12 research outputs found
Uncovering Key Metabolic Determinants of the Drug Interactions Between Trimethoprim and Erythromycin in Escherichia coli
Understanding interactions between antibiotics used in combination is an important theme in microbiology. Using the interactions between the antifolate drug trimethoprim and the ribosome-targeting antibiotic erythromycin in Escherichia coli as a model, we applied a transcriptomic approach for dissecting interactions between two antibiotics with different modes of action. When trimethoprim and erythromycin were combined, the transcriptional response of genes from the sulfate reduction pathway deviated from the dominant effect of trimethoprim on the transcriptome. We successfully altered the drug interaction from additivity to suppression by increasing the sulfate level in the growth environment and identified sulfate reduction as an important metabolic determinant that shapes the interaction between the two drugs. Our work highlights the potential of using prioritization of gene expression patterns as a tool for identifying key metabolic determinants that shape drug-drug interactions. We further demonstrated that the sigma factor-binding protein gene crl shapes the interactions between the two antibiotics, which provides a rare example of how naturally occurring variations between strains of the same bacterial species can sometimes generate very different drug interactions
Uncovering Key Metabolic Determinants of the Drug Interactions Between Trimethoprim and Erythromycin in Escherichia coli
Understanding interactions between antibiotics used in combination is an important theme in microbiology. Using the interactions between the antifolate drug trimethoprim and the ribosome-targeting antibiotic erythromycin in Escherichia coli as a model, we applied a transcriptomic approach for dissecting interactions between two antibiotics with different modes of action. When trimethoprim and erythromycin were combined, the transcriptional response of genes from the sulfate reduction pathway deviated from the dominant effect of trimethoprim on the transcriptome. We successfully altered the drug interaction from additivity to suppression by increasing the sulfate level in the growth environment and identified sulfate reduction as an important metabolic determinant that shapes the interaction between the two drugs. Our work highlights the potential of using prioritization of gene expression patterns as a tool for identifying key metabolic determinants that shape drug-drug interactions. We further demonstrated that the sigma factor-binding protein gene crl shapes the interactions between the two antibiotics, which provides a rare example of how naturally occurring variations between strains of the same bacterial species can sometimes generate very different drug interactions
On the Use of Metabolic Control Analysis in the Optimization of Cyanobacterial Biosolar Cell Factories
Oxygenic photosynthesis will have
a key role in a sustainable future. It is therefore significant that
this process can be engineered in organisms such as cyanobacteria
to construct cell factories that catalyze the (sun)light-driven conversion
of CO<sub>2</sub> and water into products like ethanol, butanol, or
other biofuels or lactic acid, a bioplastic precursor, and oxygen
as a byproduct. It is of key importance to optimize such cell factories
to maximal efficiency. This holds for their light-harvesting capabilities
under, for example, circadian illumination in large-scale photobioreactors.
However, this also holds for the “dark” reactions of
photosynthesis, that is, the conversion of CO<sub>2</sub>, NADPH,
and ATP into a product. Here, we present an analysis, based on metabolic
control theory, to estimate the optimal capacity for product formation
with which such cyanobacterial cell factories have to be equipped.
Engineered l-lactic acid producing <i>Synechocystis</i> sp. PCC6803 strains are used to identify the relation between production
rate and enzymatic capacity. The analysis shows that the engineered
cell factories for l-lactic acid are fully limited by the
metabolic capacity of the product-forming pathway. We attribute this
to the fact that currently available promoter systems in cyanobacteria
lack the genetic capacity to a provide sufficient expression in single-gene
doses
Growth-mediated negative feedback shapes quantitative antibiotic response
Dose-response relationships are a general concept for quantitatively describing biological systems across multiple scales, from the molecular to the whole-cell level. A clinically relevant example is the bacterial growth response to antibiotics, which is routinely characterized by dose-response curves. The shape of the dose-response curve varies drastically between antibiotics and plays a key role in treatment, drug interactions, and resistance evolution. However, the mechanisms shaping the dose-response curve remain largely unclear. Here, we show in Escherichia coli that the distinctively shallow dose-response curve of the antibiotic trimethoprim is caused by a negative growth-mediated feedback loop: Trimethoprim slows growth, which in turn weakens the effect of this antibiotic. At the molecular level, this feedback is caused by the upregulation of the drug target dihydrofolate reductase (FolA/DHFR). We show that this upregulation is not a specific response to trimethoprim but follows a universal trend line that depends primarily on the growth rate, irrespective of its cause. Rewiring the feedback loop alters the dose-response curve in a predictable manner, which we corroborate using a mathematical model of cellular resource allocation and growth. Our results indicate that growth-mediated feedback loops may shape drug responses more generally and could be exploited to design evolutionary traps that enable selection against drug resistance
Effect of consecutive rare codons on the recombinant production of human proteins in Escherichia coli
In Escherichia coli, the expression of heterologous genes for the production of recombinant proteins can be challenging due to the codon bias of different organisms. The rare codons AGG and AGA are among the rarest in E. coli. In this work, by using the human gene RioK2 as case study, we found that the presence of consecutive AGG-AGA led to a premature stop, which may be caused by an event of -1 frameshift. We found that translational problems caused by consecutive AGG-AGA are sequence dependent, in particular, in sequences that contain multiple rare AGG or AGA codons elsewhere. Translational problems can be alleviated by different strategies, including codon harmonization, codon optimization, or by substituting the consecutive AGG-AGA codons by more frequent arginine codons. Overall, our results furthered our understanding about the relationship between consecutive rare codons and translational problems. Such information will aid the design of DNA sequence for the production of recombinant proteins
Culturing Synechocystis sp. Strain PCC 6803 with N 2
Investigating the physiology of cyanobacteria cultured under a diel light regime is relevant for a better understanding of the resulting growth characteristics and for specific biotechnological applications that are foreseen for these photosynthetic organisms.Here, we present the results of a multi-omics study of the model cyanobacterium Synechocystis sp. PCC 6803, cultured in a lab-scale photobioreactor in physiological conditions relevant for large-scale culturing. The culture is sparged with N2 and CO2, leading to an anoxic environment during the dark period.Growth follows the availability of light. Metabolite analysis performed with 1H Nuclear Magnetic Resonance analysis, shows that amino acids involved in nitrogen and sulfur assimilation show elevated levels in the light. Most protein levels, analyzed through mass spectrometry, remain rather stable. However, several high-light-response proteins and stress-response proteins show distinct changes at the onset of the light period.Microarray-based transcript analysis finds common patterns of approximately 56 % of the transcriptome following the diel regime. These oscillating transcripts can be grouped coarsely into genes that are up-regulated and down-regulated in the dark period, respectively.The accumulated glycogen is degraded in the anaerobic environment in the dark. A small part is degraded gradually, reflecting basic maintenance requirements of the cells in darkness. Surprisingly, the largest part is degraded rapidly in a short time span at the end of the dark period. This degradation could allow rapid formation of metabolic intermediates at the end of the dark period, preparing the cells for the resumption of growth at the start of the light period. IMPORTANCE: Industrial scale biotechnological applications are anticipated for cyanobacteria. We simulated large-scale high cell-density culturing of Synechocystis sp. PCC 6803 under a diel light regime in a lab-scale photobioreactor. In BG-11 medium, Synechocystis grows only in the light. Metabolite analysis groups the collected samples according to the light and dark condition, respectively. Proteome analysis suggests that the majority of enzyme-activity regulation is not hierarchical, but rather through enzyme activity regulation. An abrupt light-on condition induces high-light-stress proteins. Transcript analysis shows distinct patterns for the light and dark periods.Glycogen gradually accumulates in the light and is rapidly consumed in the last quarter of the dark period. This suggests that the circadian clock primes the cellular machinery for immediate resumption of growth in the light
Nonhierarchical Flux Regulation Exposes the Fitness Burden Associated with Lactate Production in <i>Synechocystis</i> sp. PCC6803
Cyanobacteria are
mostly engineered to be sustainable cell-factories
by genetic manipulations alone. Here, by modulating the concentration
of allosteric effectors, we focus on increasing product formation
without further burdening the cells with increased expression of enzymes.
Resorting to a novel 96-well microplate cultivation system for cyanobacteria,
and using lactate-producing strains of <i>Synechocystis</i> PCC6803 expressing different l-lactate dehydrogenases (LDH),
we titrated the effect of 2,5-anhydro-mannitol supplementation. The
latter acts in cells as a nonmetabolizable analogue of fructose 1,6-bisphosphate,
a known allosteric regulator of one of the tested LDHs. In this strain
(SAA023), we achieved over 2-fold increase of lactate productivity.
Furthermore, we observed that as carbon is increasingly deviated during
growth toward product formation, there is an increased fixation rate
in the population of spontaneous mutants harboring an impaired production
pathway. This is a challenge in the development of green cell factories,
which may be countered by the incorporation in biotechnological processes
of strategies such as the one pioneered here