Metagenomic and metaproteomic analyses of microbial amino acid metabolism during Cantonese soy sauce fermentation.

Cantonese soy sauce is an important type of traditional Chinese brewed soy sauce that was developed in southern China, mainly in Guangdong. Due to the long fermentation period and complex microbiota in Cantonese soy sauce, there are few reports on the microbial metaproteomics of Cantonese soy sauce. In this study, integrative metagenomic and metaproteomic analyzes were used to identify the changes in the dominant microbiota and amino acid synthesis-related enzymes and metabolism during Cantonese soy sauce fermentation. Metagenomic analysis revealed that Tetragenococcus halophilus, Weissella confusa, Weissella paramesenteroides, Enterobacter hormaechei, and Aspergillus oryzae were the dominant microbiota. Using the Top 15 dominant microbiota identified by metagenomics as the database, LTQ Orbitrap Velos Pro ETD mass spectrometry was used to obtain metaproteomic information about the microbes in the soy sauce, and the results indicated that the active enzymes involved in the metabolism of amino acids were secreted by microorganisms such as A. oryzae, T. halophilus, and Zygosaccharomyces rouxii. During the Cantonese soy sauce fermentation process. Among them, early fermentation (0-15d) was dominated by A. oryzae and T. halophilus, mid-term fermentation (60-90d) was dominated by Z. rouxii, A. oryzae, and T. halophilus, and late fermentation (90-120d) was dominated by A. oryzae, Z. rouxii, and T. halophilus. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the main enzymes involved in the metabolism of umami amino acids were aspartate aminotransferase, citrate synthase, aconitase, and isocitrate dehydrogenase, which were produced by Z. rouxii and A. oryzae during early fermentation (0-15 d) and the middle fermentation stage (60-90 d). This study constructed a regulatory network of enzymes potentially involved in the metabolism of flavor amino acids, which provided a theoretical basis for studying the amino acid metabolism of Cantonese soy sauce.

Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production.

Echium plantagineum L. (Boraginaceae) is an invasive species in Australia and contains medicinal shikonins in its roots. In this study, the hairy root lines of E. plantagineum were established using Agrobacterium rhizogenes strain ATCC15834 and confirmed by the amplification of the rolB gene. Results showed significant difference in shikonin production between the hairy root lines in the 1/2B5 and M9 media. The biomass of the lines in the 1/2B5 medium was fivefold of that in the M9 medium. However, the components of detected shikonins were similar in these two liquid media. By contrast, different accumulation profiles appeared in the hairy root lines. HPLC analysis revealed the presence of nine possible related compounds, including shikonins, and acetylshikonin was the most abundant shikonin derivative. The content of acetylshikonin in the 1/2B5 medium (36.25 mg/L on average) was twofold of that in the M9 medium. Our results showed that the hairy root cultures of E. plantagineum can be used in enhancing the production of potential pharmaceutical compounds, such as acetylshikonin.

Design, synthesis and biological evaluation of benzoylacrylic acid shikonin ester derivatives as irreversible dual inhibitors of tubulin and EGFR.

In this study, a series of shikonin derivatives combined with benzoylacrylic had been designed and synthesized, which showed an inhibitory effect on both tubulin and the epidermal growth factor receptor (EGFR). In vitro EGFR and cell growth inhibition assay demonstrated that compound PMMB-317 exhibited the most potent anti-EGFR (IC50 = 22.7 nM) and anti-proliferation activity (IC50 = 4.37 µM) against A549 cell line, which was comparable to that of Afatinib (EGFR, IC50 = 15.4 nM; A549, IC50 = 6.32 µM). Our results on mechanism research suggested that, PMMB-317 could induce the apoptosis of A549 cells in a dose- and time-dependent manner, along with decrease in mitochondrial membrane potential (MMP), production of ROS and alterations in apoptosis-related protein levels. Also, PMMB-317 could arrest cell cycle at G2/M phase to induce cell apoptosis, and inhibit the EGFR activity through blocking the signal transduction downstream of the mitogen-activated protein MAPK pathway and the anti-apoptotic kinase AKT pathway; typically, such results were comparable to those of afatinib. In addition, PMMB-317 could suppress A549 cell migration through the Wnt/ß-catenin signaling pathway in a dose-dependent manner. Additionally, molecular docking simulation revealed that, PMMB-317 could simultaneously combine with EGFR protein (5HG8) and tubulin (1SA0) through various forces. Moreover, 3D-QSAR study was also carried out, which could optimize our compound through the structure-activity relationship analysis. Furthermore, the in vitro and in vivo results had collectively confirmed that PMMB-317 might serve as a promising lead compound to further develop the potential therapeutic anticancer agents.

Identification of new shikonin derivatives as STAT3 inhibitors.

The signal transducer and activator of transcription 3 is a constitutively activated oncogenic protein in various human tumors and represents a valid target for anticancer drug design. In this study, we have achieved a new type of STAT3 inhibitors based on structural modifications on shikonin scaffold, guided by computational modelling. By tests, PMMB-187 exhibited a more outstanding profile than shikonin on a small panel of human breast cancer cells, especially for the MDA-MB-231 cells. For the cellular mechanisms research, PMMB-187 was found to induce cell apoptosis in MDA-MB-231 cells, associated with the reduction of mitochondrial membrane potential, production of ROS and alteration of the levels of apoptosis-related proteins. Furthermore, PMMB-187 inhibited constitutive/inducible STAT3 activation, transcriptional activity, nuclear translocation and downstream target genes expression in STAT3-dependent breast cancer cells MDA-MB-231. Besides, no obvious inhibitory effect on activation of STAT1 and STAT5 was observed with PMMB-187 treatment. Most notably, the in vivo studies further revealed that PMMB-187 could dramatically suppress the MDA-MB-231 cells xenografted tumor growth. The in vitro and in vivo results collectively suggest that PMMB-187 may serve as a promising lead compound for the further development of potential therapeutic anti-neoplastic agents.

Transcriptome analysis explores genes related to shikonin biosynthesis in Lithospermeae plants and provides insights into Boraginales' evolutionary history.

Shikonin and its derivatives extracted from Lithospermeae plants' red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in "shikonin downstream biosynthesis" and "methyl jasmonate biosynthesis" were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.