Cordyceps militaris is an entomopathogenic fungus which is often used in Asia as a traditional medicine developed from age-old wisdom. Presently, cordycepin from C. militaris is a great interest in medicinal applications. However, cellular growth of C. militaris and the association with cordycepin production remain poorly understood. To explore the metabolism of C. militaris as potential cell factories in medical and biotechnology applications, this study developed a high-quality genome-scale metabolic model of C. militaris, iNR1329, based on its genomic content and physiological data. The model included a total of 1329 genes, 1821 biochemical reactions, and 1171 metabolites among 4 different cellular compartments. Its in silico growth simulation results agreed well with experimental data on different carbon sources. iNR1329 was further used for optimizing the growth and cordycepin overproduction using a novel approach, POPCORN, for rational design of synthetic media. In addition to the high-quality GEM iNR1329, the presented POPCORN approach was successfully used to rationally design an optimal synthetic medium with C:N ratio of 8:1 for enhancing 3.5-fold increase in cordycepin production. This study thus provides a novel insight into C. militaris physiology and highlights a potential GEM-driven method for synthetic media design and metabolic engineering application. The iNR1329 and the POPCORN approach are available at the GitHub repository: https://github.com/sysbiomics/Cordyceps_militaris-GEM.

Lactiplantibacillus pentosus 9D3, a prominent gamma-aminobutyric acid (GABA)-producing bacteria isolated from Thai pickled weed was characterized for its safety and probiotic properties via whole-genome analysis and in vitro testing. The whole-genome sequence of L. pentosus 9D3 was determined using a hybrid-sequencing approach, combining PacBio and Illumina technologies. A 3.81-Mbp genome of L. pentosus 9D3 consisting of one 3.65-Mbp chromosome and six plasmids (1.9–71.9 Kbp) was identified with an estimated GC content of 46.09% and 3,456 predicted genes. The strain was confirmed to be Lactiplantibacillus pentosus according to the high average nucleotide identity value of >95% and digital DNA–DNA hybridization scores of >70% to the L. pentosus type strain. Comparative genome analysis with other L. pentosus strains showed that the GABA-producing capability was specific to the strain 9D3. Genes related to GABA biosynthesis and transport were identified on a plasmid, pLPE-70K, indicating the acquired nature of this property. The safety of L. pentosus 9D3 was demonstrated through the lack of genes related to the production of toxins, biogenic amines, and antimicrobial drugs. Although the strain exhibited resistance to ampicillin and chloramphenicol, none of the antimicrobial resistance (AMR) genes were associated with mobile elements, i.e., plasmids and prophages. Therefore, the strain is considered to have low risk of transferring the AMR genes to other, potentially pathogenic bacteria. In addition, L. pentosus 9D3 showed good survivability in the gastrointestinal tract environment and was able to adhere to the intestinal cell in vitro. Therefore, L. pentosus 9D3 is concluded to be safe, with the potential to be used as a probiotic, exerting its health benefit through GABA production in the food system. The GABA-producing capability of the strain in vivo is the subject of further investigation.

The cellular metabolic adaptations ofCordyceps militarishave been progressively studied. In particular, the cordycepin pathway is of interest in medicinal applications. Even though the metabolic pathways for cordycepin production are known to be related to different carbon sources, the regulatory mechanisms at a systems level are poorly characterized. To explore the regulatory mechanisms, this study therefore aimed to investigate the global metabolic response to cordycepin production inC. militaristhrough transcriptome analysis and genome-scale network-driven analysis. Here, transcriptome analysis of 16,805 expressed genes inC. militarisstrain TBRC6039 grown on different carbon sources was performed. Of these genes, 2,883 were significantly differentially expressed genes, uncovering sucrose- and glucose-mediated changes in the transcriptional regulation of central carbon metabolism inC. militaris, which was shown using the CmSNF1 mechanism as an example. After applying genome-scale metabolic network-driven analysis, reporter metabolites and key metabolic subnetworks involving adenosine, cordycepin and methionine were proposed through the up-regulation of cordycepin biosynthetic genes. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific culture conditions during cordycepin overproduction.

The gut microbiome plays a major role in the maintenance of human health. Characterizing the taxonomy and metabolic functions of the human gut microbiome is necessary for enhancing health. Here, we analyzed the metagenomic sequencing, assembly and construction of a meta-gene catalogue of the human gut microbiome with the overall aim of investigating the taxonomy and metabolic functions of the gut microbiome in Thai adults. As a result, the integrative analysis of 16S rRNA gene and whole metagenome shotgun (WMGS) sequencing data revealed that the dominant gut bacterial families were Lachnospiraceae and Ruminococcaceae of the Firmicutes phylum. Consistently, across 3.8 million (M) genes annotated from 163.5 gigabases (Gb) of WMGS sequencing data, a significant number of genes associated with carbohydrate metabolism of the dominant bacterial families were identified. Further identification of bacterial community-wide metabolic functions promisingly highlighted the importance of Roseburia and Faecalibacterium involvement in central carbon metabolism, sugar utilization and metabolism towards butyrate biosynthesis. This work presents an initial study of shotgun metagenomics in a Thai population-based cohort in a developing Southeast Asian country.