a potential for agarase production in E. coli and industrial applications.

Identification, Molecular Cloning of IL-1ß and Its Expression Profile during Nocardia seriolae Infection in Largemouth Bass, Micropterus salmoides.

In the present study, IL-1ß cDNA was identified and analyzed from largemouth bass (Micropterus salmoides). Full length IL-1ß mRNA was obtained using Rapid Amplification of cDNA Ends (RACE), which contains 78 bp 3'-UTR, a 455 bp 5'-UTR, and an open reading frame (ORF) of 702 bp coding for 233 amino acid residues. The molecular weight and theoretical isoelectric point of largemouth bass IL-1ß protein was predicted to be 26.7 kDa and 6.08 respectively. A largemouth bass IL-1ß phylogenetic analysis showed a close relation to the IL-1ßs of striped trumpeter (Latris lineata), Chinese perch (Siniperca chuatsi), and Japanese sea bass (Lateolabrax japonicus). Peptidoglycan upregulated IL-1ß in the spleen and head kidney, while lipopolysaccharide upregulated detectable levels of IL-1ß in the spleen only. Largemouth bass, challenged with Nocardia seriolae (1.0 × 106 cfu/mL), showed a significant increase in IL-1ß at 3 and 5 days post infection (dpi) in the spleen, while in the head kidney significant expression was found at 2 and 3 dpi, peaking at 3 dpi. Furthermore, tumor necrosis factor α (TNF-α) showed significantly higher expression in the spleen at 3 and 5 dpi, and in the head kidney at 1 and 3 dpi, with expression decreasing at 5 dpi in both tissues.

Characterization and expression of the antifungal protein from Monascus pilosus and its distribution among various Monascus species.

Monascus species are traditionally used for food preservation. This study used the disc diffusion method to verify the antifungal activity of protein extracted from Monascus pilosus BCRC38072 against 15 fungal pathogens. An antifungal protein, designated as MAFP1, was successfully purified and confirmed through N-terminal sequencing. To further explore the antifungal gene, a mafp1 gene that is similar to that of PgAFP from Penicillium chrysogenum was cloned from M. pilosus BCRC38072. According to the N-terminal sequencing and in silico analysis, the signal peptide was assumed to have 18 amino acids and the mature MAFP1 to contain 58 peptides. Moreover, the mafp1 gene was recognized in Monascus ruber, Monascus barkeri, Monascus floridanus, and Monascus lunisporas through polymerase chain reaction and DNA sequencing and showed high homology. By contrast, the mafp1 gene was absent in Monascus kaoliang, Monascus purpureus, and Monascus sanguineus. In addition, the mafp1 gene with N-terminal polyhistidine fusion was overexpressed in Escherichia coli. However, the antifungal activity of recombinant MAFP1 was significantly lower than that of native MAFP1. According to the properties of MAFP1, Monascus species may have food preservation applications.

Immunoprotection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Lactococcus garvieae against Lactococcosis in tilapia.

In this study, the gene encoding 40 kDa GAPDH of L. garvieae was determined and overexpressed by using the Escherichia coli expression system. Analysis results indicated that the sequences of GAPDH of L. garvieae nucleotide and its amino acid are highly homologous (80.4-100%) to several products of GAPDH from L. garvieae and other Streptococcus-related bacteria. According to Western blotting results, rabbit antiserum and tilapia infection serum reacted strongly to the recombinant GAPDH protein. In another experiment, tilapia were immunized intraperitoneally with formalin-killed L. garvieae whole cells, recombinant GAPDH (50 µg fish(-1)) from L. garvieae or both. ISA 763A was used as an adjuvant for vaccine and saline was used as a negative control. The fish challenged at 4 weeks after immunization with GAPDH+WC+ISA had the highest survival rate at 100%, followed by fish immunized with WC+ISA or GAPDH+ISA, which had RPS values of 87.5% and 50%, respectively. Additionally, specific antibody responses against L. garvieae whole cells and GAPDH were based on enzyme-linked immunosorbent assay. Following 4 weeks of immunization, the specific antibody level of all vaccine groups significantly increased, except for antibody responses against L. garvieae GAPDH of those immunized with formalin-killed L. garvieae whole cells. Our results further demonstrated that GAPDH from L. garvieae protected tilapia from experimental L. garvieae infection, implying the potential use of L. garvieae GAPDH as a vaccine against L. garvieae.

Comparison of genetic characteristics and pathogenicity of Lactococcus garvieae isolated from aquatic animals in Taiwan.

Seventy-six Taiwanese bacterial isolates including 74 from diseased, cultured, aquatic animals (54 grey mullet Mugil cephalus, 3 basket mullet Chelon alatus, 2 tilapia Oreochromis niloticus, 1 grouper Epinephelus coioides, 2 yellowfin seabream Acanthopagrus latus, 1 Borneo mullet Chelon macrolepis, 1 bullfrog Rana catesbeiana, 1 Japanese eel Anguilla japonica, and 9 giant freshwater prawns Macrobrachium rosenbergii), 1 wild-caught seafood species (squid muscle collected from a restaurant) and 1 human isolate (from a patient with a history of consuming raw squid in the previously mentioned restaurant), all collected between 1999 and 2006, were confirmed by PCR assay to be Lactococcus garvieae. The phenotypic characterization was determined by rabbit anti-KG+ and KG- serums, and 74 of the 76 Taiwanese strains displayed a KG- phenotype. The genetic characterization was investigated by pulsed-field gel electrophoresis (PFGE). Genomic DNA was digested with restriction endonucleases ApaI and SmaI and separated by PFGE. Ten different L. garvieae pulsotypes were identified. Predominant pulsotypes A1a/S1a were obtained from >96% of strains (52 of 54) from grey mullet, demonstrating a clonal dissemination of L. garvieae in grey mullet in Taiwan. In experimental challenges with grey mullet and tilapia, L. garvieae pulsotypes A1/S1 and A11/S11 showed higher virulence compared with other pulsotypes.

Identification of the mokH gene encoding transcription factor for the upregulation of monacolin K biosynthesis in Monascus pilosus.

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus. The monacolin K biosynthetic gene cluster, mokA-mokI, has been characterized in Monascus pilosus. The mokH gene encoding Zn(II)2Cys6 binuclear DNA binding protein is assumed to be an activator for monacolin K production. In this study, the mokH gene was cloned and driven by the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter for overexpression in M. pilosus. The transformants containing an extra copy of the mokH gene were obtained and verified by PCR and Southern hybridization. The transcripts of mokH in the transformants were expressed significantly higher than those of the wild-type strain. The transformants were stably inherited through the next generation, as determined by observation of the enhanced green fluorescent protein (EGFP). The transformant T-mokH1 also showed a 1.7-fold higher production of monacolin K than the wild-type strain in a time course analysis. Analysis of the RT-PCR products demonstrated that the monacolin K biosynthetic genes in the transformant were expressed to a greater extent than those in the wild-type strain. These results indicated that mokH upregulated the transcription of monacolin K biosynthetic genes and increased monacolin K production.

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid.

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.

Exploring the distribution of citrinin biosynthesis related genes among Monascus species.

Citrinin, a hepato-nephrotoxic compound to humans, can be produced by the food fermentation microorganisms Monascus spp. In this study, we investigated the distribution of mycotoxin citrinin biosynthesis genes in 18 Monascus strains. The results show that the acyl-transferase and keto-synthase domains of the pksCT gene encoding citrinin polyketide synthase were found in Monascus purpureus, Monascus kaoliang, and Monascus sanguineus. Furthermore, the ctnA gene, a major activator for citrinin biosynthesis, was found in M. purpureus and M. kaoliang, but was absent in M. sanguineus. The orf3 gene encoding oxygenase, located between pksCT and ctnA, was also present in M. purpureus and M. kaoliang. The pksCT gene was highly conserved in M. purpureus, M. kaoliang, and M. sanguineus, while the ctnA and orf3 genes were shown to be highly homologous in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot analyses suggest that pksCT, ctnA, and orf3 were absent or significantly different in Monascus pilosus, Monascus ruber, Monascus barkeri, Monascus floridanus, Monascus lunisporas, and Monascus pallens. A citrinin-producing phenotype was detected only in M. purpureus and M. kaoliang using high performance liquid chromatography (HPLC). These results clearly indicate that the highly conserved citrinin gene cluster in M. purpureus and M. kaoliang carry out citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the beta-tubulin gene, the citrinin gene cluster can group the species of Monascus.

Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus.

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA- mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii.

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included marked edema and necrotic lesions, with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain/heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture.

Metschnikowia bicuspidata dominates in Taiwanese cold-weather yeast infections of Macrobrachium rosenbergii.

At water temperatures below 17 degrees C, yeast infections often occurred in 6 to 11 mo old giant freshwater prawn Macrobrachium rosenbergii (8 to 12 cm) in Taiwan from May 2001 to December 2003, with a cumulative mortality of 20 to 95%. Gross signs of disease included milky hemolymph, a yellow exoskeleton, opaque muscles, and a swollen hepatopancreas (HP). Histopathology included marked edema and extensive necrotic lesions associated with large numbers of yeast aggregates and inflammation within the muscles, HP, and other internal organs such as the heart, ovary, and intestine. Yeast cell numbers isolated from various tissues ranged from 4.5 x 10(8) to 9.0 x 10(9) colony forming units (CFU) per 100 mg. From diseased prawns from 12 affected farms, the presence of Metschnikowia bicuspidata (98.4% prevalence), Saccharomyces cerevisiae (0.8% prevalence), and Candida albicans (0.8% prevalence) was confirmed by biochemical tests and sequencing of PCR products from the D1/D2 domain of 26S rDNA. Experimental infection with these isolates caused gross signs and histopathological changes similar to those observed in naturally infected prawns, and lethal doses (LD50) were 3.8 x 10(3), 2.0 x 10(3), and 4.3 x 10(3) CFU prawn-1, respectively. Although the results of this study revealed that M. bicuspidata may be the major cause of yeast infections in the giant freshwater prawns in Taiwan, this is the first time that S. cerevisiae and C. albicans are also reported as pathogens.

Systemic infection of Kudoa lutjanus n. sp. (Myxozoa: Myxosporea) in red snapper Lutjanus erythropterus from Taiwan.

A new species of Kudoa lutjanus n. sp. (Myxosporea) is described from the brain and internal organs of cultured red snapper Lutjanus erythropterus from Taiwan. The fish, 260 to 390 g in weight, exhibited anorexia and poor appetite and swam in the surface water during outbreaks. Cumulative mortality was about 1% during a period of 3 wk. The red snapper exhibited numerous creamy-white pseudocysts, 0.003 to 0.65 cm (n = 100) in diameter, in the eye, swim bladder, muscle and other internal organs, but especially in the brain. The number of pseudocysts per infected fish was not correlated with fish size or condition. Mature spores were quadrate in apical view and suboval in side view, measuring 8.2 +/- 0.59 microm in width and 7.3 +/- 0.53 microm in length. The 4 valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 3.62 +/- 0.49 microm in length and 2.2 +/- 0.49 microm in width. Mild inflammatory responses or liquefaction of host tissue were associated with K. lutjanus n. sp. infection. The junction of shell valves appeared as overlapping, straight lines. The polar filament formed 2 to 3 coils. A general PCR (polymerase chain reaction) primer for Kudoa amplified the small subunit (SSU) rDNA sequences, and the amplified gene was sequenced. It was evident from the phylogenetic tree that the 3 strains tested, AOD93020M, AOD93028M and AOD93028B, were identical and belonged to the Kudoa SS rRNA subgroup. The evolutionary tree showed that these strains form a unique clade, at a distance from other Kudoa species and myxosporeans. The spore's morphological and ultrastructural characteristics, as well as the SS rDNA properties of the isolates, were also essentially identical and served to distinguish them from representative Kudoa. It is, therefore, proposed that the strains isolated from the diseased red snapper be assigned to a new species.

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii.

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.