Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase

Saroja Narsing Rao; G Monika Kumari; D Srividya; H S Anil; M Lakshmikanth; Harishchandra Naik; A Prabhuraj

doi:10.1007/s12088-023-01056-x

Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase

Indian J Microbiol. 2023 Mar;63(1):56-64. doi: 10.1007/s12088-023-01056-x. Epub 2023 Feb 2.

Authors

Saroja Narsing Rao¹, G Monika Kumari¹, D Srividya¹, H S Anil², M Lakshmikanth^{1

3}, Harishchandra Naik¹, A Prabhuraj¹

Affiliations

¹ Pesticide Residue and Food Quality Analysis Laboratory, University of Agricultural Sciences, Raichur, 584104 Karnataka India.
² Department of Biotechnology, Dayananda Sagar College of Engineering, KS Layout, Shavige Malleswara Hills, Bengaluru-78, Karnataka India.
³ College of Agriculture, University of Agricultural Sciences, Raichur, India.

Abstract

Numerous expression systems, engineered strains, and cultivation systems have been developed globally but producing recombinant proteins in the soluble form continues to remain a challenge. Escherichia coli, a preferred host for the recombinant production of biopharmaceuticals and other proteins. Up to 75% of human proteins expressed in E. coli have only 25% in an active soluble form. The proteolytic activity of Lon encoded protease triggers the inclusion bodies leading to heterogenous secreted proteins thereby hampering downstream processing and isolation. Putrescine monooxygenases are versatile with applications in iron acquisition, pathogen control, biotransformation, bio-remediation and redox reaction are still isolated from plant and microbial sources at low yields. As a prerequisite to developing protease knockout E. coli strains, using the Cre-loxP recombination strategy we have built a full-length Lon disruption cassette (5'lon-lox66-cre-KanR-lox71-3'lon) (3368 bp) consisting of upstream and downstream regions of Lon, loxP sites, and Cre gene driven by T7 promoter to the expression of Cre recombinase and a selectable kanamycin resistance gene. Here, after the integration of the knock-out cassette into the host genome, we show the production of homogeneous protein species of recombinant Putrescine monooxygenase by using an E. coli platform strain in which Lon gene is deleted. This Lon knock-out strain secreted more homogeneous protein at a volumetric yield of 60% of the wild-type strain.

Supplementary information: The online version contains supplementary material available at 10.1007/s12088-023-01056-x.

Keywords: Cre/loxP; Lon gene disruption cassette; Marker rescue; Putrescine monooxygenase; Solubility.

© Association of Microbiologists of India 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.