Cordyceps militaris, widely recognized as a medicinal and edible mushroom in East Asia, contains a variety of bioactive compounds, including cordycepin (COR), pentostatin (PTN) and other high-value compounds. This review explores the potential of developing C. militaris as a cell factory for the production of high-value chemicals and nutrients. This review comprehensively summarizes the fermentation advantages, metabolic networks, expression elements, and genome editing tools specific to C. militaris and discusses the challenges and barriers to further research on C. militaris across various fields, including computational biology, existing DNA elements, and genome editing approaches. This review aims to describe specific and promising opportunities for the in-depth study and development of C. militaris as a new chassis cell. Additionally, to increase the practicability of this review, examples of the construction of cell factories are provided, and promising strategies for synthetic biology development are illustrated.

Background: Cordyceps militaris, a kind of edible and medicinal fungus widely accepted in East Asia, has attracted much attention as a potential cell factory for producing adenosine analogs. Despite the rapid development in gene editing techniques and genome modeling, the diversity of DNA elements in C. militaris was too short to achieve rational heterogeneous expression for metabolic engineering studies. Results: In this study, P_trpC, a kind of promoter with a relatively appropriate expression level and small size, was selected as a monomer for promoter library construction. Through in vitro BioBricks assembly, 9 overlapping P_trpC promoters with different copy numbers as well as reporter gene gfp were connected and subsequently integrated into the genome of C. militaris. Both the mRNA transcription level and the expression level of gene gfp gradually increased along with the copy number of the overlapping promoter NP_trpC and peaked at 7. In the meantime, no significant difference was found in either the biomass or morphological characteristic of engineered and wild-type strains. Conclusions: This study firstly expanded the overlapping promoter strategy used in model microorganism in C. militaris. It was a proof-of-concept in fungi synthetic biology and provide a general method to pushed the boundary of promoter engineering in edible mushroom.

Objective: To accurately verify the pathogenicity of variants of uncertain significance (VUS) in MUT and MMACHC genes through mass spectrometry and silico analysis.Methods: This multicenter retrospective study included 35 participating units (ClinicalTrials.gov ID: NCT06183138). A total of 3,071 newborns (within 7 days of birth) were sorted into carrying pathogenic/likely pathogenic (P/LP) variants and carrying VUS, non-variant groups. Differences in metabolites among the groups were calculated using statistical analyses. Changes in conservatism, free energy, and interaction force of MMUT and MMACHC variants were analyzed using silico analysis.Results: The percentage of those carrying VUS cases was 68.15% (659/967). In the MMUT gene variant, we found that C3, C3/C2, and C3/C0 levels in those carrying the P/LP variant group were higher than those in the non-variant group (p < 0.000). The conservative scores of those carrying the P/LP variant group were >7. C3, C3/C0, and C3/C2 values of newborns carrying VUS (c.1159A>C and c.1286A>G) were significantly higher than those of the non-variant group and the remaining VUS newborns (p < 0.005). The conservative scores of c.1159A>C and c.1286A>G calculated by ConSurf analysis were 9 and 7, respectively. Unfortunately, three MMA patients with c.1159A>C died during the neonatal period; their C3, C3/C0, C3/C2, and MMA levels were significantly higher than those of the controls.Conclusion: Common variants of methylmalonic acidemia in the study population were categorized as VUS. In the neonatal period, the metabolic biomarkers of those carrying the P/LP variant group of the MUT gene were significantly higher than those in the non-variant group. If the metabolic biomarkers of those carrying VUS are also significantly increased, combined with silico analysis the VUS may be elevated to a likely pathogenic variant. The results also suggest that mass spectrometry and silico analysis may be feasible screening methods for verifying the pathogenicity of VUS in other inherited metabolic diseases.

Background: This study seeks to elucidate the clinical and biochemical features of Ornithine transcarbamylase deficiency (OTCD), a pleomorphic congenital hyperammonemia disorder with a non-specific clinical phenotype. Additionally, the research aims to analyze the mutation spectrum of the OTC gene and its potential association with phenotype, as well as to perform an in silico analysis of novel OTC variants to elucidate their structure-function relationship. Methods: In this study, we conducted a retrospective analysis of the clinical and biochemical features of 12 patients with OTCD and examined their metabolite profiles. Additionally, we reviewed existing literature to explore the range of mutations in the OTC gene and their possible associations with phenotypic outcomes. Furthermore, we employed the high ambiguity-driven protein-protein docking (HADDOCK) algorithm and protein-ligand interaction profiler (PLIP) to predict the pathogenicity of these mutations and elucidate the underlying mechanisms of pathogenesis in novel variants of the OTC gene. Results: Nine cases, all of which were male, presented with early onset, while two cases, all of which were female, exhibited late onset. Additionally, one male case was asymptomatic. The ages of the patients at the time of diagnosis ranged from 1 day to 12 years. Peak plasma ammonia levels were found to be higher in patients with early onset compared to those with late onset. Molecular analyses identified a total of 12 different mutations, including two novel mutations (V323G and R320P). In silico analysis indicated a potential difference in affinity between wild-type and mutant OTCase, with V323G and R320P mutations leading to a decreased binding ability of OTCase to the substrate, potentially disrupting its function. Conclusion: This study broadened the genetic variation spectrum of OTCD and provided substantial evidence for genetic counselling to affected families. Additionally, we elucidated variant data of OTC in Chinese patients through comprehensive literature review. Given the ongoing uncertainty surrounding the genotype-phenotype correlation of OTCD, the results of our in silico analysis can contribute to a deeper understanding of this complex, rare, and severe genetic disorder.

Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in the EFNB1 gene, which encodes the ephrin-B1 protein involved in craniofacial and neural development. This study aims to investigate the effects of a de novo heterozygous EFNB1 missense variant c.451G > A (p.Gly151Ser) using in silico methods. The whole-exome analysis in a child with CFNS harbored a heterozygous missense variant c.451G > A (p.Gly151Ser) in the EFNB1 gene. The clinical data of the child were collected. The child's physical growth and neurodevelopmental progress were followed up within the first year of life. Our study reveals the pathogenicity of the c.451G > A (p.Gly151Ser) mutation in the EFNB1 gene and its impact on protein function through AlphaFold3 Protein Modeling, HADDOCK Protein Docking, and Molecular Dynamics Simulations. A child with CFNS presented with craniofacial asymmetry, frontal bossing, hypertelorism, and polydactyly. Genetic testing identified the pathogenic variant. Structural modeling revealed altered local topology, increased rigidity, and disruption of hydrophobic interactions at the mutation site. Docking and MD simulations showed that the mutation impaired EFNB1-EPHB2 interactions at the dimer (DIM) interface by reducing stability and binding energy. Conversely, enhanced binding affinity was observed at the tetramer (TET) interface due to strengthened van der Waals and electrostatic interactions. Overall, the mutation destabilized global structure while modulating binding dynamics in an interface-specific manner. This study highlights the potential pathogenicity of the c.451G > A variant and underscores the need for further research into EFNB1-associated disorders.

The broad substrate specificity of enzymes, while advantageous for catalytic diversity, often leads to undesired side reactions and reduced product yields in engineered metabolic pathways. To address this challenge, we developed a programmable protein scaffold based on a self-assembled γPFD-SpyCatcher hydrogel for the in vivo co-immobilization of SpyTag-cyclized cascade enzymes, enabling the co-immobilization of cascade enzymes in a spatially organized manner. Enzymes with broad substrate specificities were linearly fused with SpyTags, facilitating their spatial organization on the nanoscaffold within engineered E. coli to ensure directed catalytic flux. Using this strategy, the yields of pinene and caffeoyl-CoA were enhanced by 5.8-fold (reaching 94.5 mg/L) and 2.4-fold (reaching 78.6 mg/L), respectively, compared to free enzyme systems. This work establishes an effective approach to mitigate the limitations posed by broad enzyme specificity and demonstrates its potential for applications in synthetic biology and industrial biotechnology.