Plant endophytic fungi exhibit diverse biotransformation pathways of mogrosides and show great potential application in siamenoside I production.

Fungal endophytes, as an untapped resource of glycoside hydrolase biocatalysts, need to be further developed. Mogroside V, the primary active compound in Siraitia grosvenorii fruit, can be converted into other various bioactive mogrosides by selective hydrolysis of glucose residues at C3 and C24 positions. In present study, 20 fungal strains were randomly selected from our endophytic fungal strain library to assess their capability for mogroside V transformation. The results revealed that relatively high rate (30%) endophytic fungal strains exhibited transformative potential. Further analysis indicated that endophytic fungi could produce abundant mogrosides, and the pathways for biotransforming mogroside V showed diverse. Among the given fungal endophytes, Aspergillus sp. S125 almost completely converted mogroside V into the end-products mogroside II A and aglycone within just 2 days of fermentation; Muyocopron sp. A5 produced rich intermediate products, including siamenoside I, and the end-product mogroside II E. Subsequently, we optimized the fermentation conditions for Aspergillus sp. S125 and Muyocopron sp. A5 to evaluate the feasibility of large-scale mogroside V conversion. After optimization, Aspergillus sp. S125 converted 10 g/L of mogroside V into 4.5 g/L of mogroside II A and 3.6 g/L of aglycone after 3 days of fermentation, whereas Muyocopron sp. A5 selectively produced 4.88 g/L of siamenoside I from 7.5 g/L of mogroside V after 36 h of fermentation. This study not only identifies highly effective biocatalytic candidates for mogrosides transformation, but also strongly suggests the potential of plant endophytic fungi as valuable resources for the biocatalysis of natural compounds.

Heat stress enhanced perylenequinones biosynthesis of Shiraia sp. Slf14(w) through nitric oxide formation.

Perylenequinones (PQs) are a class of natural polyketides used as photodynamic therapeutics. Heat stress (HS) is an important environmental factor affecting secondary metabolism of fungi. This study investigated the effects of HS treatment on PQs biosynthesis of Shiraia sp. Slf14(w) and the underlying molecular mechanism. After the optimization of HS treatment conditions, the total PQs amount reached 577 ± 34.56 mg/L, which was 20.89-fold improvement over the control. Also, HS treatment stimulated the formation of intracellular nitric oxide (NO). Genome-wide analysis of Shiraia sp. Slf14(w) revealed iNOSL and cNOSL encoding inducible and constitutive NOS-like proteins (iNOSL and cNOSL), respectively. Cloned iNOSL in Escherichia coli BL21 showed higher nitric oxide synthase (NOS) activity than cNOSL, and the expression level of iNOSL under HS treatment was observably higher than that of cNOSL, suggesting that iNOSL is more responsible for NO production in the HS-treated strain Slf14(w) and may play an important role in regulating PQs biosynthesis. Moreover, the putative biosynthetic gene clusters for PQs and genes encoding iNOSL and nitrate reductase (NR) in the HS-treated strain Slf14(w) were obviously upregulated. PQs biosynthesis and efflux stimulated by HS treatment were significantly inhibited upon the addition of NO scavenger, NOS inhibitor, and NR inhibitor, indicating that HS-induced NO, as a signaling molecule, triggered promoted PQs biosynthesis and efflux. Our results provide an effective strategy for PQs production and contribute to the understanding of heat shock signal transduction studies of other fungi.Key points• PQs titer of Shiraia sp. Slf14(w) was significantly enhanced by HS treatment.• HS-induced NO was first reported to participate in PQs biosynthetic regulation.• Novel inducible and constitutive NOS-like proteins (iNOSL and cNOSL) were obtained and their NOS activities were determined.

Endophytic fungi from Dongxiang wild rice (Oryza rufipogon Griff.) show diverse catalytic potential for converting glycyrrhizin.

Endophytic fungi inhabiting niche environments are novel biocatalyst resources that need to be exploited urgently. In this study, 63 endophytic fungi isolated from Dongxiang wild rice (Oryza rufipogon Griff.) were tested to assess their potentials to transform glycyrrhizin (GL) into glycyrrhetinic acid monoglucuronide (GAMG) or glycyrrhetinic acid (GA), of which 12 strains were shown to have ß-d-glucuronidase activity. Based on morphological characteristics and rDNA ITS sequence analysis, the strains S59, L138, L55 and R57 with high GL molar conversion rates (55%, 45%, 65% and 89%) were further identified as Microsphaeropsis arundinis S59, Penicillium rubens L138, Aspergillus flavus L55 and Eupenicillium javanicum R57, respectively. These four strains with four different types of GL conversion processes were identified, i.e., (1) GL → GAMG in M. arundinis S59, (2) GL → GAMG and GA in A. flavus L55, (3) GL → GA in P. rubens L138, and (4) GL → GAMG → GA in E. javanicum R57, in which the bioconversion type (4) is reported for the first time. The study not only provided abundant and diverse ß-d-glucuronidase resources that can be used for GL bioconversion, especially for GAMG biosynthesis from endophytic fungi, but also expanded our knowledge of potential roles of endophytes as new biocatalysts in biotransformation.

Use of Non-Saccharomyces Yeast Co-Fermentation with Saccharomyces cerevisiae to Improve the Polyphenol and Volatile Aroma Compound Contents in Nanfeng Tangerine Wines.

This study attempted to improve the polyphenol and volatile aroma compound contents in Nanfeng tangerine wines using non-Saccharomyces yeast and Saccharomyces cerevisiae. The effects of fermentation with pure cultures of Candida ethanolica, Hanseniaspora guilliermondii and Hanseniaspora thailandica, as well as in sequential and mixed inoculations (1:1 or 1:100 ratio) with S. cerevisiae in Nanfeng tangerine wines were evaluated. C. ethanolica was found to produce the most polyphenols (138.78 mg/L) during pure fermentation, while H. guilliermondii produced the most volatile aroma compounds (442.34 mg/L). The polyphenol content produced during sequential fermentation with S. cerevisiae and H. guilliermondii (140.24 mg/L) or C. ethanolica (140.21 mg/L) was significantly higher than other co-fermentations. Meanwhile, the volatile aroma compounds were found to be more abundant in S. cerevisiae/H. guilliermondii mixed fermentation (1:1 ratio) (588.35 mg/L) or S. cerevisiae/H. guilliermondii sequential fermentation (549.31 mg/L). Thus, S. cerevisiae/H. guilliermondii sequential fermentation could considerably boost the polyphenol and volatile aroma component contents in Nanfeng tangerine wines. The findings of this study can be used to drive strategies to increase the polyphenol content and sensory quality of tangerine wines and provide a reference for selecting the co-fermentation styles for non-Saccharomyces yeast and S. cerevisiae in fruit wine fermentation.

Concurrent production of glycyrrhetic acid 3-O-mono-ß-d-glucuronide and lignocellulolytic enzymes by solid-state fermentation of a plant endophytic Chaetomium globosum.

Glycyrrhetic acid 3-O-mono-ß-d-glucuronide (GAMG) as an important derivative of glycyrrhizin (GL) shows stronger biological activities and higher sweetness than GL. The biotransformation process is considered as an efficient strategy for GAMG production, due to its mild reaction, high production efficiency and environmentally friendly status. In this study, licorice straw was used for the first time as a medium for GAMG and lignocellulosic enzyme production via solid-state fermentation (SSF) of endophytic fungus Chaetomium globosum DX-THS3. The fermentation conditions including particle size, temperature, seed age, inoculum size, and moisture of substrate were optimized. Furthermore, additional nitrogen sources and carbon sources were screened for GAMG production by C. globosum DX-THS3 of SSF. Under optimal fermentation conditions, the percent conversion of glycyrrhizin reached 90% in 15 days, whereas the control needed 35 days to achieve the same result. The productivity of optimization (P = 2.1 mg/g/day) was 2.33-fold that of non-optimization (P = 0.9 mg/g/day). Meanwhile, high activities of filter paper enzyme (FPase) (245.80 U/g), carboxymethyl cellulase (CMCase) (33.67 U/g), xylanase (83.44 U/g), and ß-glucuronidase activity (271.42 U/g) were obtained faster than those in the control during SSF. Our study provides a novel and efficient strategy for GAMG production and indicates C. globosum DX-THS3 as a potential producer of lignocellulolytic enzymes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40643-021-00441-y.

Purification and characterization of a novel ß-glucuronidase precisely converts glycyrrhizin to glycyrrhetinic acid 3-O-mono-ß-D-glucuronide from plant endophytic Chaetomium globosum DX-THS3.

Glycyrrhetinic acid monoglucuronide (GAMG) is an innovative functional sweetener with higher sweetness and stronger pharmacological activity than glycyrrhizin (GL). A novel ß-glucuronidase (cg-GUS) was firstly screened from plant endophytic fungus Chaetomium globosum DX-THS3. The cg-GUS demonstrated the specify and highly transform glycyrrhizin (GL) to generate GAMG, and the maximum activity of ß-glucuronidase at 45 °C and pH 6.0, displaying excellent thermostability and pH-stability. The Km and Vmax values of cg-GUS were 0.134 mM and 236.42 mM/min/mg, respectively, which showed the high chemical bond selectivity and biotransformation efficiency of cg-GUS. Meanwhile, the cg-GUS gene (1896 bp) was analyzed, and Gly-345, Ser-539, Gly-563, Ala-579, Ser-581 and Glu-619 in GH2 catalytic domain of cg-GUS are potential mutation position for result in high-efficient and substrate-specify of cg-GUS. Our results were indicated that cg-GUS is a biocatalyst for production of GAMG and potent application in food and medicinal industry.

Microparticle-enhanced Chaetomium globosum DX-THS3 ß-d-glucuronidase production by controlled fungal morphology in submerged fermentation.

Glycyrrhetinic acid monoglucuronide (GAMG) is a novel and low-calorie sweetener that is widely applied in the food industry. This study aimed to enhance the production of fungal ß-d-glucuronidase (GUS) via a novel fermentation technique by evaluating the effects of the various microparticles on Chaetomium globosum DX-THS3 GUS production. Results showed that the silica microparticle greatly affected the morphology of DX-THS3 strain relative to the other microparticles. Microbial structure imaging results showed that the smallest average diameter of fungal pellets was achieved (0.7 ± 0.1 mm) by adding 10 g/L (600 mesh) of silica. The diameter of the control was 3.0 ± 0.5 mm in shake flask fermentation. The GUS activity and biomass of DX-THS3 reached 680 U/mL and 4.2 g/L, respectively, with the use of 10 g/L of silica microparticles, whereas those of the control were 210 U/mL and 2.8 g/L via shake flask fermentation. The findings in this study may provide a potential strategy for designing the morphology of filamentous fungi using microparticles in the industrial production of GAMG.

A Novel Strategy for Efficient Agaro-Oligosaccharide Production Based on the Enzymatic Degradation of Crude Agarose in Flammeovirga pacifica WPAGA1.

To avoid conflict between biofuel and food resource production, marine macroalgae (main algal polysaccharides) have been suggested as potent feedstock for biofuel production. Flammeovirga pacifica WPAGA1, a typical marine polysaccharide-degrading bacterium, can utilize crude agarose as the sole carbon source. Transcriptomic analysis was performed to further investigate the metabolic pathway of environmental-friendly utilization of crude agarose in F. pacifica WPAGA1. All these enzymes were overexpressed in Escherichia coli BL21(DE3), and the purified enzymes were characterized in vitro. As a result, the pathway of crude agarose which is desulfurized and hydrolyzed by enzymes to produce fermentable sugar is clear. Interestingly, sole neoagarobiose (~450 mg/L) was produced from crude agarose as a feedstock using engineered E. coli BL21(DE3). This study firstly reveals the metabolic pathway of crude agarose in strain WPAGA1 and establishes a novel and environmental-friendly strategy for neoagarobiose production using crude agarose as cost-effective and non-food-based feedstock.

Diversities and potential biogeochemical impacts of mangrove soil viruses.

BACKGROUND: Mangroves are ecologically and economically important forests of the tropics. As one of the most carbon-rich biomes, mangroves account for 11% of the total input of terrestrial carbon into oceans. Although viruses are considered to significantly influence local and global biogeochemical cycles, little information is available regarding the community structure, genetic diversity and ecological roles of viruses in mangrove ecosystems. METHODS: Here, we utilised viral metagenomics sequencing and virome-specific bioinformatics tools to study viral communities in six mangrove soil samples collected from different mangrove habitats in Southern China. RESULTS: Mangrove soil viruses were found to be largely uncharacterised. Phylogenetic analyses of the major viral groups demonstrated extensive diversity and previously unknown viral clades and suggested that global mangrove viral communities possibly comprise evolutionarily close genotypes. Comparative analysis of viral genotypes revealed that mangrove soil viromes are mainly affected by marine waters, with less influence coming from freshwaters. Notably, we identified abundant auxiliary carbohydrate-active enzyme (CAZyme) genes from mangrove viruses, most of which participate in biolysis of complex polysaccharides, which are abundant in mangrove soils and organism debris. Host prediction results showed that viral CAZyme genes are diverse and probably widespread in mangrove soil phages infecting diverse bacteria of different phyla. CONCLUSIONS: Our results showed that mangrove viruses are diverse and probably directly manipulate carbon cycling by participating in biomass recycling of complex polysaccharides, providing the knowledge essential in revealing the ecological roles of viruses in mangrove ecosystems.

Two-Stage Crystallization Combining Direct Succinimide Synthesis for the Recovery of Succinic Acid From Fermentation Broth.

Succinic acid is an important chemical and raw material widely used in medicine, food, biodegradable materials, fine chemicals, and other industrial fields. However, traditional methods for purifying succinic acid from fermentation broth are costly, poorly efficient, and harmful to the environment. In this study, an efficient method for purifying succinic acid from the fermentation broth of Escherichia coli NZN111 was developed through crystallization and co-crystallization with urea. First, the filtrate was collected by filtering the fermentation broth, and pH was adjusted to 2.0 by supplementing sulfuric acid. Crystallization was carried out at 8°C for 4 h to obtain succinic acid crystals. The recovery rate and purity of succinic acid were 73.4% and over 99%, respectively. Then, urea was added to the remaining solution with a mass ratio of urea to residual succinic acid of 4:1 (m urea /m SA ). The second crystallization was carried out at pH 2 and 4°C for 12 h to obtain succinic acid-urea co-crystal. The recovery rate of succinic acid residue was 92.0%. The succinic acid-urea crystal was further mixed with phosphorous acid (4.2% of the mass of succinic acid co-crystal) and maintained at 195°C for 6 h to synthesize succinimide, and the yield was >80%. This novel and efficient purification process was characterized by the significantly reduced urea consumption, and high succinic acid recovery (totally 95%), and high succinimide synthesis yield (80%). Thus, this study potentially provided a novel and efficient strategy for the industrial production of succinic acid and succinimide.

Metagenomics Investigation of Agarlytic Genes and Genomes in Mangrove Sediments in China: A Potential Repertory for Carbohydrate-Active Enzymes.

Monosaccharides and oligosaccharides produced by agarose degradation exhibit potential in the fields of bioenergy, medicine, and cosmetics. Mangrove sediments (MGSs) provide a special environment to enrich enzymes for agarose degradation. However, representative investigations of the agarlytic genes in MGSs have been rarely reported. In this study, agarlytic genes in MGSs were researched in detail from the aspects of diversity, abundance, activity, and location through deep metagenomics sequencing. Functional genes in MGSs were usually incomplete but were shown as results, which could cause virtually high number of results in previous studies because multiple fragmented sequences could originate from the same genes. In our work, only complete and nonredundant (CNR) genes were analyzed to avoid virtually high amount of the results. The number of CNR agarlytic genes in our datasets was significantly higher than that in the datasets of previous studies. Twenty-one recombinant agarases with agarose-degrading activity were detected using heterologous expression based on numerous complete open-reading frames, which are rarely obtained in metagenomics sequencing of samples with complex microbial communities, such as MGSs. Aga2, which had the highest crude enzyme activity among the 21 recombinant agarases, was further purified and subjected to enzymatic characterization. With its high agarose-degrading activity, resistance to temperature changes and chemical agents, Aga2 could be a suitable option for industrial production. The agarase ratio with signal peptides to that without signal peptides in our MGS datasets was lower than that of other reported agarases. Six draft genomes, namely, Clusters 1-6, were recovered from the datasets. The taxonomic annotation of these genomes revealed that Clusters 1, 3, 5, and 6 were annotated as Desulfuromonas sp., Treponema sp., Ignavibacteriales spp., and Polyangiaceae spp., respectively. Meanwhile, Clusters 2 and 4 were potential new species. All these genomes were first reported and found to have abilities of degrading various important polysaccharides. The metabolic pathway of agarose in Cluster 4 was also speculated. Our results showed the capacity and activity of agarases in the MGS microbiome, and MGSs exert potential as a repertory for mining not only agarlytic genes but also almost all genes of the carbohydrate-active enzyme family.

Genome Sequencing Reveals the Complex Polysaccharide-Degrading Ability of Novel Deep-Sea Bacterium Flammeovirga pacifica WPAGA1.

Flammeovirga pacifica strain WPAGA1 is a Gram-negative, polysaccharide-degrading bacterium isolated from the marine sediment of the West Pacific Ocean. This strain is a cosmopolitan marine bacterium that uses complex polysaccharides as exclusive source of carbon and energy and plays a key role in the marine carbon cycle. Genome sequence analysis of strain WPAGA1 revealed that the assembled fine genome contains 6,610,326 bp with 32.89% G+C content, 5036 open reading frames (ORFs) and abundant genomic elements. Amongst these ORFs, 1022 genes encoding carbohydrate enzymes were found in the F. pacifica WPAGA1 genome. In addition, abundant putative enzymes involved in degrading polysaccharide were found. These enzymes include amylase, xylosidase, cellulase, alginate lyase, pectate lyase, rhamnogalacturonan lyase, chitinase, carrageenase, heparinase and fucosidase. To further investigate the use of these polysaccharides in strain WPAGA1, a schematic of various polysaccharide-degrading metabolic pathways were deduced from the genome sequence. This study showed that strain WPAGA1 may serve as a potential candidate for research of glycometabolism and have potential biotechnological and industrial applications and play key roles in the marine carbon cycle.