Dissecting Holistic Metabolic Acclimatization of Mucor circinelloides WJ11 Defective in Carotenoid Biosynthesis.

Mucor circinelloides WJ11 is a lipid-producing strain with industrial potential. A holistic approach using gene manipulation and bioprocessing development has improved lipid production and the strain's economic viability. However, the systematic regulation of lipid accumulation and carotenoid biosynthesis in M. circinelloides remains unknown. To dissect the metabolic mechanism underlying lipid and carotenoid biosynthesis, transcriptome analysis and reporter metabolites identification were implemented between the wild-type (WJ11) and ΔcarRP WJ11 strains of M. circinelloides. As a result, transcriptome analysis revealed 10,287 expressed genes, with 657 differentially expressed genes (DEGs) primarily involved in amino acid, carbohydrate, and energy metabolism. Integration with a genome-scale metabolic model (GSMM) identified reporter metabolites in the ΔcarRP WJ11 strain, highlighting metabolic pathways crucial for amino acid, energy, and nitrogen metabolism. Notably, the downregulation of genes associated with carotenoid biosynthesis and acetyl-CoA generation suggests a coordinated relationship between the carotenoid and fatty acid biosynthesis pathways. Despite disruptions in the carotenoid pathway, lipid production remains stagnant due to reduced acetyl-CoA availability, emphasizing the intricate metabolic interplay. These findings provide insights into the coordinated relationship between carotenoid and fatty acid biosynthesis in M. circinelloides that are valuable in applied research to design optimized strains for producing desired bioproducts through emerging technology.

Light-Exposed Metabolic Responses of Cordyceps militaris through Transcriptome-Integrated Genome-Scale Modeling.

The genome-scale metabolic model (GSMM) of Cordyceps militaris provides a comprehensive basis of carbon assimilation for cell growth and metabolite production. However, the model with a simple mass balance concept shows limited capability to probe the metabolic responses of C. militaris under light exposure. This study, therefore, employed the transcriptome-integrated GSMM approach to extend the investigation of C. militaris's metabolism under light conditions. Through the gene inactivity moderated by metabolism and expression (GIMME) framework, the iPS1474-tiGSMM model was furnished with the transcriptome data, thus providing a simulation that described reasonably well the metabolic responses underlying the phenotypic observation of C. militaris under the particular light conditions. The iPS1474-tiGSMM obviously showed an improved prediction of metabolic fluxes in correlation with the expressed genes involved in the cordycepin and carotenoid biosynthetic pathways under the sucrose culturing conditions. Further analysis of reporter metabolites suggested that the central carbon, purine, and fatty acid metabolisms towards carotenoid biosynthesis were the predominant metabolic processes responsible in light conditions. This finding highlights the key responsive processes enabling the acclimatization of C. militaris metabolism in varying light conditions. This study provides a valuable perspective on manipulating metabolic genes and fluxes towards the target metabolite production of C. militaris.

Functional genomics and systems biology of Cordyceps species for biotechnological applications.

The use of Cordyceps species for the manufacture of natural products has been established; however, the tremendous advances observed in recent years in genetic engineering and molecular biology have revolutionized the optimization of Cordyceps as cell factories and drastically expanded the biotechnological potential of these fungi. Here, we present a review of systems and synthetic biology studies of Cordyceps and their implications for fungal biology and industrial applications. We summarize the current status of synthetic biology for enhancing targeted metabolites in Cordyceps species, such as cordycepin, adenosine, polysaccharide, and pentostatin. Progress in the systems and synthetic biology of Cordyceps provides a strategy for comprehensively comprehensive controlling efficient cell factories of natural bioproducts and novel synthetic biology toolbox for targeted engineering.

Revealing holistic metabolic responses associated with lipid and docosahexaenoic acid (DHA) production in Aurantiochytrium sp. SW1.

Aurantiochytrium sp. SW1, a marine thraustochytrid, has been regarded as a potential candidate as a docosahexaenoic acid (DHA) producer. Even though the genomics of Aurantiochytrium sp. are available, the metabolic responses at a systems level are largely unknown. Therefore, this study aimed to investigate the global metabolic responses to DHA production in Aurantiochytrium sp. through transcriptome and genome-scale network-driven analysis. Of a total of 13,505 genes, 2527 differentially expressed genes (DEGs) were identified in Aurantiochytrium sp., unravelling the transcriptional regulations behinds lipid and DHA accumulation. The highest number of DEG were found for pairwise comparison between growth phase and lipid accumulating phase where a total of 1435 genes were down-regulated with 869 genes being up-regulated. These uncovered several metabolic pathways that contributing in DHA and lipid accumulation including amino acid and acetate metabolism which involve in the generation of crucial precursors. Upon applying network-driven analysis, hydrogen sulphide was found as potential reporter metabolite that could be associated with the genes related to acetyl-CoA synthesis for DHA production. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific cultivation phases during DHA overproduction in Aurantiochytrium sp. SW1.

Whole-genome sequence analysis for evaluating the safety and probiotic potential of Lactiplantibacillus pentosus 9D3, a gamma-aminobutyric acid (GABA)-producing strain isolated from Thai pickled weed.

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.

Enhancing Genome-Scale Model by Integrative Exometabolome and Transcriptome: Unveiling Carbon Assimilation towards Sphingolipid Biosynthetic Capability of Cordyceps militaris.

Cordyceps militaris is an industrially important fungus, which is often used in Asia as traditional medicine. There has been a published genome-scale metabolic model (GSMM) of C. militaris useful for predicting its growth behaviors; however, lipid metabolism, which plays a vital role in cellular functions, remains incomplete in the GSMM of C. militaris. A comprehensive study on C. militaris was thus performed by enhancing GSMM through integrative analysis of metabolic footprint and transcriptome data. Through the enhanced GSMM of C. militaris (called iPC1469), it contained 1469 genes, 1904 metabolic reactions and 1229 metabolites. After model evaluation, in silico growth simulation results agreed well with the experimental data of the fungal growths on different carbon sources. Beyond the model-driven integrative data analysis, interestingly, we found key metabolic responses in alteration of lipid metabolism in C. militaris upon different carbon sources. The sphingoid bases (e.g., sphinganine, sphingosine, and phytosphingosine) and ceramide were statistically significant accumulated in the xylose culture when compared with other cultures; this study suggests that the sphingolipid biosynthetic capability in C. militaris was dependent on the carbon source assimilated for cell growth; this finding provides a comprehensive basis for the sphingolipid biosynthesis in C. militaris that can help to further redesign its metabolic control for medicinal and functional food applications.

Whole genome analysis and elucidation of docosahexaenoic acid (DHA) biosynthetic pathway in Aurantiochytrium sp. SW1.

Aurantiochytrium sp., a fungoid marine protist that belongs to Stramenophila has proven its potential in the production of polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acids (DHA). In this study, genomic characterisation of a potential producer for commercial production of DHA, Aurantiochytrium sp. SW1 has been carried out via whole genome sequencing analysis. The genome size of this strain is 60.89 Mb, with a total of 11,588 protein-coding genes. Among these, 9,127 genes could be functionally annotated into a total of 7,248 (62.5 %) from UniProt, 6,554 (56.6 %) from KEGG and 8,643 (74.6 %) genes from eggNOG protein database. The highest proportion of genes belongs to the protein family of metabolism were further assigned into 11 metabolic categories. The highest number of genes belonging to lipid metabolism (321 genes) followed by carbohydrate metabolism (290 genes), metabolism of cofactors and vitamins (197 genes) and amino acid metabolism (188 genes). Further analysis into the biosynthetic pathway for DHA showed evidence of all genes involved in PKS (polyketide synthase)-like PUFA synthase pathway and incomplete fatty acid synthase-elongase/desaturase pathway. Analysis of PUFA synthase showed the presence of up to ten tandem acyl carrier protein (ACP) domains which might have contributed to high DHA production in this organism. In addition, a hybrid system incorporating elements of FAS, Type I PKS and Type II PKS systems were found to be involved in the biosynthetic pathways of fatty acids in Aurantiochytrium sp. SW1. This study delivers an important reference for future research to enhance the lipid, especially DHA production in Aurantiochytrium sp, SW1 and establishment of this strain as an oleaginous thraustochytrid model.

Probing Genome-Scale Model Reveals Metabolic Capability and Essential Nutrients for Growth of Probiotic Limosilactobacillus reuteri KUB-AC5.

Limosilactobacillus reuteri KUB-AC5 displays the hallmark features of probiotic properties for food and feed industries. Optimization of cultivation condition for the industrial production is important to reach cell concentration and cost reduction. Considering the strain-specific growth physiology, metabolic capability, and essential nutrients of L. reuteri KUB-AC5, the genome-scale metabolic model (GSMM) of L. reuteri KUB-AC5 was developed. Hereby, the GSMM of iTN656 was successfully constructed which contained 656 genes, 831 metabolites, and 953 metabolic reactions. The iTN656 model could show a metabolic capability under various carbon sources and guide potentially 14 essential single nutrients (e.g., vitamin B complex and amino acids) and 2 essential double nutrients (pairwise glutamine-glutamate and asparagine-aspartate) for L. reuteri KUB-AC5 growth through single and double omission analysis. Promisingly, the iTN656 model was further integrated with transcriptome data suggesting that putative metabolic routes as preferable paths e.g., sucrose uptake, nucleotide biosynthesis, urea cycle, and glutamine transporter for L. reuteri KUB-AC5 growth. The developed GSMM offers a powerful tool for multi-level omics analysis, enabling probiotic strain optimization for biomass overproduction on an industrial scale.

Analyzing Predominant Bacterial Species and Potential Short-Chain Fatty Acid-Associated Metabolic Routes in Human Gut Microbiome Using Integrative Metagenomics.

Gut microbiome plays an essential role in host health, and there is interest in utilizing diet to modulate the composition and function of microbial communities. Copra meal hydrolysate (CMH) is commonly used as a natural additive to enhance health. However, the gut microbiome is largely unknown at species level and is associated with metabolic routes involving short-chain fatty acids (SCFAs). In this study, we aimed to analyze, using integrative metagenomics, the predominant species and metabolic routes involved in SCFAs production in the human gut microbiome after treatment with CMH. The effect of CMH treatment on the Thai gut microbiome was demonstrated using 16S rRNA genes with whole-metagenome shotgun (WMGS) sequencing technology. Accordingly, these results revealed that CMH has potentially beneficial effects on the gut microbiome. Twelve predominant bacterial species, as well as their potential metabolic routes, were involved in cooperative microbiome networks under sugar utilization (e.g., glucose, mannose, or xylose) and energy supply (e.g., NADH and ATP) in relation to SCFAs biosynthesis. These findings suggest that CMH may be used as a potential prebiotic diet for modulating and maintaining the gut microbiome. To our knowledge, this is the first study to reveal the predominant bacterial species and metabolic routes in the Thai gut microbiome after treatment with potential prebiotics.

Analysis of Human Gut Microbiome: Taxonomy and Metabolic Functions in Thai Adults.

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.

Metabolic Responses of Carotenoid and Cordycepin Biosynthetic Pathways in Cordyceps militaris under Light-Programming Exposure through Genome-Wide Transcriptional Analysis.

Cordyceps militaris is currently exploited for commercial production of specialty products as its biomass constituents are enriched in bioactive compounds, such as cordycepin. The rational process development is important for economically feasible production of high quality bioproducts. Light is an abiotic factor affecting the cultivation process of this entomopathogenic fungus, particularly in its carotenoid formation. To uncover the cell response to light exposure, this study aimed to systematically investigate the metabolic responses of C. militaris strain TBRC6039 using integrative genome-wide transcriptome and genome-scale metabolic network (GSMN)-driven analysis. The genome-wide transcriptome analysis showed 8747 expressed genes in the glucose and sucrose cultures grown under light-programming and dark conditions. Of them, 689 differentially expressed genes were significant in response to the light-programming exposure. Through integration with the GSMN-driven analysis using the improved network (iRT1467), the reporter metabolites, e.g., adenosine-5'-monophosphate (AMP) and 2-oxoglutarate, were identified when cultivated under the carotenoid-producing condition controlled by light-programming exposure, linking to up-regulations of the metabolic genes involved in glyoxalase system, as well as cordycepin and carotenoid biosynthesis. These results indicated that C. militaris had a metabolic control in acclimatization to light exposure through transcriptional co-regulation, which supported the cell growth and cordycepin production in addition to the accumulation of carotenoid as a photo-protective bio-pigment. This study provides a perspective in manipulating the metabolic fluxes towards the target metabolites through either genetic or physiological approaches.

Probing Carbon Utilization of Cordyceps militaris by Sugar Transportome and Protein Structural Analysis.

Beyond comparative genomics, we identified 85 sugar transporter genes in Cordyceps militaris, clustering into nine subfamilies as sequence- and phylogenetic-based functional classification, presuming the versatile capability of the fungal growths on a range of sugars. Further analysis of the global gene expression patterns of C. militaris showed 123 genes were significantly expressed across the sucrose, glucose, and xylose cultures. The sugar transporters specific for pentose were then identified by gene-set enrichment analysis. Of them, the putative pentose transporter, CCM_06358 gene, was highest expressed in the xylose culture, and its functional role in xylose transport was discovered by the analysis of conserved structural motifs. In addition, a battery of molecular modeling methods, including homology modeling, transport pathway analysis, residue interaction network combined with molecular mechanics Poisson-Boltzmann surface area simulation (MM-PBSA), was implemented for probing the structure and function of the selected pentose transporter (CCM_06358) as a representative of sugar transportome in C. militaris. Considering the network bottlenecks and structural organizations, we further identified key amino acids (Phe38 and Trp441) and their interactions with other residues, contributing the xylose transport function, as verified by binding free energy calculation. The strategy used herein generated remarkably valuable biological information, which is applicable for the study of sugar transportome and the structure engineering of targeted transporter proteins that might link to the production of bioactive compounds derived from xylose metabolism, such as cordycepin.

Optimizing cultivation of Cordyceps militaris for fast growth and cordycepin overproduction using rational design of synthetic media.

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.

Alternative metabolic routes in channeling xylose to cordycepin production of Cordyceps militaris identified by comparative transcriptome analysis.

The responsive mechanism of C. militaris TBRC7358 on xylose utilization was investigated by comparative analysis of transcriptomes, growth kinetics and cordycepin productions. The result showed that the culture grown on xylose exhibited high production yield of cordycepin on dry biomass. Comparing xylose to other carbon sources, a set of significantly up-regulated genes in xylose were enriched in pentose and glucuronate interconversion, and cordycepin biosynthesis. After validating up-regulated genes using quantitative real-time PCR, interestingly, putative alternative 3'-AMP-associated metabolic route on cordycepin biosynthesis was identified. Through reporter metabolites analysis of C. militaris, significant metabolites (e.g., AMP, glycine and L-glutamate) were identified guiding involvement of growth and cordycepin production. These findings suggested that there was a cooperative mechanism in transcriptional control of the supplying precursors pool directed towards the cordycepin biosynthesis through main and putative alternative metabolic routes for leverage of cell growth and cordycepin production on xylose of C. militaris strain TBRC7358.

Uncovering global metabolic response to cordycepin production in Cordyceps militaris through transcriptome and genome-scale network-driven analysis.

The cellular metabolic adaptations of Cordyceps militaris have 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 in C. militaris through transcriptome analysis and genome-scale network-driven analysis. Here, transcriptome analysis of 16,805 expressed genes in C. militaris strain 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 in C. 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.

Genome-scale metabolic network of Cordyceps militaris useful for comparative analysis of entomopathogenic fungi.

The first genome-scale metabolic network of Cordyceps militaris (iWV1170) was constructed representing its whole metabolisms, which consisted of 894 metabolites and 1,267 metabolic reactions across five compartments, including the plasma membrane, cytoplasm, mitochondria, peroxisome and extracellular space. The iWV1170 could be exploited to explain its phenotypes of growth ability, cordycepin and other metabolites production on various substrates. A high number of genes encoding extracellular enzymes for degradation of complex carbohydrates, lipids and proteins were existed in C. militaris genome. By comparative genome-scale analysis, the adenine metabolic pathway towards putative cordycepin biosynthesis was reconstructed, indicating their evolutionary relationships across eleven species of entomopathogenic fungi. The overall metabolic routes involved in the putative cordycepin biosynthesis were also identified in C. militaris, including central carbon metabolism, amino acid metabolism (glycine, l-glutamine and l-aspartate) and nucleotide metabolism (adenosine and adenine). Interestingly, a lack of the sequence coding for ribonucleotide reductase inhibitor was observed in C. militaris that might contribute to its over-production of cordycepin.

Cyanobacterial Biofuels: Strategies and Developments on Network and Modeling.

Cyanobacteria, the phototrophic microorganisms, have attracted much attention recently as a promising source for environmentally sustainable biofuels production. However, barriers for commercial markets of cyanobacteria-based biofuels concern the economic feasibility. Miscellaneous strategies for improving the production performance of cyanobacteria have thus been developed. Among these, the simple ad hoc strategies resulting in failure to optimize fully cell growth coupled with desired product yield are explored. With the advancement of genomics and systems biology, a new paradigm toward systems metabolic engineering has been recognized. In particular, a genome-scale metabolic network reconstruction and modeling is a crucial systems-based tool for whole-cell-wide investigation and prediction. In this review, the cyanobacterial genome-scale metabolic models, which offer a system-level understanding of cyanobacterial metabolism, are described. The main process of metabolic network reconstruction and modeling of cyanobacteria are summarized. Strategies and developments on genome-scale network and modeling through the systems metabolic engineering approach are advanced and employed for efficient cyanobacterial-based biofuels production.

Sequence- and Structure-Based Functional Annotation and Assessment of Metabolic Transporters in Aspergillus oryzae: A Representative Case Study.

Aspergillus oryzae is widely used for the industrial production of enzymes. In A. oryzae metabolism, transporters appear to play crucial roles in controlling the flux of molecules for energy generation, nutrients delivery, and waste elimination in the cell. While the A. oryzae genome sequence is available, transporter annotation remains limited and thus the connectivity of metabolic networks is incomplete. In this study, we developed a metabolic annotation strategy to understand the relationship between the sequence, structure, and function for annotation of A. oryzae metabolic transporters. Sequence-based analysis with manual curation showed that 58 genes of 12,096 total genes in the A. oryzae genome encoded metabolic transporters. Under consensus integrative databases, 55 unambiguous metabolic transporter genes were distributed into channels and pores (7 genes), electrochemical potential-driven transporters (33 genes), and primary active transporters (15 genes). To reveal the transporter functional role, a combination of homology modeling and molecular dynamics simulation was implemented to assess the relationship between sequence to structure and structure to function. As in the energy metabolism of A. oryzae, the H(+)-ATPase encoded by the AO090005000842 gene was selected as a representative case study of multilevel linkage annotation. Our developed strategy can be used for enhancing metabolic network reconstruction.