A Berberine Bridge Enzyme-like Oxidase Mediates the Cage-like Acresorbicillinol C Biosynthesis.

Oxosorbicillinol and cage-like acresorbicillinol C are bioactive sorbicillinoids produced by Acremonium chrysogenum. We found that a berberine bridge enzyme-like oxidase AcsorD was responsible for their biosynthesis by gene deletion and heterologous expression. AcsorD catalyzed oxidation of sorbicillinol to form oxosorbicillinol in in vitro assays, which was successively condensed with sorbicillinol to form acresorbicillinol C spontaneously. Finally, site-directed mutation revealed that Tyr525 was the key residue in the catalysis of the oxidation reaction and unlocking cage-like acresorbicillinol C production.

Caesalpinflavans D-F and caesalpinnone B, hybrid flavan-chalcones and normonoterpene-chalcone heterodimer from Caesalpinia digyna.

Three undescribed hybrid flavan-chalcones, caesalpinflavans D-F, and an unreported normonoterpene-chalcone heterodimer, caesalpinnone B, along with three known biflavonoids were isolated from the twigs and leaves of Caesalpinia digyna. Their structures were elucidated based on extensive spectroscopic analysis and quantum chemical calculations. Caesalpinflavan F was identified as a bis-(hybrid flavan-chalcone), its natural occurrence was supported by HPLC-IT-TOF-MS analysis. The condensation of caesalpinflavan B with acetone was possibly a key step in the biosynthesis of caesalpinflavan F. Caesalpinnone B represents an unprecedented meroterpenoid featuring a cyclobutane central framework, which was derived from chalcone and normonoterpenoid via a key [2 + 2] cyclization reaction. Biological evaluation revealed that compounds caesalpinflavan D, oxytrodiflavanone A, and caesalpinnone B exhibited moderate cytotoxicity against HL-60, SMMC-7721, SW480, A-549 and/or MDA-MB-231 cell lines with IC50 values ranging from 8.051 ± 0.673 to 24.26 ± 0.61 µM. This study provided evidence for further research and possible utilization of C. digyna in the future.

Exploring the Role of Active Functional Microbiota in Flavor Generation by Integrated Metatranscriptomics and Metabolomics during Niulanshan Baijiu Fermentation.

Active functional microbiota for producing volatile flavors is critical to Chinese baijiu fermentation. Microbial communities correlated with the volatile metabolites are generally explored using DNA-based sequencing and metabolic analysis. However, the active functional microbiota related to the volatile flavor compounds is poorly understood. In this study, an integrated metatranscriptomic and metabolomics analysis was employed to unravel the metabolite profiles comprehensively and the contributing active functional microbiota for flavor generation during Niulanshan baijiu fermentation. A total of 395, 83, and 181 compounds were annotated using untargeted metabolomics, including LC-MS, GC-MS, and HS-SPME-GC-MS, respectively. Significant variances were displayed in the composition of compounds among different time-point samples according to the heatmaps and orthogonal partial least-square discriminant analysis. The correlation between the active microbiota and the volatile flavors was analyzed based on the bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) model. Six bacterial genera, including Streptococcus, Lactobacillus, Pediococcus, Campylobacter, Yersinia, and Weissella, and five fungal genera of Talaromyces, Aspergillus, Mixia, Rhizophagus, and Gloeophyllum were identified as the active functional microbiota for producing the volatile flavors. In summary, this study revealed the active functional microbial basis of unique flavor formation and provided novel insights into the optimization of Niulanshan baijiu fermentation.

Metatranscriptomics Unravel Composition, Drivers, and Functions of the Active Microorganisms in Light-Flavor Liquor Fermentation.

The microbial community in the fermented pit determines the quantity and quality of light-flavor liquor. Genetic diversity and the potential functions of the microbial community are often analyzed by DNA-based omics sequencing. However, the features of the active microbial community have not been systematically studied. Here, metatranscriptomic analysis was performed to elucidate the active microbial composition, drivers, and their functions in light-flavor liquor fermentation. Bacterial genera, Lactobacillus, Streptococcus, Pediococcus, Thermotoga, and Faecalibacterium, and fungal genera, Saccharomyces, Talaromyces, Aspergillus, Clavispora, Rhizophagus, Cyberlindnera, and Wickerhamomyces, were the dominant active microorganisms during the fermentation process. Additionally, they dominated the three-stage fermentation successively. Redundancy analysis showed that pH, ethanol, moisture, and starch were the main driving forces of microbial succession. Among the genes for the respective carbohydrate-active enzyme families, those for the glycoside hydrolase family 23, the glycosyltransferase family 2, the carbohydrate-binding module family 50, the polysaccharide lyase family 4, the auxiliary activity family 1, and the carbohydrate esterase family 9 showed the highest expression level. Additionally, the highly expressed enzymes and their contributed microorganisms were found in the key KEGG pathways, including carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Based on these data, a functional model of carbohydrate hydrolysis, ethanol production, and flavor generation were proposed. Taken together, Saccharomyces, Lactobacillus, Wickerhamomyces, Pediococcus, Candida, and Faecalibacterium were suggested as the core active microorganisms. Overall, our findings provide new insights into the composition, drivers, and functions of the active microorganisms, which is crucial for improving the quality of light-flavor liquor. IMPORTANCE There is an urgent need for discovering the diversity and functions of the active microbial community in solid-state fermentation, especially in the pit of Chinese distilled liquor fermentation. Although the genetic composition of the microbial community has been clarified frequently by DNA-based sequencing, the composition and functions of the active microbial community have not been systematically revealed so far. Therefore, analysis of RNA-based data is crucial for discovering the functional microbial community. In this study, we employed metatranscriptomic analysis to elucidate the active microbial composition, successive drivers, and their functions in light-flavor liquor fermentation. The strategy can be broadly useful for discovering the active microbial community and exploring their functions in other types of flavor distilled liquor or other ecosystems. This study provides new insights into the understanding of the active microbial community composition and its functions.

Sorbicillinoid Derivatives with the Radical Scavenging Activities from the Marine-Derived Fungus Acremonium chrysogenum C10.

Sorbicillinoids are a class of structurally diverse hexaketide metabolites with good biological activities. To explore new structural sorbicillinoids and their bioactivities, the marine-derived fungus Acremonium chrysogenum C10 was studied. Three new sorbicillinoid derivatives, acresorbicillinols A-C (1-3), along with five known ones, trichotetronine (4), trichodimerol (5), demethyltrichodimerol (6), trichopyrone (7) and oxosorbicillinol (8), were isolated. The structures of new sorbicillinoids were elucidated by analysis of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS). The absolute configurations of compounds 1-3 were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Compound 3 exhibited a strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, with the IC50 value ranging from 11.53 ± 1.53 to 60.29 ± 6.28 µM in 24 h. Additionally, compounds 2 and 3 showed moderate activities against Staphylococcus aureus and Cryptococcus neoformans, with IC50 values of 86.93 ± 1.72 and 69.06 ± 10.50 µM, respectively. The boundary of sorbicillinoid biosynthetic gene cluster in A. chrysogenum was confirmed by transcriptional analysis, and the biosynthetic pathway of compounds 1-8 was also proposed. In summary, our results indicated that A. chrysogenum is an important reservoir of sorbicillinoid derivatives, and compound 3 has the potential for new natural agents in DPPH radical scavenging.

Improvement of the CRISPR-Cas9 mediated gene disruption and large DNA fragment deletion based on a chimeric promoter in Acremonium chrysogenum.

Acremonium chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. chrysogenum.