Holistic transcriptional responses of Cordyceps militaris to different culture temperatures.

Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes, 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris.

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.

Uncovering global lipid accumulation routes towards docosahexaenoic acid (DHA) production in Aurantiochytrium sp. SW1 using integrative proteomic analysis.

Aurantiochytrium sp., a marine thraustochytrid possesses a remarkable ability to produce lipid rich in polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA). Although gene regulation underlying lipid biosynthesis has been previously reported, proteomic analysis is still limited. In this study, high DHA accumulating strain Aurantiochytrium sp. SW1 has been used as a study model to elucidate the alteration in proteome profile under different cultivation phases i.e. growth, nitrogen-limitation and lipid accumulation. Of the total of 5146 identified proteins, 852 proteins were differentially expressed proteins (DEPs). The largest number of DEPs (488 proteins) was found to be uniquely expressed between lipid accumulating phase and growth phase. Interestingly, there were up-regulated proteins involved in glycolysis, glycerolipid, carotenoid and glutathione metabolism which were preferable metabolic routes towards lipid accumulation and DHA production as well as cellular oxidative defence. Integrated proteomic and transcriptomic data were also conducted to comprehend the gene and protein regulation underlying the lipid and DHA biosynthesis. A significant up-regulation of acetyl-CoA synthetase was observed which suggests alternative route of acetate metabolism for acetyl-CoA producer. This study presents the holistic routes underlying lipid accumulation and DHA production in Aurantiochytrium sp. SW1 and other relevant thraustochytrid.

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.

Construction of Light-Responsive Gene Regulatory Network for Growth, Development and Secondary Metabolite Production in Cordyceps militaris.

Cordyceps militaris is an edible fungus that produces many beneficial compounds, including cordycepin and carotenoid. In many fungi, growth, development and secondary metabolite production are controlled by crosstalk between light-signaling pathways and other regulatory cascades. However, little is known about the gene regulation upon light exposure in C. militaris. This study aims to construct a gene regulatory network (GRN) that responds to light in C. militaris. First, a genome-scale GRN was built based on transcription factor (TF)-target gene interactions predicted from the Regulatory Sequence Analysis Tools (RSAT). Then, a light-responsive GRN was extracted by integrating the transcriptomic data onto the genome-scale GRN. The light-responsive network contains 2689 genes and 6837 interactions. From the network, five TFs, Snf21 (CCM_04586), an AT-hook DNA-binding motif TF (CCM_08536), a homeobox TF (CCM_07504), a forkhead box protein L2 (CCM_02646) and a heat shock factor Hsf1 (CCM_05142), were identified as key regulators that co-regulate a large group of growth and developmental genes. The identified regulatory network and expression profiles from our analysis suggested how light may induce the growth and development of C. militaris into a sexual cycle. The light-mediated regulation also couples fungal development with cordycepin and carotenoid production. This study leads to an enhanced understanding of the light-responsive regulation of growth, development and secondary metabolite production in the fungi.

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.