scholarly journals Development of a new gene expression vector for Thermus thermophilus using a silica-inducible promoter

2020 ◽  
Author(s):  
Yasuhiro Fujino ◽  
Shuichiro Goda ◽  
Yuri Suematsu ◽  
Katsumi Doi
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Heterologous Gene Expression ◽  
Thermus Thermophilus ◽  
Expression System ◽  
Active Form ◽  
Inducible Promoter ◽  
Additional Treatment ◽  
Selection Marker ◽  
Thermostable Enzymes

Abstract Background Thermostable enzymes are commonly produced in mesophilic hosts for research and bioengineering purposes. However, these hosts do not overexpress the active forms of some biologically functional thermoenzymes. Therefore, an efficient thermophilic expression system is needed. Thermus thermophilus contains an easily manipulable genome and is therefore among the best candidate microbes for a “hot” expression system. We previously identified a strong and inducible promoter that was active in T. thermophilus under supersaturated silica conditions. Here, we report a new heterologous gene expression system based on a silica-inducible promoter in T. thermophilus.Results A Thermus sp. A4 gene encoding thermostable β-galactosidase was cloned as a reporter gene into the expression vector pSix1, which contains a selection marker that confers thermostable resistance to hygromycin and a 600-bps DNA region containing a putative silica-inducible promoter. β-Galactosidase activity was 11-fold higher in the presence than in the absence of 10 mM silicic acid. SDS-PAGE revealed a prominent band corresponding to β-galactosidase, and this enzyme was expressed as an active and soluble protein (yield: 27 mg/L) in Thermus but as an inclusion body in Escherichia coli. Deletion of the promoter region improved the yield of the target protein, possibly by avoiding plasmid instability due to homologous recombination. Finally, we developed an expression vector containing the pSix1 backbone and a 100-bps DNA region corresponding to the silica-inducible promoter. We used this vector to successfully express the active form of glutamate dehydrogenase from Pyrobaculum islandicum (PisGDH) without additional treatment (yield: 9.5 mg/L), whereas the expression of active PisGDH in E. coli required heat treatment.Conclusion We successfully expressed the thermoenzymes β-galactosidase and PisGDH in T. thermophilus and achieved the highest known protein expression levels in this species. These thermoenzymes were expressed in active and soluble forms. Our results validate the use of our silica-inducible expression system as a novel strategy for the intracellular overexpression of thermostable proteins.

scholarly journals A Derepression System Based on the Bacillus subtilis Sporulation Pathway Offers Dynamic Control of Heterologous Gene Expression

2007 ◽  
Vol 73 (7) ◽  
pp. 2390-2393 ◽  
Author(s):  
Reindert Nijland ◽  
Jan-Willem Veening ◽  
Oscar P. Kuipers
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Dynamic Control ◽  
Inducible Promoter ◽  
Heterologous Gene ◽  
Gene Regulatory

ABSTRACT By rewiring the sporulation gene-regulatory network of Bacillus subtilis, we generated a novel expression system relying on derepression. The gene of interest is placed under the control of the abrB promoter, which is active only when Spo0A is absent, and Spo0A is controlled via an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter.

scholarly journals Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426

2013 ◽  
Vol 79 (17) ◽  
pp. 5151-5158 ◽  
Author(s):  
Hirokazu Suzuki ◽  
Ken-ichi Yoshida ◽  
Toshihisa Ohshima
Keyword(s):  
Gene Expression ◽  
Promoter Region ◽  
Plant Biomass ◽  
Heterologous Gene Expression ◽  
Expression Profiles ◽  
Expression System ◽  
Multicopy Plasmid ◽  
Thermostable Enzymes ◽  
Content Type ◽  
Geobacillus Kaustophilus

ABSTRACTThermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activitiesin vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophileGeobacillus kaustophilusHTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using β-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed inEscherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions toG. kaustophilusstrains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications ofG. kaustophilus.

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

Plants ◽  
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.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

scholarly journals Construction and Characterization of a Gradually Inducible Expression Vector for Halobacterium salinarum, Based on thekdpPromoter

2012 ◽  
Vol 78 (7) ◽  
pp. 2100-2105 ◽  
Author(s):  
Dorthe Kixmüller ◽  
Jörg-Christian Greie
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Expression System ◽  
Expression Patterns ◽  
Inducible Expression ◽  
Halobacterium Salinarum ◽  
Expression Vectors ◽  
Content Type ◽  
Inducible Expression System

ABSTRACTGradually inducible expression vectors which are governed by variations of growth conditions are powerful tools for gene expression of conditionally lethal mutants. Furthermore, controlled expression allows monitoring of overproduction of proteins at various stages in their expressing hosts. ForHalobacterium salinarum, which is often used as a paradigm for halophilic archaea, such an inducible expression system is not available to date. Here we show that thekdppromoter (Pkdp), which facilitates gene expression upon K+limitation, can be used to establish such a system for molecular applications. Pkdpfeatures a rather high expression rate, with an approximately 50-fold increase that can be easily varied by K+concentrations in the growth medium. Besides the construction of an expression vector, our work describes the characterization of expression patterns and, thus, offers a gradually inducible expression system to the scientific community.

scholarly journals Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture

2000 ◽  
Vol 182 (24) ◽  
pp. 7029-7034 ◽  
Author(s):  
Artem Khlebnikov ◽  
Øystein Risa ◽  
Tove Skaug ◽  
Trent A. Carrier ◽  
J. D. Keasling
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
High Capacity ◽  
Inducible Promoter ◽  
Transport Systems ◽  
Specific Expression ◽  
Homogeneous Population ◽  
E Coli ◽  
Transport Control ◽  
Inducible Gene

ABSTRACT The arabinose-inducible promoter PBAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, thearaE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-d-thiogalactopyranoside-induciblePtac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independentaraE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (PBAD ) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGHmutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.

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