Summary
Aro80, a member of the Zn2Cys6 family proteins, activates expression of the ARO9 and ARO10 genes involved in catabolism of aromatic amino acids in response to aromatic amino acids that act as ...inducers. ARO9 and ARO10 are also under the control of nitrogen catabolite repression, but the direct roles for GATA factors, Gat1 and Gln3, in this regulation have not yet been elucidated. Here, we demonstrate that Aro80 is constitutively bound to its target promoters and activated by inducers at the level of transactivation. Although Aro80 also binds to its own promoter, ARO80 expression is induced only by rapamycin, but not by tryptophan. We show that Aro80 is absolutely required for Gat1 binding to the ARO9, ARO10 and ARO80 promoters upon rapamycin treatment. Gln3 binding to these promoters shows a partial requirement for Aro80. Rapamycin‐dependent Gat1 and Gln3 binding to the Aro80 target promoters is not affected by tryptophan availability, suggesting that transactivation activity of Aro80 is not necessary for the recruitment of GATA factors. Rapamycin‐dependent induction of Aro80 target genes also requires PP2A phosphatase complex, but not Sit4 phosphatase, acting downstream of TORC1.
Cellulosic biomass is considered as a promising alternative to fossil fuels, but its recalcitrant nature and high cost of cellulase are the major obstacles to utilize this material. Consolidated ...bioprocessing (CBP), combining cellulase production, saccharification, and fermentation into one step, has been proposed as the most efficient way to reduce the production cost of cellulosic bioethanol. In this study, we developed a cellulolytic yeast consortium for CBP, based on the surface display of cellulosome structure, mimicking the cellulolytic bacterium, Clostridium thermocellum.
We designed a cellulolytic yeast consortium composed of four different yeast strains capable of either displaying a scaffoldin (mini CipA) containing three cohesin domains derived from C. thermocellum, or secreting one of the three types of cellulases, C. thermocellum CelA (endoglucanase) containing its own dockerin, Trichoderma reesei CBHII (exoglucanase) fused with an exogenous dockerin from C. thermocellum, or Aspergillus aculeatus BGLI (β-glucosidase). The secreted dockerin-containing enzymes, CelA and CBHI, were randomly assembled to the surface-displayed mini CipA via cohesin-dockerin interactions. On the other hand, BGLI was independently assembled to the cell surface since we newly found that it already has a cell adhesion characteristic. We optimized the cellulosome activity and ethanol production by controlling the combination ratio among the four yeast strains. A mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/l ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/l).
We produced cellulosic ethanol using a cellulolytic yeast consortium, which is composed of cells displaying mini cellulosomes generated via random assembly of CelA and CBHII to a mini CipA, and cells displaying BGLI independently. One of the advantages of this system is that ethanol production can be easily optimized by simply changing the combination ratio of the different populations. In addition, there is no limitation on the number of enzymes to be incorporated into this cellulosome structure. Not only cellulases used in this study, but also any other enzymes, including cellulases and hemicellulases, could be applied just by fusing dockerin domains to the enzymes.
Compared to the existing noninvasive methods, temporal interference (TI) current stimulation is an emerging noninvasive neuromodulation technique that can improve the ability to focus an electrical ...field on a target nerve. Induced TI field distribution depends on the anatomical structure of individual neurons, and thus the electrode and current optimization to enhance the field focus must reflect these factors. The current study presents a TI field optimization framework for focusing the stimulation energy on the target tibial nerve through extensive electrical simulations, factoring in individual anatomical differences. We conducted large-scale in-silico experiments using realistic models based on magnetic resonance images of human subjects to evaluate the effectiveness of the proposed methods for tibial nerve stimulation considering overactive bladder (OAB) treatment. The electrode position and current intensity were optimized for each subject using an automated algorithm, and the field-focusing performance was evaluated based on the maximum intensity of the electric fields induced at the target nerve compared with the electric fields in the neighboring tissues. Using the proposed optimization framework, the focusing ability increased by 12% when optimizing the electrode position. When optimizing both the electrode position and current, this capability increased by 11% relative to electrode position optimization alone. These results suggest the significance of optimizing the electrode position and current intensity for focusing TI fields at the target nerve. Our electrical simulation-based TI optimization framework can be extended to enable personalized peripheral nerve stimulation therapy to modulate peripheral nerves.
•ARO9 and ARO10 genes are activated by heat shock in an Aro80-dependent manner.•PKA, PKC, and HOG pathways do not affect the heat shock activation of Aro80.•Heat shock increases intracellular ...concentrations of aromatic amino acids.•Amino acid permeases do not affect the heat shock induction of ARO9 and ARO10.
In Saccharomyces cerevisiae, transcription of ARO9 and ARO10 genes, involved in the catabolism of aromatic amino acids, is activated by Aro80 transcription factor in response to aromatic amino acids. Here we show that the transcription of ARO9 and ARO10 is also induced by heat shock in an Aro80-dependent manner. However, heat shock-related signaling pathways including PKA, PKC, and HOG pathways are not involved in the heat shock activation of Aro80. We elucidate that heat-induced increase in aromatic amino acid influx can lead to the inducer-dependent activation of Aro80 upon heat shock. Known aromatic amino acid permeases play an insignificant role in the heat-induced expression of ARO9 and ARO10, suggesting that an increase in plasma membrane fluidity might be responsible for the influx of aromatic amino acids during heat shock stress.
This study is part of three-dimensional(3D) heat transfer analysis program developmental process. The program is being developed without it's own built in 3D-modeller. So 3D-model must be created ...from another 3D-modeller such as generic CAD programs and imported to the developed program. After that, according to the 3D-geometric data form imported model, 3D-mesh created for numerical calculation. But the 3D-model created from another 3D-modeller is likely to have errors in it's geometric data such as mismatch of position between vertexes or surfaces. these errors make it difficult to create 3D-mesh for calculation. These errors are must be detected and cured in the pre-process before creating 3D-mesh. So, in this study four kinds of filters and functions are developed and tested. Firstly, 'vertex error filter' is developed for detecting and curing for position data errors between vertexes. Secondly, 'normal vector error filter' is developed for errors of surface's normal vector in 3D-model. Thirdly, 'intersection filter' is developed for extracting and creating intersection surface between adjacent objects. fourthly, 'polygon-line filter' is developed for indicating outlines of object in 3D-model. the developed filters and functions were tested on several shapes of 3D-models. and confirmed applicability. these developed filters and functions will be applied to the developed program and tested and modified continuously for less errors and more accuracy.
Purpose: Heat transfers phenomena are described by the second order partial differential equation and its boundary conditions. In a three-dimensional structure of a building, the heat transfer ...phenomena generally include more than one material, and thus, become complicate. The analytic solutions are useful to understand heat transfer phenomena, but they can hardly be applied in engineering or design problems. Engineers and designers have generally been forced to use numerical methods providing reliable results. Finite volume methods with the unstructured grid system is only the suitable means of the analysis for the complex and arbitrary domains. Method: To obtain an numerical solution, a discretization method, which approximates the differential equations, and the interpolation methods for temperature and heat flux between two or more materials are required. The discretization methods are applied to small domains in space and time, and these numerical solutions form the descretized equations provide approximated solutions in both space and time. The accuracy of numerical solutions is dependent on the quality of discretizations and size of cells used. The higher accuracy, the higher numerical resources are required. The balance between the accuracy and difficulty of the numerical methods is critical for the success of the numerical analysis. A simple and easy interpolation methods among multiple materials are developed. The linear equations are solved with the BiCGSTAB being a effective matrix solver. Result: This study provides an overview of discretization methods, boundary interface, and matrix solver for the 3-dimensional numerical heat transfer including two materials.
Summary
Aro
80, a member of the
Zn
2
Cys
6
family proteins, activates expression of the
ARO9
and
ARO10
genes involved in catabolism of aromatic amino acids in response to aromatic amino acids that ...act as inducers.
ARO9
and
ARO10
are also under the control of nitrogen catabolite repression, but the direct roles for
GATA
factors,
Gat
1 and
Gln
3, in this regulation have not yet been elucidated. Here, we demonstrate that
Aro
80 is constitutively bound to its target promoters and activated by inducers at the level of transactivation. Although
Aro
80 also binds to its own promoter,
ARO80
expression is induced only by rapamycin, but not by tryptophan. We show that
Aro
80 is absolutely required for
Gat
1 binding to the
ARO9
,
ARO10
and
ARO80
promoters upon rapamycin treatment.
Gln
3 binding to these promoters shows a partial requirement for
Aro
80. Rapamycin‐dependent
Gat
1 and
Gln
3 binding to the
Aro
80 target promoters is not affected by tryptophan availability, suggesting that transactivation activity of
Aro
80 is not necessary for the recruitment of
GATA
factors. Rapamycin‐dependent induction of
Aro
80 target genes also requires
PP
2
A
phosphatase complex, but not
Sit
4 phosphatase, acting downstream of
TORC
1.
Shinorine, a mycosporine-like amino acid (MAA), is a small molecule sunscreen produced in some bacteria. In this study, by introducing shinorine biosynthetic genes from cyanobacteria Nostoc ...punctiform into Saccharomyces cerevisiae, we successfully constructed yeast strains capable of producing shinorine. Sedoheptulose 7-phosphate (S7P), an intermediate of the pentose phosphate pathway, is a key substrate for shinorine biosynthesis. To increase the S7P pool, xylose, which is assimilated via the pentose phosphate pathway, was used as a carbon source after introducing xylose assimilation genes from Scheffersomyces stipitis into the shinorine-producing strain. The resulting xylose-fermenting strain produced a trace amount of shinorine when cells were grown in glucose, but shinorine production was dramatically increased by adding xylose in the medium. Shinorine production was further improved by modulating the pentose phosphate pathway through deleting TAL1 and overexpressing STB5 and TKL1. The final engineered strain JHYS17–4 produced 31.0 mg/L (9.62 mg/g DCW) of shinorine in the optimized medium containing 8 g/L of xylose and 12 g/L of glucose, demonstrating that S. cerevisiae is a promising host to produce this natural sunscreen material.
A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, ...the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U₄C ARO₉ promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U₄C ARO₉ promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.
A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, ...the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U.sub.4C.sub.ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U.sub.4C.sub.ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that gamma-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.
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