Figure 2.

Strategies have been developed to maintain plasmids and strain phenotypes stability. (a) TA system; it consists of a stable toxin protein in genome interacting with a less stable antidote molecule in plasmid. If losing the antidote-containing plasmid, the toxin protein cannot be repressed by antidote and the host will be killed by the toxin. (b) RNA-based interactions; the essential gene is under the control of a regulatory promoter, which can be repressed by a corresponding inhibitor. A plasmid-transcribed antisense RNA (asRNA) can interact with the promoter sequence of this inhibitor. When the plasmid is present, the transcribed asRNA can bind with the promoter sequence of inhibitor and repress the expression of the inhibitor. Then the inhibition on the essential gene will be relieved. With the successfully expressed essential gene, the cells can grow well. However, when the plasmid is absent, the inhibitor cannot the repressed by asRNA and the expression of this inhibitor will repress the essential gene, which will cause growth defects on cells. (c) operator-repressor titration (ORT); it has been devised as a plasmid maintenance approach based on competitive binding of repressor on DNA-binding operator copies between plasmids and the chromosome. In the existence of a plasmid containing the same operator, most of the repressors will bind to the operators in the plasmid and only a small fraction of repressors will bind to the operators in the genome, because of the competitive binding between repressors and operators. Thus, the inhibition efficiency on the essential gene will decrease and it can be expressed to help the cell growth. However, if no plasmid is present, all the repressors will bind to the operator located in the genome and inhibit the expression of the essential gene. Without the essential gene, the cells will be unbale to grow. (d) auxotrophy complementation; this system is to optimize the process of traditional auxotrophic strain construction. In the optimized process, first, the essential gene was expressed in a temperature-sensitive intermediate plasmid and the essential gene in genome was deleted. Then, the production plasmid containing pathway gene and the deleted essential gene were transferred into host with the intermediate plasmid. When increasing the incubating temperature to 42 °C, the intermediate plasmid will be degraded and only the production plasmid was kept in the cell, resulting an auxotrophic strain. In this strain, if the plasmid is lost or mutated during fermentation, the cells will be unbale to grow due to the loss of essential gene.