US-12624363-B2 - Plasmid addiction system to drive desired gene expression

Abstract

The present invention relates to a Plasmid Addiction System for the stabilization of expression plasmids encoding proteins of interest. The invention uses a succinate cycle optimization to ensure the expression of plasmid(s) of interest. By ensuring that plasmids of interest contain genes necessary in the succinate cycle, the system ensures that the passage of the plasmid to daughters and therefore improves the efficiency of production and expression of genes and/or products of interest.

Inventors

• Matthew de la Pena Mattozzi
• Daniel Kim
• Sonya Clarkson

Assignees

• SUMITOMO CHEMICAL COMPANY, LIMITED

Dates

Publication Date

20260512

Application Date

20220701

Claims (10)

1. 1 . A process for producing a recombinant protein of interest in a transformed bacterial host, comprising: a) introducing one or more recombinant plasmids into said bacterial host cell, wherein said one or more recombinant plasmids comprise a cloned DNA sequence comprising all or part of at least two genes each encoding a functional essential succinate pathway enzyme that is integral to the survival of the host cell and the one or more recombinant plasmids also encode at least one gene of interest coding for a protein product, and wherein the one or more recombinant plasmids are devoid of an antibiotic resistance gene; b) selecting surviving colonies of the transformed host containing the one or more recombinant plasmids; and, c) using the surviving colonies for fermenting bacterial colonies to allow the expression of said protein product; and wherein the at least two genes are sucAB and sucCD, and wherein the bacterial host is a mutant with eliminated activity of sucAD or sucABCD.
2. 2 . The process of claim 1 , wherein the recombinant protein product additionally comprises an amino acid sequence that will optimize purification, isolation or tagging.
3. 3 . The process of claim 1 , wherein said essential genes are operably linked to a promoter.
4. 4 . The process of claim 3 , wherein said promoter linked to said essential gene is inducible.
5. 5 . The process of claim 4 , wherein said inducible promoter is independent of any other inducible promoter controlling a foreign DNA sequence.
6. 6 . The process of claim 1 , wherein said cell is selected from the group comprising: a Escherichia coli cell; Corynebacterium spp., Vibrio spp.; Escherichia spp.; Enterobacter spp.; Citrobacter spp.; Erwinia 0 spp.; Bacillus spp.; Pseudomonas spp.; Cyanobacteria spp.; Salmonella spp. and Klebsiella spp.
7. 7 . The process of claim 6 , wherein said one or more recombinant plasmids additionally contain a second gene of interest coding for a protein product.
8. 8 . The process of claim 1 , wherein the one or more recombinant plasmids are two recombinant plasmids.
9. 9 . The process of claim 1 , wherein the one or more recombinant plasmids are up to four recombinant plasmids.
10. 10 . The process of claim 9 , wherein the one or more recombinant plasmids are four recombinant plasmids.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a Division of U.S. application Ser. No. 16/036,261, filed Jul. 16, 2018, entitled “PLASMID ADDICTION SYSTEM TO DRIVE DESIRED GENE EXPRESSION”, which claims priority to U.S. Provisional Patent Application No. 62/697531, filed Jul. 13, 2018, entitled PLASMID ADDICTION SYSTEM TO DRIVE DESIRED GENE EXPRESSION, and to U.S. Provisional Patent Application No. 62/535,596, filed Jul. 21, 2017, entitled PLASMID ADDICTION SYSTEM TO DRIVE DESIRED GENE EXPRESSION. The entire contents of these applications are incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (C149770027US02-SEQ-ZJG.xml; Size: 81,435 bytes; and Date of Creation: Jun. 30, 2022) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION The field of the invention relates to methods and processes useful in maintaining extrachromosomal elements of interest in a microbial production strain using genes from the succinate pathway to ensure inclusion and expression of the elements in daughter cells. More specifically, it relates to the use of a plasmid addiction system that ensures that modified microbial cells will maintain plasmids carrying genes involved in producing desired expression products. BACKGROUND OF THE INVENTION The present invention is directed to a method of manipulating microbial cells in culture to maintain at least one extrachromosomal element of interest containing at least one gene of interest. Typically, this extrachromosomal element is a plasmid, though phages, prophages, phagemids, cosmids, bacterial artificial chromosomes (BACs) also contain extrachromosomal elements to contain transgenes of heterologous interest. Though naturally occurring in bacteria, not all wild type plasmids contain genetic information that is required to maintain the viability of the host cell in normal conditions. However, plasmids can contain genetic information that provides selective advantages to the host under specific environmental challenges such as antibiotic resistance or resistance to noxious compounds present in the environment. However, in those situations where adverse environmental conditions are not present, the presence of the plasmid is, in fact, a metabolic burden upon the cell (Nordstrom and Austin, 1989). In other words, the metabolic activity required to maintain plasmids exerts a small but real metabolic cost to the host cell relative to those cells not carrying the plasmid in question. This metabolic burden is why many daughter cells tend to ‘lose’ the plasmid of interest over time if they can continue to exist or reproduce without it. This process of loss or limited replication of the extrachromosomal element(s) also leads to diminished efficiency in those experiments that require the presence of a plasmid genetic component to produce a product of interest and therefore cultures with significant amounts of daughter cells that do not have the plasmid(s) of interest provide a reduced efficiency for the experiment being conducted. This is particularly acute in those fermentation experiments that rely upon economies of scale and consistent production of a molecule of interest to make their cost targets. Daughter cells deficient in the desired plasmids or extrachromosomal elements represent a media and energy sink in overall production and contribute to the economic benefits of fermentation costs. In the biotechnology industry, plasmids and similar extrachromosomal elements have become very important tools in the genetic engineering of microbes and in the expression of proteins of interest and commercial synthetic biology. Such elements can be manipulated and designed to force the host cell to carry them forward or perish. (Balbas 2001: Baba 2006). In this sense, the cells become irreversibly ‘addicted’ to maintaining the extrachromosomal element in the cell despite the consequent metabolic burden (hence the term, Plasmid Addiction System or “PAS”). With such a system in hand the researcher can then focus on driving the host cell culture not just to maintain and express the PAS system genes, but to express all the genes contained on such an extrachromosomal element. According to the current invention, this can entail the expression of a number of genes and potential gene products of interest in microbial systems. Plasmid Addiction Systems and Alternatives Given the power of such techniques to drive the expression of proteins of interest, it is not surprising that a variety of approaches have been developed to ensure the stable maintenance of plasmids in cells (Nordstrom and Austin, 1989). This includes: (i) site-specific recombination systems functioning as plasmid maintenance systems for high-copy plasmid systems (Grindley et al., 2006); (ii) active partition systems (Funnell and Slavcev, 2004); and, as mentioned above, (iii) plasmid addiction systems (PAS), like the invention