Antibiotic-free maintenance of multiple plasmids in Escherichia coli using conditional complementation

Abstract

A key sustainability concern in biotechnology is the heavy reliance on antibiotics and antibiotic resistance genes (ARGs) for plasmid selection and maintenance. This is leading to increasing regulatory pressure to develop antibiotic-free biomanufacturing processes. Currently, there are limited options enabling antibiotic-free use of multiple plasmids, which is often required in processes with advanced genetic complexity. Here, we use conditional complementation of two essential genes in Escherichia coli to enable ARG-free selection and maintenance of two plasmids in non-supplemented media. First, we develop orthogonal strains containing an essential gene under the control of an inducible promoter; as a result, the growth of each strain is dependent on the corresponding inducer or the presence of a complementing plasmid. We characterize these strains, demonstrating straightforward, antibiotic-free plasmid transformations and robust plasmid maintenance in non-supplemented media. Next, by combining these modifications, we create strains enabling maintenance of two plasmids in non-supplemented media without requiring ARGs. We demonstrate that by using two orthogonal inducible promoters, it is even possible to create a strain where one or two complementing plasmids can be used. By expressing model genes of interest, we show the productivity of these strains is equivalent or superior to conventional, antibiotic-dependent strains under selective conditions. This method offers a robust, antibiotic-free solution for multi-plasmid biosynthetic pathways, enabling improved sustainability without imposing any technical restrictions or compromising handling.