Improving the production of recombinant L-Asparaginase-II in Escherichia coli by co-expressing catabolite repressor activator (cra) gene

Abstract

Identification of a single genetic target for microbial strain improvement is difficult due to the complexity of the genetic regulatory network. Hence, a more practical approach is to identify bottlenecks in the regulatory networks that control critical metabolic pathways. The present work focuses on enhancing cellular physiology by increasing the metabolic flux through the central carbon metabolic pathway. Global regulator cra (catabolite repressor activator), a DNA-binding transcriptional dual regulator was selected for the study as it controls the expression of a large number of operons that modulate central carbon metabolism. To upregulate the activity of central carbon metabolism, the cra gene was co-expressed using a plasmid-based system. Co-expression of cra led to a 17% increase in the production of model recombinant protein L-Asparaginase-II. A pulse addition of 0.36% of glycerol every two hours post-induction, further increased the production of L-Asparaginase-II by 35% as compared to the control strain expressing only recombinant protein. This work exemplifies that upregulating the activity of central carbon metabolism by tuning the expression of regulatory genes like cra can relieve the host from cellular stress and thereby promote the growth as well as expression of recombinant hosts.

Keywords:

• Asparaginase
• cellular stress response
• co-expression
• cra
• Recombinant protein

Acknowledgements

The authors acknowledge the P.G. of Biosciences & Biotechnology and P.G. of Environmental Science of Fakir Mohan University, Balasore, Odisha, India for all the support and cooperation to carry out the research. DM acknowledges Biju Patnaik Research Fellowship offered by Science & Technology Department, Govt. of Odisha.

Author’ contributions

SM participated in experimental design, manuscript writing and supervised the overall work. DM carried out all the experimental work and data analysis. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The source of data used for insilico study has been appropriately mentioned in the main manuscript. All other data generated in this study are mentioned in the methods section.

Additional information

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.