Search

Xylomyrocins, a unique group of nonribosomal peptide secondary metabolites, were discovered in Paramyrothecium and Colletotrichum spp. fungi by employing a combination of high-resolution tandem mass spectrometry (HRMS/MS)–based chemometrics, comparative genome mining, gene disruption, stable isotope feeding, and chemical complementation techniques. These polyol cyclodepsipeptides all feature an unprecedented d -xylonic acid moiety as part of their macrocyclic scaffold. This biosynthon is derived from d -xylose supplied by xylooligosaccharide catabolic enzymes encoded in the xylomyrocin biosynthetic gene cluster, revealing a novel link between carbohydrate catabolism and nonribosomal peptide biosynthesis. Xylomyrocins from different fungal isolates differ in the number and nature of their amino acid building blocks that are nevertheless incorporated by orthologous nonribosomal peptide synthetase (NRPS) enzymes. Another source of structural diversity is the variable choice of the nucleophile for intramolecular macrocyclic ester formation during xylomyrocin chain termination. This nucleophile is selected from the multiple available alcohol functionalities of the polyol moiety, revealing a surprising polyspecificity for the NRPS terminal condensation domain. Some xylomyrocin congeners also feature N- methylated amino acid residues in positions where the corresponding NRPS modules lack N- methyltransferase (M) domains, providing a rare example of promiscuous methylation in the context of an NRPS with an otherwise canonical, collinear biosynthetic program.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10 µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

Dietary intervention with plant protein is one of the main methods that is used to lessen the symptoms of malnutrition. Supplementary soy protein to undernourished weaning rats for 6 weeks significantly increased their body weight gain. After the intervention, the level of total short-chain fatty acids (SCFAs) was restored to 1,512.7 μg/g, while the level was only 637.1 μg/g in the 7% protein group. The amino acids (valine, isoleucine, phenylalanine, and tryptophan) increased in the colon, and vitamin B6 metabolism was significantly influenced in undernourished rats. The tryptophan and glycine-serine-threonine pathways were elevated, leading to an increase in the level of tryptophan and 5-hydroxytryptophan (5-HTP) in the serum. In addition, the relative abundance of Lachnospiraceae_NK4A136_group and Lactobacillus increased, while Enterococcus and Streptococcus decreased compared to undernourished rats. Overall, soy protein improved the growth of rats with malnutrition in early life by regulating gut microbiota and metabolites in the colon and serum.

Increasing attention has been focused on plant-derived peptides because of their potential bioactivities. In this study, bioactive peptides were released from extruded adzuki bean protein by simulated gastrointestinal digestion. A peptide (KQS-1) sequenced as KQSESHFVDAQPEQQQR was separated and identified using ultrafiltration, pre-high-performance liquid chromatography (HPLC), and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). KQS-1 was shown to exert significant anti-inflammatory effects in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages by reducing the production of IL-1, IL-6, TNF-α, and MCP-1 to 38.31, 6.07, 43.96, and 41.74%, respectively. The involved signaling pathways were identified by transcriptome analysis. Overall, 5236 differentially expressed genes (DEGs) were identified. Gene ontology (GO) functions demonstrated that DEGs were significantly related to the NF-κB pathway. In conclusion, KQS-1 prevented the activation and expression of NF-κB/caspase-1 by upstream and downstream factors. These findings highlight the bioactivity of adzuki bean peptides.

Sweet sorghum [Sorghum bicolor (L.) Moench] accumulates large amounts of sugars in the stem parenchyma and can be used to manufacture sucrose and alcohol. Soluble acid invertase (SAI) plays an important role in sucrose accumulation in the sweet sorghum stem. Characterization of SAI genes and development of functional markers are important for marker-assisted selection in breeding programs. Here, the full-length genomic DNA sequence of a SAI gene (SAI-1) was cloned by in silico cloning and experimentally validated. The SAI-1 gene comprises three exons and two introns, totaling 4,966 bp, with an open reading frame of 2,025 bp encoding a polypeptide of 674 amino acids. The protein encoded by SAI-1 (molecular weight 73.4 kDa) contains a glycosyl hydrolase family 32 conserved domain, which catalyzes the hydrolysis of sucrose. Four allelic variants at the SAI-1 locus, SAI-1a, SAI-1b, SAI-1c and SAI-1d (GenBank accession nos. JX535515, JX535516, JX535517 and JX535518), were identified in 165 sweet sorghum cultivars. A co-dominant marker, SBX1, was developed to discriminate these variants. The mean Brix value from cultivars with a 141-bp (SAI-1c) PCR product was higher than in cultivars with a 136-bp (SAI-1b) product, and the mean Brix value from cultivars with a 136-bp (SAI-1b) product was significantly higher than those with a 132-bp (SAI-1a) product. The results showed that a domain of ATTGA repeat sequences in the second intron might be an important regulatory site, and that the functional marker SBX1 was closely related to the Brix value, and could therefore be used in sweet sorghum breeding programs.

Background and Purpose: Aspergillus genus biosynthesize a large number of metabolites with wide-ranging bioactivities, but how to target the active compounds quickly and accurately? In this work, we provide a strategy to fast identify bioactive metabolites in fungi with similar morphology but different antimicrobial activities. Materials and Methods: Combined with High Performance Liquid Chromatography (HPLC) profiles and bioactivity screening of fungal extracts, three Aspergillus sp. strains YSN038, YSN052 and YSN064 were studied. The bacteria Bacillus pumilus , Bacillus subtilis , Staphylococcus aureus , Kocuria rhizophila , Escherichia coli and Ralstonia solanacearum were used for antibiotic assay. Results: Three strains showed the similar morphology, but the crude extracts of YSN038 and YSN064 displayed antibacterial activities against B. pumilus, B. subtilis, S. aureus and K. rhizophila, whereas YSN052 had not. The compound contributed to antibacterial activity in vitro was rapidly identified, isolated and characterized. Compound 3 connected with specific HPLC peak should be the antibiotic substance by analysis of HPLC profiles, and was confirmed after the following antimicrobial tests. The compound was elucidated as butyrolactone I based on nuclear magnetic resonance(NMR) and mass spectrometry (MS) data, and showed various biological activities. Conclusions: Our study has significant scope on targeting antibacterial metabolites. The described method could also be used as a rapid and cost-effective tool for screening other bioactivity products. As such, this article could offer a fast approach to isolate drug-lead compounds from microorganisms.

A highly N -phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.

Glyphosate, a powerful nonselective herbicide, acts as an inhibitor of the activity of the enzyme 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by the aroA gene involved in aromatic amino acid biosynthesis. An Escherichia coli mutant AKM4188 was constructed by insertion a kanamycin cassette within the aroA coding sequence. The mutant strain is an aromatic amino acids auxotroph and fails to grow on M9 minimal media due to the inactive aroA. A DNA metagenomic library was constructed with samples from a glyphosate-polluted area and was screened by using the mutant AKM4188 as recipient. Three plasmid clones, which restored growth to the aroA mutant in M9 minimal media supplemented with chloramphenicol, kanamycin, and 50 mM: glyphosate, were obtained from the DNA metagenomic library. One of them, which conferred glyphosate tolerance up to 150 mM: , was further characterized. The cloned fragment encoded a polypeptide, designated RD, sharing high similarity with other Class II EPSPS proteins. A His-tagged RD fusion protein was produced into E. coli to characterize the enzymatic properties of the RD EPSP protein.

This study aimed to investigate the effects of different concentrations of sodium bicarbonate (NaHCO3) on the accumulation of flavonoids, total phenolics and d-chiro-inositol (DCI), as well as the antioxidant and α-glucosidase inhibitory activities, in tartary buckwheat sprouts. Treatment with low concentrations of NaHCO3 (0.05, 0.1, and 0.2%) resulted in an increase in flavonoids, total phenolic compounds and DCI concentrations, and improved DPPH radical-scavenging and α-glucosidase inhibition activities compared with the control (0%). The highest levels of total flavonoids (26.69mg/g DW), individual flavonoids (rutin, isoquercitrin, quercetin, and kaempferol), total phenolic compounds (29.31mg/g DW), DCI (12.56mg/g DW), as well as antioxidant and α-glucosidase inhibition activities, were observed in tartary buckwheat sprouts treated with 0.05% NaHCO3 for 96h. These results indicated that appropriate treatment with NaHCO3 could improve the healthy benefits of tartary buckwheat sprouts.

A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media. 12 unique sites, which can tolerate a 5-aa insertion, were identified. In all of the 12 sites, only F295/T296 site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799. The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp.DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296lc. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296lc, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

In order to explore the feasibility of planting sweet sorghum in sugarcane growing area to prolong milling duration for bioethanol production, 15 varieties were sown monthly from March to September in Liuzhou of Central Guangxi Zhuang Autonomous Region. Yields of fresh stem, grain and leave were documented. The results showed that all varieties grew well when sown from March to August, but could not get mature when sown after late September. The high fresh stem yields were observed for the varieties Sart and PT3-S, 79.28 t/hm2 and 78.58 t/hm2 for single growing season, and 157.95 t/hm2 and 155.25 t/hm2 for two growing seasons. Ripening began from the end of June to late December, making the feed stock available for ethanol production from July to the end of December, even January next year.

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.