Enhanced production of recombinant proteins in Corynebacterium glutamicum by constructing a bicistronic gene expression system

Corynebacterium glutamicum is an important workhorse for industrial production of diversiform bioproducts. Precise regulation of gene expression is crucial for metabolic balance and enhancing production of target molecules. Auto-inducible promoters, which can be activated without expensive inducers, are ideal regulatory tools for industrial-scale application. However, few auto-inducible promoters have been identified and applied in C. glutamicum. Here, a hyperosmotic stress inducible gene expression system was developed and used for metabolic engineering of C. glutamicum. The promoter of NCgl1418 (PNCgl1418) that was activated by the two-component signal transduction system MtrA/MtrB was found to exhibit a high inducibility under hyperosmotic stress conditions. A synthetic promoter library was then constructed by randomizing the flanking and space regions of PNCgl1418, and mutant promoters exhibiting high strength were isolated via fluorescence activated cell sorting (FACS)-based high-throughput screening. The hyperosmotic stress inducible gene expression system was applied to regulate the expression of lysE encoding a lysine exporter and repress four genes involved in lysine biosynthesis (gltA, pck, pgi, and hom) by CRISPR interference, which increased the lysine titer by 64.7% (from 17.0 to 28.0 g/L) in bioreactors. The hyperosmotic stress inducible gene expression system developed here is a simple and effective tool for gene auto-regulation in C. glutamicum and holds promise for metabolic engineering of C. glutamicum to produce valuable chemicals and fuels.

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Development of A Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium glutamicum

Iranian Journal of Biotechnology ◽

10.21859/ijb.1746 ◽

2018 ◽

Vol 16 (1) ◽

pp. 42-48 ◽

Cited By ~ 2

Author(s):

Huimin Jia ◽

Hedan Li ◽

Lirong Zhang ◽

Daqing Xu

Keyword(s):

Gene Expression ◽

Corynebacterium Glutamicum ◽

Expression System ◽

Heterologous Proteins ◽

Gene Expression System ◽

Sec Pathway ◽

Novel Gene ◽

Secretory Production

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Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽

10.3390/plants10030524 ◽

2021 ◽

Vol 10 (3) ◽

pp. 524

Author(s):

Bingqi Wu ◽

Zhiting Chen ◽

Xiaohui Xu ◽

Ronghua Chen ◽

Siwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Nicotiana Benthamiana ◽

Heterologous Gene Expression ◽

Expression System ◽

Functional Characterization ◽

Transient Gene Expression ◽

High Mobility ◽

Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

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Artificial complementary chromatic acclimation gene expression system in Escherichia coli

Microbial Cell Factories ◽

10.1186/s12934-021-01621-3 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Dwi Ariyanti ◽

Kazunori Ikebukuro ◽

Koji Sode

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Light Exposure ◽

Expression System ◽

Red Light ◽

Green Light ◽

Light Illumination ◽

Multiple Gene ◽

Gene Expression System ◽

Chromatic Acclimation

Abstract Background The development of multiple gene expression systems, especially those based on the physical signals, such as multiple color light irradiations, is challenging. Complementary chromatic acclimation (CCA), a photoreversible process that facilitates the control of cellular expression using light of different wavelengths in cyanobacteria, is one example. In this study, an artificial CCA systems, inspired by type III CCA light-regulated gene expression, was designed by employing a single photosensor system, the CcaS/CcaR green light gene expression system derived from Synechocystis sp. PCC6803, combined with G-box (the regulator recognized by activated CcaR), the cognate cpcG2 promoter, and the constitutively transcribed promoter, the PtrcΔLacO promoter. Results One G-box was inserted upstream of the cpcG2 promoter and a reporter gene, the rfp gene (green light-induced gene expression), and the other G-box was inserted between the PtrcΔLacO promoter and a reporter gene, the bfp gene (red light-induced gene expression). The Escherichia coli transformants with plasmid-encoded genes were evaluated at the transcriptional and translational levels under red or green light illumination. Under green light illumination, the transcription and translation of the rfp gene were observed, whereas the expression of the bfp gene was repressed. Under red light illumination, the transcription and translation of the bfp gene were observed, whereas the expression of the rfp gene was repressed. During the red and green light exposure cycles at every 6 h, BFP expression increased under red light exposure while RFP expression was repressed, and RFP expression increased under green light exposure while BFP expression was repressed. Conclusion An artificial CCA system was developed to realize a multiple gene expression system, which was regulated by two colors, red and green lights, using a single photosensor system, the CcaS/CcaR system derived from Synechocystis sp. PCC6803, in E. coli. The artificial CCA system functioned repeatedly during red and green light exposure cycles. These results demonstrate the potential application of this CCA gene expression system for the production of multiple metabolites in a variety of microorganisms, such as cyanobacteria.

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260. Controlled Nonviral Gene Delivery and Expression Using Stable Neural Stem Cell Line Transfected with a Hypoxia-Inducible Gene Expression System

Molecular Therapy ◽

10.1016/s1525-0016(16)37701-2 ◽

2010 ◽

Vol 18 ◽

pp. S99

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Gene Delivery ◽

Cell Line ◽

Expression System ◽

Nonviral Gene Delivery ◽

Stem Cell Line ◽

Gene Expression System ◽

Neural Stem Cell Line ◽

Inducible Gene

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Use of a Beet necrotic yellow vein virus RNA-5-derived replicon as a new tool for gene expression

Journal of General Virology ◽

10.1099/vir.0.80720-0 ◽

2005 ◽

Vol 86 (2) ◽

pp. 463-467 ◽

Cited By ~ 19

Author(s):

Laure Schmidlin ◽

Didier Link ◽

Jérôme Mutterer ◽

Hubert Guilley ◽

David Gilmer

Keyword(s):

Gene Expression ◽

Recombinant Proteins ◽

Expression System ◽

Green Fluorescence Protein ◽

Yellow Vein ◽

Expression Levels ◽

Virus Rna ◽

New Gene ◽

Infected Cells ◽

Fluorescence Protein

A new gene-expression system based on RNA-5 of Beet necrotic yellow vein virus (BNYVV) was constructed to allow the expression of recombinant proteins in virally infected cells. Replication and expression levels of the RNA-5-based replicon containing the green fluorescence protein (GFP) gene were compared with those obtained with the well-characterized RNA-3-derived replicon (Rep-3). When RNA-3 and/or RNA-4 BNYVV RNAs were added to the inoculum, the expression levels of RNA-5-encoded GFP were considerably reduced. To a lesser extent, RNA-3-derived GFP expression was also affected by the presence of RNA-4 and -5. Both RNA-3- and RNA-5-derived molecules were able to express proteins within the same infected cells. Together with Rep-3, the RNA-5-derived replicon thus provides a new tool for the co-expression of different recombinant proteins. In Beta macrocarpa, Rep-5-GFP was able to move in systemic tissues in the presence of RNA-3 and thus provides a new expression system that is not restricted to the inoculated leaves.

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