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6. Additional file 1 of Signal amplification and optimization of riboswitch-based hybrid inputs by modular and titratable toehold switches

Author

Yunhee Hwang, Kim, Seong Gyeong, Sungho Jang, Jongmin Kim, and Jung, Gyoo Yeol

Abstract

Additional file 1: Table S1. Strains and plasmids used in this study. Table S2. Oligonucleotides used in this study. Table S3. Measured OD600, GFP fluorescence, and specific fluorescence. Figure S1. Overall schematics of the toehold switch. Switch RNA is hiding the RBS (ribosome binding site) in the hairpin structure. When trigger RNA binds complementarily to the toehold domain within switch RNA [A], the switch RNA stem starts to unwind, exposing RBS and the start codon to activate translation of the reporter gene. Figure S2. (a) Dose-response curves for trN1-swN1with various IPTG (1, 10, 100, 1000 μM) and coenzyme B12 (0, 0.1, 0.3, 1, 3, 10, 30 μM) concentrations. (b) Dose-response curves for modified trN1-swN1 strain in which the promoter for the riboswitch was changed to J23119 with various IPTG (1, 10, 100, 1000 μM) and coenzyme B12 (0, 0.1, 0.3, 1, 3, 10, 30 μM) concentrations. (c) EC50 values for (a). (d) EC50 Values for (b). Error bars indicate standard deviations from triplicate measurements. Figure S3. Histograms of the toehold switch-based modulators measured by flow cytometry. The fold-changes of average fluorescence in the presence and absence of coenzyme B12 are plotted in Fig. 3b. The strain NC (negative control) refers to BL21 star (DE3) without toehold switch-based modulator. Figure S4. Performance of toehold switch-based modulator in E. coli BL21(DE3). (a) Fluorescence measurements for BL21-trN1-swN1 and BL21-trN3-swN3 with and without coenzyme B12 (30 μM). (b) Fold-change of GFP reporters for BL21-trN1-swN1 and BL21-trN3-swN3. Error bars indicate standard deviations from triplicate measurements.

Published
2021
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7. Synthetic biosensor accelerates evolution by rewiring carbon metabolism toward a specific metabolite

Author

National Research Foundation of Korea, Ministry of Science, ICT and Future Planning (South Korea), Seoul National University, Seok, Joo Yeon, Han, Yonghee, Yang, Jae-Seong, Yang, Jina, Lim, Hyun Gyu, Kim, Seong Gyeong, Seo, Sang Woo, Jung, Gyoo Yeol, National Research Foundation of Korea, Ministry of Science, ICT and Future Planning (South Korea), Seoul National University, Seok, Joo Yeon, Han, Yonghee, Yang, Jae-Seong, Yang, Jina, Lim, Hyun Gyu, Kim, Seong Gyeong, Seo, Sang Woo, and Jung, Gyoo Yeol

Abstract

Proper carbon flux distribution between cell growth and production of a target compound is important for biochemical production because improper flux reallocation inhibits cell growth, thus adversely affecting production yield. Here, using a synthetic biosensor to couple production of a specific metabolite with cell growth, we spontaneously evolve cells under the selective condition toward the acquisition of genotypes that optimally reallocate cellular resources. Using 3-hydroxypropionic acid (3-HP) production from glycerol in Escherichia coli as a model system, we determine that mutations in the conserved regions of proteins involved in global transcriptional regulation alter the expression of several genes associated with central carbon metabolism. These changes rewire central carbon flux toward the 3-HP production pathway, increasing 3-HP yield and reducing acetate accumulation by alleviating overflow metabolism. Our study provides a perspective on adaptive laboratory evolution (ALE) using synthetic biosensors, thereby supporting future efforts in metabolic pathway optimization.

Published
2021

12. Optimization of hexanoic acid production in recombinant Escherichia coli by precise flux rebalancing.

Author

Kim, Seong Gyeong, Jang, Sungho, Lim, Jae Hyung, Jeon, Byoung Seung, Kim, Jungyeon, Kim, Kyoung Heon, Sang, Byoung-In, and Jung, Gyoo Yeol

Subjects
*CARBOXYLIC acid derivatives, *ESCHERICHIA coli, *ACETYLCOENZYME A, *BUTYRIC acid, *METABOLIC flux analysis, *GENE expression in bacteria
Abstract

The aim of this study is to demonstrate that rebalancing of metabolic fluxes at acetyl-CoA branch node can substantially improve the titer and productivity of hexanoic acid in recombinant Escherichia coli strains. First, a hexanoic acid-producing E. coli strain was constructed by expressing genes encoding β-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Megasphaera sp. MH in a butyric acid producer strain. Next, metabolic flux was optimized at the acetyl-CoA branch node by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Four synthetic 5′-untranslated regions were designed for atoB using UTR Designer to modulate the expression level of the gene. Notably, the productivity of the optimized strain (14.7 mg/L/h) was the highest among recombinant E. coli strains in literature when using a similar inoculum size for fermentation. These results show that fine-tuning the expression level of atoB is critical for production of hexanoic acid. [ABSTRACT FROM AUTHOR]

Published
2017
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15. Improving the Electrical Properties of Lanthanum Silicate Films on Ge Metal Oxide Semiconductor Capacitors by Adopting Interfacial Barrier and Capping Layers

Author

Choi, Yu Jin, primary, Lim, Hajin, additional, Lee, Suhyeong, additional, Suh, Sungin, additional, Kim, Joon Rae, additional, Jung, Hyung-Suk, additional, Park, Sanghyun, additional, Lee, Jong Ho, additional, Kim, Seong Gyeong, additional, Hwang, Cheol Seong, additional, and Kim, HyeongJoon, additional

Published
2014
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