Genetic Strategies for alleviating metabolic bottlenecks to enhance recombinant protein production in Escherichia Coli

Abstract

newlineE. coli is a popular choice for making proteins due to its genetics and growth speed. Protein production in E. coli causes stress, affecting metabolic functions like energy production and ribosome synthesis. To improve protein production, global regulators can be targeted to maintain energy supply and cellular health. Glycerol is used as a carbon source, but it leads to stress in E. coli compared to glucose. Cra is a global regulator that controls carbon metabolism pathways in E. coli. Engineering Cra can enhance glycerol metabolism, growth, and protein production. The study focuses on using in-silico methods to identify and engineer Cra for improved protein production. In-silico methods are used to study cra gene expression under metabolic stress before modifying central carbon metabolism genes. Systems biology tools help understand cellular physiology for enhancing recombinant protein production. GenExpDB and EcoCyc databases are used to analyze cra gene expression and its regulation on key metabolic pathways. Alterations in cra gene expression impact downstream metabolic pathways, affecting growth and productivity in recombinant cultures. In the post-induction phase, a metabolic stress condition affects cell growth and productivity by downregulating the cra gene and central carbon metabolic pathways. Co-expressing cra gene from a plasmid system enhances growth and protein production by diverting carbon flux and activating metabolic pathways. The cra gene was cloned in pBAD33 vector and co-transformed with L-Asparaginase-II in E. coli strains W3110 and BW25113. Different glycerol concentrations were used as carbon sources to test cra gene expression effects on protein yield. Arabinose was used as an inducer for cra gene expression under the PBAD promoter. Batch and fed-batch cultures showed increased protein yield with optimal cra gene induction levels. pH profiles and residual glycerol concentrations were monitored during cultures. BW25113 strain showed higher protein yield compared to W3110 stra