109 results on '"De Mey M"'
Search Results
2. Transport kinetics of ectoine, an osmolyte produced by Brevibacterium epidermis
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Onraedt, A., De Mey, M., Walcarius, B., Soetaert, W., and Vandamme, E. J.
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- 2006
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3. The Cognitive Turn:Sociological and Psychological Perspectives on Science
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Woolgar, S, de Mey, M, Schinn, T, and Fuller, S
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- 2016
4. THE PSYCHOLOGY OF NEGATION AND ATTENTION
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De Mey, M.
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- 1972
5. Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)
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Waegeman, H. (author), Beauprez, J. (author), Moens, H. (author), Maertens, J. (author), De Mey, M. (author), Foulquié-Moreno, M.R. (author), Heijnen, J.J. (author), Charlier, D. (author), Soetaert, W. (author), Waegeman, H. (author), Beauprez, J. (author), Moens, H. (author), Maertens, J. (author), De Mey, M. (author), Foulquié-Moreno, M.R. (author), Heijnen, J.J. (author), Charlier, D. (author), and Soetaert, W. (author)
- Abstract
Background: Gene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3). Results: The deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ?arcA?iclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ?iclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ?arcA ?iclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a comparison, BT/Biotechnology, Applied Sciences
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- 2011
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6. Crack detection and inpainting for virtual restoration of paintings: The case of the Ghent Altarpiece
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Cornelis, B., primary, Ružić, T., additional, Gezels, E., additional, Dooms, A., additional, Pižurica, A., additional, Platiša, L., additional, Cornelis, J., additional, Martens, M., additional, De Mey, M., additional, and Daubechies, I., additional
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- 2013
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7. Microbial metabolomics: Past, present and future methodologies
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Mashego, M.R. (author), Rumbold, K. (author), De Mey, M. (author), Vandamme, E. (author), Soetaert, W. (author), Heijnen, J.J. (author), Mashego, M.R. (author), Rumbold, K. (author), De Mey, M. (author), Vandamme, E. (author), Soetaert, W. (author), and Heijnen, J.J. (author)
- Abstract
Applied Sciences
- Published
- 2007
8. Spatiogram features to characterize pearls in paintings
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Platisa, L., primary, Cornells, B., additional, Ruzic, T., additional, Pizurica, A., additional, Dooms, A., additional, Martens, M., additional, De Mey, M., additional, and Daubechies, I., additional
- Published
- 2011
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9. Designer bugs — A platform for the production of tailor-made chemicals from renewable resources
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De Mey, M., primary, Beauprez, J., additional, Lequeux, G., additional, Maertens, J., additional, Waegeman, H., additional, and Soetaert, W., additional
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- 2010
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10. Metabolic characterisation of E. coli citrate synthase and phosphoenolpyruvate carboxylase mutants in aerobic cultures.
- Author
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Maeseneire, S. L., De Mey, M., Vandedrinck, S., and Vandamme, E. J.
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ESCHERICHIA coli ,PROTEINS ,EXCRETION ,RECOMBINANT proteins ,GLUCOSE - Abstract
E. coli is still one of the most commonly used hosts for protein production. However, when it is grown with excess glucose, acetate accumulation occurs. Elevated acetate concentrations have an inhibitory effect on growth rate and recombinant protein yield, and thus elimination of acetate formation is an important aim towards industrial production of recombinant proteins. Here we examine if over-expression of citrate synthase ( gltA) or phosphoenolpyruvate carboxylase ( ppc) can eliminate acetate production. Knock-out as well as over-expression mutants were constructed and characterized. Knocking out ppc or gltA decreased the maximum cell density by 14% and increased the acetate excretion by 7%, respectively decreased it by 10%. Over-expression of ppc or gltA increased the maximum cell dry weight by 91% and 23%, respectively. No acetate excretion was detected at these increased cell densities (35 and 23 g/l, respectively). [ABSTRACT FROM AUTHOR]
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- 2006
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11. Granny, the Naked emperor and the second cognitive revolution
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de Gelder, B., Fuller, S., De Mey, M., Shinn, T., and TS Social and Behavioral Sciences
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- 1989
12. Science policy and the scientific picture of the world
- Author
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Rip, Arie, Callebaut, W., De Mey, M., and University of Twente
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IR-57452 - Published
- 1979
13. Cognitive science and science dynamics. Philosophical and epistemological issues for information science
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De Mey, M., primary
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- 1984
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14. Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)
- Author
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Charlier Daniel, Heijnen Joseph J, Foulquié-Moreno Maria R, De Mey Marjan, Maertens Jo, Moens Helena, Beauprez Joeri, Waegeman Hendrik, and Soetaert Wim
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Microbiology ,QR1-502 - Abstract
Abstract Background Gene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3). Results The deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ΔarcAΔiclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ΔiclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ΔarcA ΔiclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a comparison of the genome sequences of the two strains showed that BL21 possesses two mutations in the promoter region of iclR and rare codons are present in arcA implying a lower tRNA acceptance. Both phenomena presumably result in a reduced ArcA and IclR synthesis in BL21, which contributes to the similar physiology as observed in E. coli K12 ΔarcAΔiclR. Conclusions The deletion of arcA results in a decrease of repression on transcription of TCA cycle genes under glucose abundant conditions, without significantly affecting the glyoxylate pathway activity. IclR clearly represses transcription of glyoxylate pathway genes under glucose abundance, a condition in which Crp activation is absent. Under glucose limitation, Crp is responsible for the high glyoxylate flux, but IclR still represses transcription. Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.
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- 2011
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15. Promoter knock-in: a novel rational method for the fine tuning of genes
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Cunin Raymond, Vandamme Erick J, Soetaert Wim K, Boogmans Sarah, Maertens Jo, De Mey Marjan, and Foulquié-Moreno Maria R
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Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound. An important strategy to achieve this is the modification of the expression level of specific genes. Several methods for the modification or the replacement of promoters have been proposed, but most of them involve time-consuming screening steps. We describe here a novel optimized method for the insertion of constitutive promoters (referred to as "promoter knock-in") whose strength can be compared with the native promoter by applying a promoter strength predictive (PSP) model. Results Our method was successfully applied to fine tune the ppc gene of Escherichia coli. While developing the promoter knock-in methodology, we showed the importance of conserving the natural leader region containing the ribosome binding site (RBS) of the gene of interest and of eliminating upstream regulatory elements (transcription factor binding sites). The gene expression was down regulated instead of up regulated when the natural RBS was not conserved and when the upstream regulatory elements were eliminated. Next, three different promoter knock-ins were created for the ppc gene selecting three different artificial promoters. The measured constitutive expression of the ppc gene in these knock-ins reflected the relative strength of the different promoters as predicted by the PSP model. The applicability of our PSP model and promoter knock-in methodology was further demonstrated by showing that the constitutivity and the relative levels of expression were independent of the genetic background (comparing wild-type and mutant E. coli strains). No differences were observed during scaling up from shake flask to bioreactor-scale, confirming that the obtained expression was independent of environmental conditions. Conclusion We are proposing a novel methodology for obtaining appropriate levels of expression of genes of interest, based on the prediction of the relative strength of selected synthetic promoters combined with an optimized promoter knock-in strategy. The obtained expression levels are independent of the genetic background and scale conditions. The method constitutes therefore a valuable addition to the genetic toolbox for the metabolic engineering of E. coli.
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- 2010
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16. Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering
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Soetaert Wim K, Lequeux Gaspard J, Maertens Jo, De Mey Marjan, and Vandamme Erick J
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Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Nowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of Escherichia coli as a useful tool for fine tuning gene expression is discussed here. Results A degenerated oligonucleotide sequence that encodes consensus sequences for E. coli promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent E. coli MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD600 ml to 7606.83 RFU/OD600 ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated. Conclusion For Escherichia coli, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.
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- 2007
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17. Common schemata in social cognition, ancient thought and the organization of language
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Peeters, Guido, De Mey, M, Pinxten, R, Poriau, M, Vandamme, F, De Mey, M., Pinxten, R., Poriau, M., and Vandamme, F.
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ispartof: pages:25-30 ispartof: CC77 International Workshop on the Cognitive Viewpoint pages:25-30 ispartof: C77 International Workshop on the cognitive Viewpoint location:Ghent date:24 Mar - 26 Mar 1977 status: published
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- 1977
18. Science and Technology Policy Research for Development: a Research Proposal
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Jean Paul VAN BENDEGEM, Werner Callebaut, Callebaut, W., De Mey, M., Pinxten, R., Vandamme, F., and Centre for Logic and Philosophy of Science
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- 1980
19. An empirical investigation on scientific observation
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Jean Paul VAN BENDEGEM, Leo Apostel, Dirk Batens, Callebaut, W., De Mey, M., Pinxten, R., Vandamme, F., and Centre for Logic and Philosophy of Science
20. The formal structure of action theory is deducible from generalized set theory
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Jean Paul VAN BENDEGEM, De Mey, M., Pinxten, R., Poriau, M., Vandamme, F., and Centre for Logic and Philosophy of Science
21. Fundamentals and Exceptions of the LysR-type Transcriptional Regulators.
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Demeester W, De Paepe B, and De Mey M
- Abstract
LysR-type transcriptional regulators (LTTRs) are emerging as a promising group of macromolecules for the field of biosensors. As the largest family of bacterial transcription factors, the LTTRs represent a vast and mostly untapped repertoire of sensor proteins. To fully harness these regulators for transcription factor-based biosensor development, it is crucial to understand their underlying mechanisms and functionalities. In the first part, this Review discusses the established model and features of LTTRs. As dual-function regulators, these inducible transcription factors exude precise control over their regulatory targets. In the second part of this Review, an overview is given of the exceptions to the "classic" LTTR model. While a general regulatory mechanism has helped elucidate the intricate regulation performed by LTTRs, it is essential to recognize the variations within the family. By combining this knowledge, characterization of new regulators can be done more efficiently and accurately, accelerating the expansion of transcriptional sensors for biosensor development. Unlocking the pool of LTTRs would significantly expand the currently limited range of detectable molecules and regulatory functions available for the implementation of novel synthetic genetic circuitry.
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- 2024
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22. Delaying production with prokaryotic inducible expression systems.
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De Baets J, De Paepe B, and De Mey M
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- Bacteria metabolism, Bacteria genetics, Metabolic Engineering methods, Gene Expression Regulation, Bacterial, Promoter Regions, Genetic, Fermentation, Quorum Sensing
- Abstract
Background: Engineering bacteria with the purpose of optimizing the production of interesting molecules often leads to a decrease in growth due to metabolic burden or toxicity. By delaying the production in time, these negative effects on the growth can be avoided in a process called a two-stage fermentation., Main Text: During this two-stage fermentation process, the production stage is only activated once sufficient cell mass is obtained. Besides the possibility of using external triggers, such as chemical molecules or changing fermentation parameters to induce the production stage, there is a renewed interest towards autoinducible systems. These systems, such as quorum sensing, do not require the extra interference with the fermentation broth to start the induction. In this review, we discuss the different possibilities of both external and autoinduction methods to obtain a two-stage fermentation. Additionally, an overview is given of the tuning methods that can be applied to optimize the induction process. Finally, future challenges and prospects of (auto)inducible expression systems are discussed., Conclusion: There are numerous methods to obtain a two-stage fermentation process each with their own advantages and disadvantages. Even though chemically inducible expression systems are well-established, an increasing interest is going towards autoinducible expression systems, such as quorum sensing. Although these newer techniques cannot rely on the decades of characterization and applications as is the case for chemically inducible promoters, their advantages might lead to a shift in future inducible expression systems., (© 2024. The Author(s).)
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- 2024
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23. "Metabolic burden" explained: stress symptoms and its related responses induced by (over)expression of (heterologous) proteins in Escherichia coli.
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Snoeck S, Guidi C, and De Mey M
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- Biotechnology, Metabolic Engineering, Bacteria metabolism, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism
- Abstract
Background: Engineering bacterial strains to redirect the metabolism towards the production of a specific product has enabled the development of industrial biotechnology. However, rewiring the metabolism can have severe implications for a microorganism, rendering cells with stress symptoms such as a decreased growth rate, impaired protein synthesis, genetic instability and an aberrant cell size. On an industrial scale, this is reflected in processes that are not economically viable., Main Text: In literature, most stress symptoms are attributed to "metabolic burden", however the actual triggers and stress mechanisms involved are poorly understood. Therefore, in this literature review, we aimed to get a better insight in how metabolic engineering affects Escherichia coli and link the observed stress symptoms to its cause. Understanding the possible implications that chosen engineering strategies have, will help to guide the reader towards optimising the envisioned process more efficiently., Conclusion: This review addresses the gap in literature and discusses the triggers and effects of stress mechanisms that can be activated when (over)expressing (heterologous) proteins in Escherichia coli. It uncovers that the activation of the different stress mechanisms is complex and that many are interconnected. The reader is shown that care has to be taken when (over)expressing (heterologous) proteins as the cell's metabolism is tightly regulated., (© 2024. The Author(s).)
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- 2024
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24. Towards a rational approach to promoter engineering: understanding the complexity of transcription initiation in prokaryotes.
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Deal C, De Wannemaeker L, and De Mey M
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- Promoter Regions, Genetic genetics, Gene Expression Regulation, DNA, Sigma Factor genetics, Sigma Factor metabolism, Transcription, Genetic genetics, DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism
- Abstract
Promoter sequences are important genetic control elements. Through their interaction with RNA polymerase they determine transcription strength and specificity, thereby regulating the first step in gene expression. Consequently, they can be targeted as elements to control predictability and tuneability of a genetic circuit, which is essential in applications such as the development of robust microbial cell factories. This review considers the promoter elements implicated in the three stages of transcription initiation, detailing the complex interplay of sequence-specific interactions that are involved, and highlighting that DNA sequence features beyond the core promoter elements work in a combinatorial manner to determine transcriptional strength. In particular, we emphasize that, aside from promoter recognition, transcription initiation is also defined by the kinetics of open complex formation and promoter escape, which are also known to be highly sequence specific. Significantly, we focus on how insights into these interactions can be manipulated to lay the foundation for a more rational approach to promoter engineering., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)
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- 2024
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25. Standardization of Fluorescent Reporter Assays in Synthetic Biology across the Visible Light Spectrum.
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De Wannemaeker L, Mey F, Bervoets I, Ver Cruysse M, Baldwin GS, and De Mey M
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- Reproducibility of Results, Light, Reference Standards, Synthetic Biology, Fluorescent Dyes
- Abstract
In synthetic biology, Fluorescent reporters are frequently used to characterize the expression levels obtained from both genetic parts such as promoters and ribosome binding sites as well as from complex genetic circuits. To this end, plate readers offer an easy and high-throughput way of characterizing both the growth and fluorescence expression levels of cell cultures. However, despite the similar mode of action used in different devices, their output is not comparable due to intrinsic differences in their setup. Additionally, the generated output is expressed using arbitrary units, limiting reliable comparison of results to measurements taken within one single experiment using one specific plate reader, hampering the transferability of data across different plate readers and laboratories. This article presents an easy and accessible calibration method for transforming the device-specific output into a standardized output expressing the amount of fluorescence per well as a known equivalent fluorophore concentration per cell for fluorescent reporters spanning the visible light spectrum. This calibration method follows a 2-fold approach determining both the estimated number of cells and the equivalent chemical fluorophore concentration per well. It will contribute to the comparison of plate reader experiments between different laboratories across the world and will therefore greatly improve the reliability and exchange of both results and genetic parts between research groups.
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- 2023
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26. Taming CRISPRi: Dynamic range tuning through guide RNA diversion.
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Van Hove B, De Wannemaeker L, Missiaen I, Maertens J, and De Mey M
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- RNA, CRISPR-Cas Systems genetics, Metabolic Engineering methods
- Abstract
CRISPRi is a powerful technique to repress gene expression in a targeted and highly efficient manner. However, this potency is a double-edged sword in inducible systems, as even leaky expression of guide RNA results in a repression phenotype, complicating applications such as dynamic metabolic engineering. We evaluated three methods to enhance the controllability of CRISPRi by modulating the level of free and DNA-bound guide RNA complexes. Overall repression can be attenuated through rationally designed mismatches in the reversibility determining region of the guide RNA sequence; decoy target sites can selectively modulate repression at low levels of induction; and the implementation of feedback control not only enhances the linearity of induction, but broadens the dynamic range of the output as well. Furthermore, feedback control significantly enhances the recovery rate after induction is removed. Used in combination, these techniques enable the fine-tuning of CRISPRi to meet restrictions imposed by the target and match the input signal required for induction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2023
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27. MoBioS: Modular Platform Technology for High-Throughput Construction and Characterization of Tunable Transcriptional Biological Sensors.
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Demeester W, De Baets J, Duchi D, De Mey M, and De Paepe B
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- Fluorescence, Technology, Biosensing Techniques
- Abstract
All living organisms have evolved and fine-tuned specialized mechanisms to precisely monitor a vast array of different types of molecules. These natural mechanisms can be sourced by researchers to build Biological Sensors (BioS) by combining them with an easily measurable output, such as fluorescence. Because they are genetically encoded, BioS are cheap, fast, sustainable, portable, self-generating and highly sensitive and specific. Therefore, BioS hold the potential to become key enabling tools that stimulate innovation and scientific exploration in various disciplines. However, the main bottleneck in unlocking the full potential of BioS is the fact that there is no standardized, efficient and tunable platform available for the high-throughput construction and characterization of biosensors. Therefore, a modular, Golden Gate-based construction platform, called MoBioS, is introduced in this article. It allows for the fast and easy creation of transcription factor-based biosensor plasmids. As a proof of concept, its potential is demonstrated by creating eight different, functional and standardized biosensors that detect eight diverse molecules of industrial interest. In addition, the platform contains novel built-in features to facilitate fast and efficient biosensor engineering and response curve tuning.
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- 2023
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28. Human gut microbiota stratified by (+)-catechin metabolism dynamics reveals colon region-dependent metabolic profile.
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Li Q, Stautemas J, Omondi Onyango S, De Mey M, Duchi D, Tuenter E, Hermans N, Calders P, and Van de Wiele T
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- Humans, Colon microbiology, Bacteria genetics, Bacteria metabolism, Metabolome, Gastrointestinal Microbiome, Catechin metabolism, Microbiota
- Abstract
Catechins have proven to have several health benefits, yet a huge interindividual variability occurs. The metabolic potency of the colonic microbiota towards catechin is a key determinant of this variability. Microbiota from two donors - previously characterized as a fast and a slow converter- were incubated with (+)-catechin in vitro. The robustness of in vitro metabolic profiles was verified by well-fitted human trials. The colon region-dependent and donor-dependent patterns were reflected in both metabolic features and colonic microbiota composition. Upstream and downstream metabolites were mainly detected in the proximal and distal colons, respectively, and were considered important explanatory variables for microbiota clustering in the corresponding colon regions. Higher abundances of two catechin-metabolizing bacteria, Eggerthella and Flavonifractor were found in the distal colon compared to the proximal colon and in slow converter than fast converter. Additionally, these two bacteria were enriched in treatment samples compared to sham treatment samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2023
- Full Text
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29. Controlled processivity in glycosyltransferases: A way to expand the enzymatic toolbox.
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Guidi C, Biarnés X, Planas A, and De Mey M
- Subjects
- Glycosylation, Protein Engineering, Eukaryotic Cells metabolism, Glycosyltransferases metabolism, Carbohydrates
- Abstract
Glycosyltransferases (GT) catalyse the biosynthesis of complex carbohydrates which are the most abundant group of molecules in nature. They are involved in several key mechanisms such as cell signalling, biofilm formation, host immune system invasion or cell structure and this in both prokaryotic and eukaryotic cells. As a result, research towards complete enzyme mechanisms is valuable to understand and elucidate specific structure-function relationships in this group of molecules. In a next step this knowledge could be used in GT protein engineering, not only for rational drug design but also for multiple biotechnological production processes, such as the biosynthesis of hyaluronan, cellooligosaccharides or chitooligosaccharides. Generation of these poly- and/or oligosaccharides is possible due to a common feature of several of these GTs: processivity. Enzymatic processivity has the ability to hold on to the growing polymer chain and some of these GTs can even control the number of glycosyl transfers. In a first part, recent advances in understanding the mechanism of various processive enzymes are discussed. To this end, an overview is given of possible engineering strategies for the purpose of new industrial and fundamental applications. In the second part of this review, we focused on specific chain length-controlling mechanisms, i.e., key residues or conserved regions, and this for both eukaryotic and prokaryotic enzymes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2023
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30. Dynamic feedback regulation for efficient membrane protein production using a small RNA-based genetic circuit in Escherichia coli.
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Guidi C, De Wannemaeker L, De Baets J, Demeester W, Maertens J, De Paepe B, and De Mey M
- Subjects
- Feedback, Membrane Proteins genetics, Membrane Proteins metabolism, RNA, Bacterial genetics, Gene Expression Regulation, Bacterial, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism
- Abstract
Background: Membrane proteins (MPs) are an important class of molecules with a wide array of cellular functions and are part of many metabolic pathways. Despite their great potential-as therapeutic drug targets or in microbial cell factory optimization-many challenges remain for efficient and functional expression in a host such as Escherichia coli., Results: A dynamically regulated small RNA-based circuit was developed to counter membrane stress caused by overexpression of different MPs. The best performing small RNAs were able to enhance the maximum specific growth rate with 123%. On culture level, the total MP production was increased two-to three-fold compared to a system without dynamic control. This strategy not only improved cell growth and production of the studied MPs, it also suggested the potential use for countering metabolic burden in general., Conclusions: A dynamically regulated feedback circuit was developed that can sense metabolic stress caused by, in casu, the overexpression of an MP and responds to it by balancing the metabolic state of the cell and more specifically by downregulating the expression of the MP of interest. This negative feedback mechanism was established by implementing and optimizing simple-to-use genetic control elements based on post-transcriptional regulation: small non-coding RNAs. In addition to membrane-related stress when the MP accumulated in the cytoplasm as aggregates, the sRNA-based feedback control system was still effective for improving cell growth but resulted in a decreased total protein production. This result suggests promiscuity of the MP sensor for more than solely membrane stress., (© 2022. The Author(s).)
- Published
- 2022
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31. Unlocking the bacterial domain for industrial biotechnology applications using universal parts and tools.
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De Wannemaeker L, Bervoets I, and De Mey M
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Saccharomyces cerevisiae genetics, Synthetic Biology, Biotechnology, Metabolic Engineering
- Abstract
Synthetic biology can play a major role in the development of sustainable industrial biotechnology processes. However, the development of economically viable production processes is currently hampered by the limited availability of host organisms that can be engineered for a specific production process. To date, standard hosts such as Escherichia coli and Saccharomyces cerevisiae are often used as starting points for process development since parts and tools allowing their engineering are readily available. However, their suboptimal metabolic background or impaired performance at industrial scale for a desired production process, can result in increased costs associated with process development and/or disappointing production titres. Building a universal and portable gene expression system allowing genetic engineering of hosts across the bacterial domain would unlock the bacterial domain for industrial biotechnology applications in a highly standardized manner and, doing so, render industrial biotechnology processes more competitive compared to the current polluting chemical processes. This review gives an overview of a selection of bacterial hosts highly interesting for industrial biotechnology based on both their metabolic and process optimization properties. Moreover, the requirements and progress made so far to enable universal, standardized, and portable gene expression across the bacterial domain is discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)
- Published
- 2022
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32. The bacterial quorum sensing peptide iAM373 is a novel inducer of sarcopenia.
- Author
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De Spiegeleer A, Wynendaele E, Descamps A, Debunne N, Braeckman BP, De Mey M, Coudenys J, Crombez L, Verbeke F, Janssens Y, Janky R, Goossens E, Vlaeminck C, Duchi D, Andries V, Dumas E, Petrovic M, Van de Wiele T, Knappe D, Hoffmann R, Mouly V, Bigot A, Vereecke L, Van Immerseel F, Van Den Noortgate N, De Spiegeleer B, and Elewaut D
- Subjects
- Bacteria, Humans, Oligopeptides, Peptides, Quorum Sensing, Sarcopenia
- Published
- 2022
- Full Text
- View/download PDF
33. In Vitro Microbial Metabolism of (+)-Catechin Reveals Fast and Slow Converters with Individual-Specific Microbial and Metabolite Markers.
- Author
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Li Q, Van Herreweghen F, Onyango SO, De Mey M, and Van de Wiele T
- Subjects
- Fatty Acids, Volatile, Feces microbiology, Humans, Catechin metabolism, Gastrointestinal Microbiome, Microbiota
- Abstract
The bioavailability of catechin highly relies on gut microbiota which may determine its metabolic profile, resulting in different health outcomes. Here, we investigated in vitro (+)-catechin metabolism by human microbial communities. There were substantial interindividual differences in the metabolic profiles of (+)-catechin, with 5-(3',4'-dihydroxyphenyl)-γ-valerolactone being the major contributor. Furthermore, the microbial metabolic rate of catechin enabled stratification of 12 participants (fast, medium, and slow converters), despite the interference from the strong intrinsic interindividual variability in fecal microbiota. Correlations were established between this stratified population and microbiota features, such as ecosystem diversity. Additionally, fast converters had significantly higher prevalences of amplicon sequence variants (ASVs) with potential capacity of C-ring cleavage (ASV233_ Eggerthella and ASV402_ Eubacterium ), B-ring dihydroxylation (ASV402_ Eubacterium ), and short-chain fatty acid (SCFA)-producing ASVs. In conclusion, metabolic-capability-based stratification allows us to uncover differences in microbial composition between fast and slow converters, which could help to elucidate interindividual variabilities in the health benefits of catechins.
- Published
- 2022
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34. Biosensor-driven, model-based optimization of the orthogonally expressed naringenin biosynthesis pathway.
- Author
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Van Brempt M, Peeters AI, Duchi D, De Wannemaeker L, Maertens J, De Paepe B, and De Mey M
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, Flavanones, Metabolic Engineering methods, Biosensing Techniques methods, Biosynthetic Pathways
- Abstract
Background: The rapidly expanding synthetic biology toolbox allows engineers to develop smarter strategies to tackle the optimization of complex biosynthetic pathways. In such a strategy, multi-gene pathways are subdivided in several modules which are each dynamically controlled to fine-tune their expression in response to a changing cellular environment. To fine-tune separate modules without interference between modules or from the host regulatory machinery, a sigma factor (σ) toolbox was developed in previous work for tunable orthogonal gene expression. Here, this toolbox is implemented in E. coli to orthogonally express and fine-tune a pathway for the heterologous biosynthesis of the industrially relevant plant metabolite, naringenin. To optimize the production of this pathway, a practical workflow is still imperative to balance all steps of the pathway. This is tackled here by the biosensor-driven screening, subsequent genotyping of combinatorially engineered libraries and finally the training of three different computer models to predict the optimal pathway configuration., Results: The efficiency and knowledge gained through this workflow is demonstrated here by improving the naringenin production titer by 32% with respect to a random pathway library screen. Our best strain was cultured in a batch bioreactor experiment and was able to produce 286 mg/L naringenin from glycerol in approximately 26 h. This is the highest reported naringenin production titer in E. coli without the supplementation of pathway precursors to the medium or any precursor pathway engineering. In addition, valuable pathway configuration preferences were identified in the statistical learning process, such as specific enzyme variant preferences and significant correlations between promoter strength at specific steps in the pathway and titer., Conclusions: An efficient strategy, powered by orthogonal expression, was applied to successfully optimize a biosynthetic pathway for microbial production of flavonoids in E. coli up to high, competitive levels. Within this strategy, statistical learning techniques were combined with combinatorial pathway optimization techniques and an in vivo high-throughput screening method to efficiently determine the optimal operon configuration of the pathway. This "pathway architecture designer" workflow can be applied for the fast and efficient development of new microbial cell factories for different types of molecules of interest while also providing additional insights into the underlying pathway characteristics., (© 2022. The Author(s).)
- Published
- 2022
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35. Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor.
- Author
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Sybers D, Joka Bernauw A, El Masri D, Ramadan Maklad H, Charlier D, De Mey M, Bervoets I, and Peeters E
- Subjects
- Acyl Coenzyme A genetics, Acyl Coenzyme A metabolism, Bacterial Proteins genetics, Binding Sites, Escherichia coli drug effects, Escherichia coli metabolism, Fatty Acids metabolism, Gene Expression Regulation, Bacterial, Isopropyl Thiogalactoside pharmacology, Laurates pharmacology, Microorganisms, Genetically-Modified, Operator Regions, Genetic, Promoter Regions, Genetic, Repressor Proteins genetics, Sulfolobus acidocaldarius genetics, Archaeal Proteins genetics, Escherichia coli genetics, Genetic Engineering methods, Transcription Factors genetics
- Abstract
Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators. This work aims to design and explore the use of an archaeal TetR family regulator, FadR
Sa from Sulfolobus acidocaldarius, in a bacterial system, namely Escherichia coli. This is a challenging objective given the fundamental difference between the bacterial and archaeal transcription machinery and the lack of a native TetR-like FadR regulatory system in E. coli. The synthetic σ70 -dependent bacterial promoter proD was used as a starting point to design hybrid bacterial/archaeal promoter/operator regions, in combination with the mKate2 fluorescent reporter enabling a readout. Four variations of proD containing FadRSa binding sites were constructed and characterized. While expressional activity of the modified promoter proD was found to be severely diminished for two of the constructs, constructs in which the binding site was introduced adjacent to the -35 promoter element still displayed sufficient basal transcriptional activity and showed up to 7-fold repression upon expression of FadRSa . Addition of acyl-CoA has been shown to disrupt FadRSa binding to the DNA in vitro. However, extracellular concentrations of up to 2 mM dodecanoate, subsequently converted to acyl-CoA by the cell, did not have a significant effect on repression in the bacterial system. This work demonstrates that archaeal transcription regulators can be used to generate actuator elements for use in E. coli, although the lack of ligand response underscores the challenge of maintaining biological function when transferring parts to a phylogenetically divergent host., (Copyright © 2021 Elsevier B.V. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
36. ProD: A Tool for Predictive Design of Tailored Promoters in Escherichia coli.
- Author
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Mey F, Clauwaert J, Van Brempt M, Stock M, Maertens J, Waegeman W, and De Mey M
- Subjects
- High-Throughput Nucleotide Sequencing, Promoter Regions, Genetic, Synthetic Biology, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins metabolism
- Abstract
A major goal in synthetic biology is the engineering of synthetic gene circuits with a predictable, controlled and designed outcome. This creates a need for building blocks that can modulate gene expression without interference with the native cell system. A tool allowing forward engineering of promoters with predictable transcription initiation frequency is still lacking. Promoter libraries specific for σ
70 to ensure the orthogonality of gene expression were built in Escherichia coli and labeled using fluorescence-activated cell sorting to obtain high-throughput DNA sequencing data to train a convolutional neural network. We were able to confirm in vivo that the model is able to predict the promoter transcription initiation frequency (TIF) of new promoter sequences. Here, we provide an online tool for promoter design (ProD) in E. coli, which can be used to tailor output sequences of desired promoter TIF or predict the TIF of a custom sequence., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)- Published
- 2022
- Full Text
- View/download PDF
37. The Donor-Dependent and Colon-Region-Dependent Metabolism of (+)-Catechin by Colonic Microbiota in the Simulator of the Human Intestinal Microbial Ecosystem.
- Author
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Li Q, Van Herreweghen F, De Mey M, Goeminne G, and Van de Wiele T
- Subjects
- Adult, Biological Variation, Population, Chromatography, High Pressure Liquid, Female, Humans, Male, Metabolic Networks and Pathways, Metabolome, Metabolomics methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Catechin metabolism, Colon, Gastrointestinal Microbiome, Intestinal Mucosa metabolism, Intestinal Mucosa microbiology
- Abstract
The intestinal absorption of dietary catechins is quite low, resulting in most of them being metabolized by gut microbiota in the colon. It has been hypothesized that microbiota-derived metabolites may be partly responsible for the association between catechin consumption and beneficial cardiometabolic effects. Given the profound differences in gut microbiota composition and microbial load between individuals and across different colon regions, this study examined how microbial (+)-catechin metabolite profiles differ between colon regions and individuals. Batch exploration of the interindividual variability in (+)-catechin microbial metabolism resulted in a stratification based on metabolic efficiency: from the 12 tested donor microbiota, we identified a fast- and a slow-converting microbiota that was subsequently inoculated to SHIME, a dynamic model of the human gut. Monitoring of microbial (+)-catechin metabolites from proximal and distal colon compartments with UHPLC-MS and UPLC-IMS-Q-TOF-MS revealed profound donor-dependent and colon-region-dependent metabolite profiles with 5-(3',4'-dihydroxyphenyl)-γ-valerolactone being the largest contributor to differences between the fast- and slow-converting microbiota and the distal colon being a more important region for (+)-catechin metabolism than the proximal colon. Our findings may contribute to further understanding the role of the gut microbiota as a determinant of interindividual variation in pharmacokinetics upon (+)-catechin ingestion.
- Published
- 2021
- Full Text
- View/download PDF
38. Improving the performance of machine learning models for biotechnology: The quest for deus ex machina.
- Author
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Mey F, Clauwaert J, Van Huffel K, Waegeman W, and De Mey M
- Subjects
- Biotechnology, Machine Learning
- Abstract
Machine learning is becoming an integral part of the Design-Build-Test-Learn cycle in biotechnology. Machine learning models learn from collected datasets such as omics data and predict a defined outcome, which has led to both production improvements and predictive tools in the field. Robust prediction of the behavior of microbial cell factories and production processes not only greatly increases our understanding of the function of such systems, but also provides significant savings of development time. However, many pitfalls when modeling biological data - bad fit, noisy data, model instability, low data quantity and imbalances in the data - cause models to suffer in their performance. Here we provide an accessible, in-depth analysis on the problems created by these pitfalls, as well as means of their detection and mediation, with a focus on supervised learning. Assessing the state of the art, we show that, currently, in-depth analyses of model performance are often absent and must be improved. This review provides a toolbox for the analysis of model robustness and performance, and simultaneously proposes a standard for the community to facilitate future work. It is further accompanied by an interactive online tutorial on the discussed issues., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
39. Predictive design of sigma factor-specific promoters.
- Author
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Van Brempt M, Clauwaert J, Mey F, Stock M, Maertens J, Waegeman W, and De Mey M
- Subjects
- Base Sequence, Fluorescence, Gene Library, Genotype, Models, Genetic, Reproducibility of Results, Transcription Initiation, Genetic, Bacillus subtilis genetics, Escherichia coli genetics, Promoter Regions, Genetic, Sigma Factor metabolism
- Abstract
To engineer synthetic gene circuits, molecular building blocks are developed which can modulate gene expression without interference, mutually or with the host's cell machinery. As the complexity of gene circuits increases, automated design tools and tailored building blocks to ensure perfect tuning of all components in the network are required. Despite the efforts to develop prediction tools that allow forward engineering of promoter transcription initiation frequency (TIF), such a tool is still lacking. Here, we use promoter libraries of E. coli sigma factor 70 (σ
70 )- and B. subtilis σB -, σF - and σW -dependent promoters to construct prediction models, capable of both predicting promoter TIF and orthogonality of the σ-specific promoters. This is achieved by training a convolutional neural network with high-throughput DNA sequencing data from fluorescence-activated cell sorted promoter libraries. This model functions as the base of the online promoter design tool (ProD), providing tailored promoters for tailored genetic systems.- Published
- 2020
- Full Text
- View/download PDF
40. Metabolic engineering for glycoglycerolipids production in E. coli: Tuning phosphatidic acid and UDP-glucose pathways.
- Author
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Orive-Milla N, Delmulle T, de Mey M, Faijes M, and Planas A
- Subjects
- Glycolipids genetics, Mycoplasma genitalium enzymology, Phosphatidic Acids genetics, Uridine Diphosphate Glucose genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Escherichia coli genetics, Escherichia coli metabolism, Glycolipids biosynthesis, Glycosyltransferases genetics, Glycosyltransferases metabolism, Metabolic Engineering, Mycoplasma genitalium genetics, Phosphatidic Acids metabolism, Uridine Diphosphate Glucose metabolism
- Abstract
Glycolipids are target molecules in biotechnology and biomedicine as biosurfactants, biomaterials and bioactive molecules. An engineered E. coli strain for the production of glycoglycerolipids (GGL) used the MG517 glycolipid synthase from M. genitalium for glucosyl transfer from UDPGlc to diacylglycerol acceptor (Mora-Buyé et al., 2012). The intracellular diacylglycerol pool proved to be the limiting factor for GGL production. Here we designed different metabolic engineering strategies to enhance the availability of precursor substrates for the glycolipid synthase by modulating fatty acids, acyl donor and phosphatidic acid biosynthesis. Knockouts of tesA, fadE and fabR genes involved in fatty acids degradation, overexpression of the transcriptional regulator FadR, the acyltransferases PlsB and C, and the pyrophosphatase Cdh for phosphatidic acid biosynthesis, as well as the phosphatase PgpB for conversion to diacylglycerol were explored with the aim of improving GGL titers. Among the different engineered strains, the ΔtesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Attempts to boost UDPGlc availability by overexpressing the uridyltransferase GalU or knocking out the UDP-sugar diphosphatase encoding gene ushA did not further improve GGL titers. Most of the strains produced GGL containing a variable number of glucosyl units from mono-to tetra-saccharides. Interestingly, the strains co-expressing Cdh showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs) whereas the strains with a fadR knockout presented a higher amount of unsaturated acyl chains. In all cases, GGL production altered the lipidic composition of the E. coli membrane, observing that GGL replace phosphatidylethanolamine to maintain the overall membrane charge balance., (Copyright © 2020 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
41. Mapping and refactoring pathway control through metabolic and protein engineering: The hexosamine biosynthesis pathway.
- Author
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Coussement P, Bauwens D, Peters G, Maertens J, and De Mey M
- Subjects
- Hexosamines, Metabolic Networks and Pathways, Protein Engineering, Biosynthetic Pathways, Metabolic Engineering
- Abstract
Microorganisms possess a plethora of regulatory mechanisms to tightly control the flux through their metabolic network, allowing optimal behaviour in response to environmental conditions. However, these mechanisms typically counteract metabolic engineering efforts to rewire the metabolism with a view to overproduction. Hence, overcoming flux control is key in the development of microbial cell factories, illustrated in this contribution using the strictly controlled hexosamine biosynthesis pathway. The hexosamine biosynthesis pathway has recently garnered attention as gateway for the industrial biotechnological production of numerous mono-, oligo- and polysaccharidic compounds, composed of, i.a., glucosamine, N-acetylglucosamine, and neuraminic acid and with a vast application potential in the health, comsetics, and agricultural sector. First, the various alternative pathways in eukaryotes and prokaryotes are discussed. Second, the main regulatory mechanisms on transcriptional, translational and post-translational control, and the strategies to circumvent these pathway bottlenecks are highlighted. These efforts can serve as an inspiration to tackle regulatory control when optimizing any microbial cell factory., (Copyright © 2020. Published by Elsevier Inc.)
- Published
- 2020
- Full Text
- View/download PDF
42. Modulating transcription through development of semi-synthetic yeast core promoters.
- Author
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Decoene T, De Maeseneire SL, and De Mey M
- Subjects
- Gene Library, Metabolic Engineering methods, Peptide Elongation Factor 1 genetics, Saccharomyces cerevisiae Proteins genetics, Synthetic Biology methods, Gene Expression Regulation, Fungal, Promoter Regions, Genetic genetics, Saccharomyces cerevisiae genetics, Transcription, Genetic
- Abstract
Altering gene expression regulation by promoter engineering is a very effective way to fine-tune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today's introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
- Full Text
- View/download PDF
43. Editorial overview: Tissue, cell and pathway engineering.
- Author
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Peeters E and De Mey M
- Published
- 2019
- Full Text
- View/download PDF
44. Chimeric LysR-Type Transcriptional Biosensors for Customizing Ligand Specificity Profiles toward Flavonoids.
- Author
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De Paepe B, Maertens J, Vanholme B, and De Mey M
- Subjects
- Apigenin metabolism, Flavanones metabolism, Gene Expression Regulation, Bacterial, Luteolin metabolism, Metabolic Engineering, Transcription Factors metabolism, Biosensing Techniques, Flavonoids metabolism
- Abstract
Transcriptional biosensors enable key applications in both metabolic engineering and synthetic biology. Due to nature's immense variety of metabolites, these applications require biosensors with a ligand specificity profile customized to the researcher's needs. In this work, chimeric biosensors were created by introducing parts of a donor regulatory circuit from Sinorhizobium meliloti, delivering the desired luteolin-specific response, into a nonspecific biosensor chassis from Herbaspirillum seropedicae. Two strategies were evaluated for the development of chimeric LysR-type biosensors with customized ligand specificity profiles toward three closely related flavonoids, naringenin, apigenin, and luteolin. In the first strategy, chimeric promoter regions were constructed at the biosensor effector module, while in the second strategy, chimeric transcription factors were created at the biosensor detector module. Via both strategies, the biosensor repertoire was expanded with luteolin-specific chimeric biosensors demonstrating a variety of response curves and ligand specificity profiles. Starting from the nonspecific biosensor chassis, a shift from 27.5% to 95.3% luteolin specificity was achieved with the created chimeric biosensors. Both strategies provide a compelling, faster, and more accessible route for the customization of biosensor ligand specificity, compared to de novo design and construction of each biosensor circuit for every desired ligand specificity.
- Published
- 2019
- Full Text
- View/download PDF
45. Combinatorial Assembly of Multigene Pathways by Combining Single-Strand Assembly with Golden Gate Assembly.
- Author
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Bauwens D, Coussement P, Maertens J, and De Mey M
- Subjects
- Biological Assay, Escherichia coli genetics, Escherichia coli metabolism, Fluorescent Antibody Technique, Gene Library, High-Throughput Screening Assays, Lycopene metabolism, Metabolic Engineering, Metabolic Networks and Pathways, Transformation, Genetic, Cloning, Molecular methods, Genetic Engineering methods, Genetic Vectors genetics
- Abstract
Biotechnological production routes for fine and bulk chemicals are progressively explored and developed. Yet this development is hampered by the many constraints determining the metabolic sweet spot, such as optimal expression levels, metabolic stress, feedback regulation, etc.In this regard, we introduce a novel, highly reliable, and rapid single-strand assembly (SSA) methods for combinatorial pathway engineering. In this contribution, SSA is elucidated which enables one to modulate the expression via promoter and/or RBS randomization. Moreover, a new combinatorial multigene pathway assembly scheme based on single-strand assembly (SSA) methods and Golden Gate Assembly is introduced, exploiting the strengths of both assembly techniques.
- Published
- 2019
- Full Text
- View/download PDF
46. Exploring of the feature space of de novo developed post-transcriptional riboregulators.
- Author
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Peters G, Maertens J, Lammertyn J, and De Mey M
- Subjects
- Escherichia coli genetics, Escherichia coli Proteins genetics, Gene Expression physiology, Gene Expression Regulation genetics, Gene Expression Regulation physiology, Genes, Regulator genetics, Nucleic Acid Conformation, RNA genetics, RNA Processing, Post-Transcriptional physiology, Repressor Proteins genetics, Metabolic Engineering methods, Protein Engineering methods, Repressor Proteins metabolism
- Abstract
Metabolic engineering increasingly depends upon RNA technology to customly rewire the metabolism to maximize production. To this end, pure riboregulators allow dynamic gene repression without the need of a potentially burdensome coexpressed protein like typical Hfq binding small RNAs and clustered regularly interspaced short palindromic repeats technology. Despite this clear advantage, no clear general design principles are available to de novo develop repressing riboregulators, limiting the availability and the reliable development of these type of riboregulators. Here, to overcome this lack of knowledge on the functionality of repressing riboregulators, translation inhibiting RNAs are developed from scratch. These de novo developed riboregulators explore features related to thermodynamical and structural factors previously attributed to translation initiation modulation. In total, 12 structural and thermodynamic features were defined of which six features were retained after removing correlations from an in silico generated riboregulator library. From this translation inhibiting RNA library, 18 riboregulators were selected using a experimental design and subsequently constructed and co-expressed with two target untranslated regions to link the translation inhibiting RNA features to functionality. The pure riboregulators in the design of experiments showed repression down to 6% of the original protein expression levels, which could only be partially explained by a ordinary least squares regression model. To allow reliable forward engineering, a partial least squares regression model was constructed and validated to link the properties of translation inhibiting RNA riboregulators to gene repression. In this model both structural and thermodynamic features were important for efficient gene repression by pure riboregulators. This approach enables a more reliable de novo forward engineering of effective pure riboregulators, which further expands the RNA toolbox for gene expression modulation., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
- Full Text
- View/download PDF
47. Standardization in synthetic biology: an engineering discipline coming of age.
- Author
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Decoene T, De Paepe B, Maertens J, Coussement P, Peters G, De Maeseneire SL, and De Mey M
- Subjects
- Biomedical Research standards, Biotechnology standards, DNA, Escherichia coli, Reproducibility of Results, Bioengineering standards, Synthetic Biology standards
- Abstract
Background: Leaping DNA read-and-write technologies, and extensive automation and miniaturization are radically transforming the field of biological experimentation by providing the tools that enable the cost-effective high-throughput required to address the enormous complexity of biological systems. However, standardization of the synthetic biology workflow has not kept abreast with dwindling technical and resource constraints, leading, for example, to the collection of multi-level and multi-omics large data sets that end up disconnected or remain under- or even unexploited., Purpose: In this contribution, we critically evaluate the various efforts, and the (limited) success thereof, in order to introduce standards for defining, designing, assembling, characterizing, and sharing synthetic biology parts. The causes for this success or the lack thereof, as well as possible solutions to overcome these, are discussed., Conclusion: Akin to other engineering disciplines, extensive standardization will undoubtedly speed-up and reduce the cost of bioprocess development. In this respect, further implementation of synthetic biology standards will be crucial for the field in order to redeem its promise, i.e. to enable predictable forward engineering.
- Published
- 2018
- Full Text
- View/download PDF
48. Development of N-acetylneuraminic acid responsive biosensors based on the transcriptional regulator NanR.
- Author
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Peters G, De Paepe B, De Wannemaeker L, Duchi D, Maertens J, Lammertyn J, and De Mey M
- Subjects
- Escherichia coli growth & development, Escherichia coli metabolism, Fluorometry, Protein Binding, Transcription, Genetic, Biosensing Techniques methods, DNA-Binding Proteins metabolism, Escherichia coli genetics, Escherichia coli Proteins metabolism, N-Acetylneuraminic Acid analysis, Promoter Regions, Genetic
- Abstract
Transcriptional biosensors have various applications in metabolic engineering, including dynamic pathway control and high-throughput screening of combinatorial strain libraries. Previously, various biosensors have been created from naturally occurring transcription factors (TFs), largely relying on native sequences without the possibility to modularly optimize their response curve. The lack of design and engineering techniques thus greatly hinders the development of custom biosensors. In view of the intended application this is detrimental. In contrast, a bottom-up approach to design tailor-made biosensors was pursued here. Novel biosensors were created that respond to N-acetylneuraminic acid (Neu5Ac), an important sugar moiety with various biological functions, by employing native and engineered promoters that interact with the TF NanR. This bottom-up approach, whereby various tuned modules, e.g., the ribosome binding site (RBS) controlling NanR translation can be combined, enabled the reliable engineering of various response curve characteristics. The latter was validated by testing these biosensors in combination with various Neu5Ac-producing pathways, which allowed to produce up to 1.4 ± 0.4 g/L extracellular Neu5Ac. In this way, the repertoire of biosensors was expanded with seven novel functional Neu5Ac-responsive biosensors., (© 2018 Wiley Periodicals, Inc.)
- Published
- 2018
- Full Text
- View/download PDF
49. Modularization and Response Curve Engineering of a Naringenin-Responsive Transcriptional Biosensor.
- Author
-
De Paepe B, Maertens J, Vanholme B, and De Mey M
- Subjects
- Bacterial Proteins genetics, Culture Media, Escherichia coli metabolism, Fluorescence, Gene Expression Regulation, Bacterial, Gene Regulatory Networks, Herbaspirillum genetics, Microorganisms, Genetically-Modified, Transcription Factors genetics, Biosensing Techniques methods, Escherichia coli genetics, Flavanones metabolism, Genetic Engineering methods
- Abstract
To monitor the intra- and extracellular environment of micro-organisms and to adapt their metabolic processes accordingly, scientists are reprogramming nature's myriad of transcriptional regulatory systems into transcriptional biosensors, which are able to detect small molecules and, in response, express specific output signals of choice. However, the naturally occurring response curve, the key characteristic of biosensor circuits, is typically not in line with the requirements for real-life biosensor applications. In this contribution, a natural LysR-type naringenin-responsive biosensor circuit is developed and characterized with Escherichia coli as host organism. Subsequently, this biosensor is dissected into a clearly defined detector and effector module without loss of functionality, and the influence of the expression levels of both modules on the biosensor response characteristics is investigated. Two collections of ten unique synthetic biosensors each are generated. Each collection demonstrates a unique diversity of response curve characteristics spanning a 128-fold change in dynamic and 2.5-fold change in operational ranges and 3-fold change in levels of Noise, fit for a wide range of applications, such as adaptive laboratory evolution, dynamic pathway control and high-throughput screening methods. The established biosensor engineering concepts, and the developed biosensor collections themselves, are of use for the future development and customization of biosensors in general, for the multitude of biosensor applications and as a compelling alternative for the commonly used LacI-, TetR- and AraC-based inducible circuits.
- Published
- 2018
- Full Text
- View/download PDF
50. A sigma factor toolbox for orthogonal gene expression in Escherichia coli.
- Author
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Bervoets I, Van Brempt M, Van Nerom K, Van Hove B, Maertens J, De Mey M, and Charlier D
- Subjects
- Bacillus subtilis, Escherichia coli metabolism, Genome, Plasmids genetics, Promoter Regions, Genetic, Escherichia coli genetics, Gene Expression Regulation, Sigma Factor metabolism
- Abstract
Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future.
- Published
- 2018
- Full Text
- View/download PDF
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