Construction of cascade circuits for dynamic temporal regulation and its application to PHB production

To maximize the production capacity and yield of microbial cell factories, metabolic pathways are generally modified with dynamic regulatory strategies, which can effectively solve the problems of low biological yield, growth retardation and metabolic imbalance. However, the strategy of dynamic regulating multiple genes in different time and order is still not effectively solved. Based on the quorum-sensing (QS) system and the principle of cascade regulation, we studied the sequence and time interval of gene expression in metabolic pathways.

The self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.

We designed and constructed a self-induced dynamic temporal regulatory cascade circuit in Escherichia coli using the QS system and dual regulatory protein cascade and found that the time intervals of the cascade circuits based on the Tra, Las system and the Lux, Tra system reached 200 min and 150 min, respectively. Furthermore, a dynamic temporal regulatory cascade circuit library with time intervals ranging from 110 to 310 min was obtained based on this circuit using promoter engineering and ribosome binding site replacement, which can provide more selective synthetic biology universal components for metabolic applications. Finally, poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of the cascade circuit library. The content of PHB increased 1.5-fold. Moreover, circuits with different time intervals and different expression orders were found to have different potentials for application in PHB production, and the preferred time-interval circuit strain C2-max was identified by screening. Conclusions: The self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.