Electrochemical biosensors based on immobilized enzymes are among the most popular and commercially successful biosensors. The literature in this field suggests that modification of electrodes with nanomaterials is an excellent method for enzyme immobilization, which can greatly improve the stability and sensitivity of the sensor. However, the poor stability, weak reproducibility, and limited lifetime of the enzyme itself still limit the requirements for the development of enzyme electrochemical biosensors for food production process monitoring. Therefore, constructing sensing technologies based on enzyme electrochemical biosensors remains a great challenge. This article outlines the construction principles of four generations of enzyme electrochemical biosensors and discusses the applications of single-enzyme systems, multi-enzyme systems, and nano-enzyme systems developed based on these principles. The article further describes methods to improve enzyme immobilization by combining different types of nanomaterials such as metals and their oxides, graphene-related materials, metal–organic frameworks, carbon nanotubes, and conducting polymers. In addition, the article highlights the challenges and future trends of enzyme electrochemical biosensors, providing theoretical support and future perspectives for further research and development of high-performance enzyme chemical biosensors.

The modification of single or multiple genes via metabolic engineering can lead to the dysregulation of central metabolism and affect bacterial growth and metabolite accumulation. Meanwhile, transcription factor engineering can trigger metabolic network reprogramming at the global or systemic level, redirecting metabolic flux toward the synthetic pathways of target metabolites. In this study, we modulated pyrimidine and carbon-nitrogen metabolism in Bacillus subtilis through transcription factor engineering to promote the synthesis of cytidine, a drug intermediate. First, cytidine synthesis was enhanced by knocking out the transcriptional regulator PyrR, which increased the cytidine titer during shake flask fermentation to 0.67 g/L. Second, mutations in the transcriptional regulator catabolite control protein A (CcpA) significantly promoted cytidine synthesis, increasing the shake flask titer to 2.03 g/L. Finally, after culture in a 5 L fermenter, the cytidine titer reached 7.65 g/L, which was 3.77-fold that of shake flask fermentation. Moreover, a cytidine yield and productivity of 0.06 g/g glucose and 0.16 g/L/h, respectively, were achieved. Subsequently, the regulatory mechanisms through which PyrR and CcpA modification affect cytidine biosynthesis were explored through multi-omics analysis. Transcriptome and metabolome analysis revealed that coordinated alterations in carbon, nitrogen, nucleotide, and amino acid metabolism were essential to promote cytidine synthesis. However, the increased cytidine production in recombinant strains was attributed to the enhancement of pyrimidine metabolism, the Phosphotransferase (PTS) system, the tricarboxylic acid (TCA) cycle, the pentose phosphate (PP) pathway, and nitrogen metabolism. These results indicate that PyrR knockdown can enhance pyrimidine metabolic pathway and promote cytidine synthesis. CcpA mutation can reprogram the central carbon-nitrogen metabolic network, change the metabolic flow to de novo synthesis pathway of pyrimidine nucleoside, increase the supply of cytidine synthesis precursors and promote the accumulation of cytidine. Overall, regulation of engineered carbon and nitrogen metabolic networks is essential for improving the efficiency of microbial cell factories.

Electrochemical biosensors based on immobilized enzymes are among the most popular and commercially successful biosensors. Literature in this field suggests that modification of electrodes with nanomaterials is an excellent method for enzyme immobilization, which can greatly improve the stability and sensitivity of the sensor. However, the poor stability, weak reproducibility, and limited lifetime of the enzyme itself still limit the requirements for the development of enzyme electrochemical biosensors for food production process monitoring. Therefore, constructing sensing technologies based on enzyme electrochemical biosensors remains a great challenge. This article outlines the construction principles of four generations of enzyme electrochemical biosensors and discusses the applications of single-enzyme systems, multi-enzyme systems and nano-enzyme systems developed based on these principles. The article further describes methods to improve enzyme immobilisation by combining different types of nanomaterials such as metals and their oxides, graphene-related materials, metal-organic frameworks, carbon nanotubes and conducting polymers. In addition, the article highlights the challenges and future trends of enzyme electrochemical biosensors, providing theoretical support and future perspectives for further research and development of high-performance enzyme chemical biosensors.

Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli.