29 research outputs found
A Modified Cre-<i>lox</i> Genetic Switch To Dynamically Control Metabolic Flow in <i>Saccharomyces cerevisiae</i>
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
A Modified Cre-<i>lox</i> Genetic Switch To Dynamically Control Metabolic Flow in <i>Saccharomyces cerevisiae</i>
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
A Highly Tunable System for the Simultaneous Expression of Multiple Enzymes in <i>Saccharomyces cerevisiae</i>
Control of the expression levels
of multiple enzymes in transgenic
yeasts is essential for the effective production of complex molecules
through fermentation. Here, we propose a tunable strategy for the
control of expression levels based on the design of terminator regions
and other gene-expression control elements in <i>Saccharomyces
cerevisiae</i>. Our genome-integrated system, which is capable
of producing high expression levels over a wide dynamic range, will
broadly enable metabolic engineering and synthetic biology. We demonstrated
that the activities of multiple cellulases and the production of ethanol
were doubled in a transgenic yeast constructed with our system compared
with those achieved with a standard expression system
Scheme of the combinatorial screening.
<p>Preparation of the 64 (4 Ă— 4 Ă— 4) possible strains. Transformation of cellulase cassettes was performed repeatedly in the order CBH2, EG2, CBH1. Screening of the cellulase activity of the 64 possible strains was also performed.</p
Relative cellulase activities of 368 combinatorially prepared transformants.
<p>Relative cellulase activity was normalized to the cellulase activity of the HR strain with Avicel cellulose as the substrate. Approximately 15% of the variants had higher cellulase activity than that of the HR strain.</p
Ethanol fermentation by the transgenic strains obtained from the combinatorial screening.
<p>Conversion of Avicel cellulose to ethanol with the external addition of β-glucosidase. The cultures described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144870#pone.0144870.g005" target="_blank">Fig 5</a> legend were used for ethanol fermentation. Symbols are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144870#pone.0144870.g005" target="_blank">Fig 5</a>.</p
Diagnostic polymerase chain reaction (PCR) assay of the variants obtained from the combinatorial screening.
<p>PCR was performed with the indicated primer sets in Materials and methods. The type of genome-integrated cellulase construct was determined from the lengths of the PCR products. PCR products of the transformants carrying the CBH1 (A, B), CBH2 (C, D), and EG2 constructs (E, F). The PCR products for the core promoter (A, C, E) and terminator regions (B, D, F) were amplified.</p
SDS-PAGE analysis of secreted cellulases.
<p>Lane 1, SW strain; Lane 2, CBH1 strain; Lane 3, CBH2 strain; Lane 4, EG2 strain; Lane 5, CBH1 + CBH2 + CBH3 with <i>TDH3pro</i> + <i>CYC1t</i> strain; Lane 6, HR strain; Lane 7, 3B5 strain; Lane 8, 2D9 strain; Lane 9, 4D4 strain.</p
Cellulase activity of the transgenic strains obtained from the combinatorial screening.
<p>Squares, diamonds, triangles, circles, and inverted triangles represent the HR, 2D9, 3B5, 4D4, and reference strains, respectively. Cellulase secretion was assessed by culturing the cells in yeast extract–peptone–dextrose medium and then measuring the cellulase activity by using Avicel cellulose as the substrate.</p
<i>Porphyromonas gingivalis</i> Gingipain-Dependently Enhances IL-33 Production in Human Gingival Epithelial Cells
<div><p>The cytokine IL-33 is constitutively expressed in epithelial cells and it augments Th2 cytokine-mediated inflammatory responses by regulating innate immune cells. We aimed to determine the role of the periodontal pathogen, <i>Porphyromonas gingivalis</i>, in the enhanced expression of IL-33 in human gingival epithelial cells. We detected IL-33 in inflamed gingival epithelium from patients with chronic periodontitis, and found that <i>P</i>. <i>gingivalis</i> increased IL-33 expression in the cytoplasm of human gingival epithelial cells <i>in vitro</i>. In contrast, lipopolysaccharide, lipopeptide, and fimbriae derived from <i>P</i>. <i>gingivalis</i> did not increase IL-33 expression. Specific inhibitors of <i>P</i>. <i>gingivalis</i> proteases (gingipains) suppressed IL-33 mRNA induction by <i>P</i>. <i>gingivalis</i> and the <i>P</i>. <i>gingivalis</i> gingipain-null mutant KDP136 did not induce IL-33 expression. A small interfering RNA for protease-activated receptor-2 (PAR-2) as well as inhibitors of phospholipase C, p38 and NF-ÎşB inhibited the expression of IL-33 induced by <i>P</i>. <i>gingivalis</i>. These results indicate that the PAR-2/IL-33 axis is promoted by <i>P</i>. <i>gingivalis</i> infection in human gingival epithelial cells through a gingipain-dependent mechanism.</p></div