Sustainability and Stability - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 821

chemicals; for a lac promoter, lactose or a chemical analog of lactose (e.g. isopropyl- b - D -
thiogalactoside) can be used. Such promoters are often used in plasmid construction to
control the synthesis of a plasmid-encoded protein. If induction of plasmid-encoded protein
synthesis from this promoter reduces cellular growth rates, then a mutation that inactivates
lac permease would prevent protein induction in that mutant cell. The lac permease protein
is necessary for the rapid uptake of the inducer. Thus, the mutant cell would grow faster than
the desired strain. Alternatively, a host cell mutation in the repressor, so that it would not
recognize the inducer, would make induction impossible.
The key feature of this category of genetic instability is that a host cell mutation imparts
a growth advantage to the mutant, so that it will eventually dominate the culture. In this
case, the mutant cell will contain unaltered plasmids but will make very little of the target,
plasmid-encoded protein.
16.5.4. Growth-Rate-Dominated Instability
The importance of all three of these factors (segregational loss of plasmid, structural alter-
ations of the plasmid, and host cell mutations) depends on the growth-rate differential of the
changed cell e plasmid system to the original host e vector system. If the altered host e vector
system has a distinct growth advantage over the original host e vector system, the altered
system will eventually dominate (i.e. genetic instability will occur).
The terms used to describe the cause of genetic instability are based on fairly subtle
distinctions. For example, if genetic instability is due to segregational instability, we would
infer that the rate of formation of plasmid-free cells is high. In this case, the number of
plasmid-free cells would be high irrespective of whether the plasmid-free cells had
a growth-rate advantage. If, on the other hand, we claimed that the genetic instability is
growth-rate dependent, we would imply that the rate of formation of plasmid-free cells is
low, but the plasmid-free cells have such a large growth-rate advantage that they outgrow
the original host e vector system. In most cases, growth-rate dependent instability and one
of the other factors (segregational loss, structural changes in the plasmid, or host cell muta-
tions) are important.
The growth-rate ratio can be manipulated to some extent by the choice of medium (e.g. the
use of selective pressure such as antibiotic supplementation to kill plasmid-free cells) and the use
of production systems that do not allow significant target-protein production during most of
the culture period. For example, an inducible promoter can be turned on only at the end of
a batch growth cycle when only one or two more cell doublings may normally occur. Before
induction, the metabolic burden imposed by the formation of the target protein is nil, and the
growth ratio of the altered to the original host e vector system is close to 1 (or less if selective
pressure is also applied). This two-phase fermentation can be done as a modified batch system,
or a multistage chemostat could be used. In a two-stage system, the first stage is optimized to
produce viable plasmid-containing cells, and production formation is induced in the second
stage. The continual resupply of fresh cells to the second stage ensures that many unaltered
cells will be present.
The problem of genetic instability is more significant in commercial operations than in
laboratory-scale experiments. The primary reason is that the culture must go through
many more generations to reach a density of 10 13 cells/l in a 10,000 l tank than in a shake flask