A Modified Cre-<i>lox</i> Genetic Switch
To Dynamically Control Metabolic Flow in <i>Saccharomyces cerevisiae</i>
- Publication date
- Publisher
Abstract
The control of metabolic flow is a prerequisite for efficient
chemical
production in transgenic microorganisms. Exogenous genes required
for the biosynthesis of target chemicals are expressed under strong
promoters, while the endogenous genes of the original metabolic pathway
are repressed by disruption or mutation. These genetic manipulations
occasionally cause harmful effects to the host. In the lactate-producing
yeast <i>Saccharomyces cerevisiae</i>, where endogenous
pyruvate decarboxylase (<i>PDC</i>) is disrupted and exogenous
lactate dehydrogenase (<i>LDH</i>) is introduced, <i>PDC</i> deletion is extremely detrimental to cell growth but
is required for efficient production of lactate. A suitable means
to dynamically control the metabolic flow from ethanol fermentation
during the growth phase to lactate fermentation during the production
phase is needed. Here, we demonstrated that this flow can be controlled
by the exclusive expression of <i>PDC</i> and <i>LDH</i> with a Cre-<i>lox</i> genetic switch. This switch was
evaluated with a gene cassette that encoded two different fluorescence
proteins and enabled changes in genotype and phenotype within 2 and
10 h, respectively. Transgenic yeast harboring this switch and the <i>PDC</i>-<i>LDH</i> cassette showed a specific growth
rate (0.45 h<sup>–1</sup>) that was almost the same as that
of wild-type (0.47 h<sup>–1</sup>). Upon induction of the genetic
switch, the transgenic yeast produced lactate from up to 85.4% of
the glucose substrate, while 91.7% of glucose went to ethanol before
induction. We thus propose a “metabolic shift” concept
that can serve as an alternative means to obtain gene products that
are currently difficult to obtain by using conventional methodologies