Design and characterization of allantoin-inducible expression systems in budding yeast DOI Creative Commons
Junyi Wang, Jun Ma, Luo Xiang-hang

et al.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 14, 2024

Abstract Background Saccharomyces cerevisiae, a eukaryotic model microorganism, has been extensively employed as host for the production of various biochemicals and recombinant proteins. The expression systems in S. cerevisiae typically rely on constitutive or galactose-regulated promoters, limited repertoire gene regulations imposes constraints productivity microbial cell factories based budding yeast. Results In this study, we designed characterized series allantoin-inducible endogenous allantoin catabolic system (DAL-related genes) cerevisiae. We first profile set DAL promoters induced by allantoin, further combined with galactose-inducible (GAL) to create highly responsive genetic switch that efficiently amplifies output signals. resulting allantoin-GAL could give ON/OFF ratio 68.6, 6.8-fold higher signal over direct DAL2p-controlled after 96 h cultivation. Subsequently, successfully demonstrated can be used effectively regulate carotenoid flocculation Conclusions summary, have constructed an effective bioproduction applications. Considering there are microorganisms directly utilize alternative nitrogen source, envision it will possible develop contamination-resistant fermentation process harnessing competitive advantage source utilization.

Language: Английский

Genetically Encoded Biosensors for Constrained Biological Functions in Probiotic Escherichia coli Nissle DOI
Shan Jiang, Haofeng Chen,

Shiyao Chen

et al.

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 8, 2025

The probiotic Escherichia coli Nissle (EcN) is an exceptional strain that has attracted significant attention not only for its clinical efficacy in the treatment and prevention of gastrointestinal disorders but also as a burgeoning microbial chassis living therapeutic applications. However, there immediate necessity to develop conditional expression systems confine activity EcN specifically tract, avoid influencing environment. Here, we constructed two genetically encoded interchangeable sensors responsive body temperature at 37 °C, small molecules such protocatechuic acid (PCA), metabolite found green tea. By employing dCpf1 targeted deactivation LacI gene, thereby coupled above sensing modules with Ptrc-lacO system achieved improved signal outputs relatively high ON/OFF ratios. Subsequently, validated biological function engineering using enhanced fluorescent protein (eGFP) animal model mice. Taken together, construction restrict functions would be applicable real-world implementation therapeutics or drug delivery.

Language: Английский

Citations

1
Design and characterization of allantoin-inducible expression systems in budding yeast DOI Creative Commons
Junyi Wang,

Jiaxue Ma,

Luo Xiang-hang

et al.

Biotechnology for Biofuels and Bioproducts, Journal Year: 2025, Volume and Issue: 18(1)

Published: Feb. 28, 2025

Saccharomyces cerevisiae has been extensively employed as a host for the production of various biochemicals and recombinant proteins. The expression systems in S. typically rely on constitutive or galactose-regulated promoters, limited repertoire gene regulations imposes constraints productivity microbial cell factories based budding yeast. In this study, we designed characterized series allantoin-inducible endogenous allantoin catabolic system (DAL-related genes) cerevisiae. We first profile set DAL promoters induced by allantoin, further combined with galactose-inducible (GAL) to create highly responsive genetic switch that efficiently amplifies output signals. resulting allantoin–GAL could give ON/OFF ratio 68.6, 6.8-fold higher signal over direct PDAL2-controlled expression. Additionally, when centromeric plasmid was used EGFP expression, increased > 67.2, surpassing levels driven DAL2 promoter. Subsequently, successfully demonstrated can be effectively regulate carotenoid flocculation summary, several from yeast developed layered utilizes promoter galactose regulon an impressive surpassed traditional It is anticipated utilizing our alternative nitrogen source might favor low-cost pharmaceuticals.

Language: Английский

Citations

1
Extending dynamic and operational range of the biosensor responding to L-carnitine by directed evolution DOI Creative Commons
Tingting Li,

Huina Dong,

Jinlong Li

et al.

Synthetic and Systems Biotechnology, Journal Year: 2025, Volume and Issue: 10(3), P. 897 - 906

Published: April 22, 2025

l-carnitine is a quaternary amine compound essential for eukaryotic metabolism. It mainly involved in the oxidative decomposition of medium-and long-chain fatty acids and provides energy body. Therefore, it widely used health care food additives. As pivotal transcriptional activator metabolism, CaiF notably activated by crotonobetainyl-CoA, key intermediate product carnitine metabolic pathway. Capitalizing on this mechanism, sophisticated biosensor was ingeniously developed. Nevertheless, worth mentioning that currently exhibits relatively restricted detection range, which results some specific limitations practical application scenarios. In paper, we constructed based developed strategy modifying biosensor. The structural configuration formulated computer-aided design, DNA binding site simulated, verified alanine scanning. Functional Diversity-Oriented Volume-Conservative Substitution Strategy sites conducted to extend dynamic range CaiFY47W/R89A, exhibited considerably expanded concentration response from 10-4 mM-10 mM, obtained. 1000-fold wider output signal intensity 3.3-fold higher control These variants may have great value improving production process.

Language: Английский

Citations

0
Development of Highly Sensitive and Specific and Genetically Encoded Biosensors for Calcifediol and Calcitriol Detection DOI

Kang Ma,

Ying Huang, Mengying Li

et al.

ACS Sensors, Journal Year: 2025, Volume and Issue: unknown

Published: May 9, 2025

1α,25-Dihydroxyvitamin D3 (calcitriol) and 25-hydroxyvitamin (calcifediol) are essential steroid metabolites that serve as key indicators of vitamin status in the human body. However, conventional detection methods, such high-performance liquid chromatography (HPLC) enzyme-linked immunosorbent assay (ELISA), costly, time-consuming, labor-intensive, limiting their accessibility for routine analysis. Here, we engineered genetically encoded biosensors by leveraging D receptor (VDR) retinoid X alpha (RXRA) to enable precise calcitriol calcifediol Saccharomyces cerevisiae. The were designed two-component modules fusing VDR ligand-binding domain S. cerevisiae GAL4 DNA-binding RXRA VP16 activation domain, facilitating ligand-dependent transcriptional activation. To enhance sensitivity, increased copy number reporter module incorporated a amplifier, improving half-maximal effective concentration (EC50) 23.3 nM 219.8 calcifediol. Furthermore, biosensor specificity was optimized screening mutations using URA3-mediated dual-selection system, yielding VDRCT mutant, which exhibited no response 10 μM demonstrated high accuracy measuring dietary supplements pharmaceutical formulations, showing strong concordance with HPLC results. These findings suggest proposed may provide cost-effective, rapid, reliable alternative food safety inspection clinical diagnostics.

Language: Английский

Citations

0
CRISPR/Cas9-based engineered Escherichia coli biosensor for sensitive and specific detection of Cd(II) in drinking water DOI

Yi‐Jun Wei,

Danyang Shi,

Tianjiao Chen

et al.

Chemosphere, Journal Year: 2024, Volume and Issue: 362, P. 142607 - 142607

Published: June 13, 2024

Language: Английский

Citations

1
Design and characterization of allantoin-inducible expression systems in budding yeast DOI Creative Commons
Junyi Wang, Jun Ma, Luo Xiang-hang

et al.

Research Square (Research Square), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 14, 2024

Abstract Background Saccharomyces cerevisiae, a eukaryotic model microorganism, has been extensively employed as host for the production of various biochemicals and recombinant proteins. The expression systems in S. cerevisiae typically rely on constitutive or galactose-regulated promoters, limited repertoire gene regulations imposes constraints productivity microbial cell factories based budding yeast. Results In this study, we designed characterized series allantoin-inducible endogenous allantoin catabolic system (DAL-related genes) cerevisiae. We first profile set DAL promoters induced by allantoin, further combined with galactose-inducible (GAL) to create highly responsive genetic switch that efficiently amplifies output signals. resulting allantoin-GAL could give ON/OFF ratio 68.6, 6.8-fold higher signal over direct DAL2p-controlled after 96 h cultivation. Subsequently, successfully demonstrated can be used effectively regulate carotenoid flocculation Conclusions summary, have constructed an effective bioproduction applications. Considering there are microorganisms directly utilize alternative nitrogen source, envision it will possible develop contamination-resistant fermentation process harnessing competitive advantage source utilization.

Language: Английский

Citations

0