The development of a heterologous gene expression system in thermophilic fungus Thermoascus aurantiacus

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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Production of foreign proteins in the yeast Pichia pastoris

Canadian Journal of Botany ◽

10.1139/b95-336 ◽

1995 ◽

Vol 73 (S1) ◽

pp. 891-897 ◽

Cited By ~ 46

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.

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Fine‐tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system

Plant Biotechnology Journal ◽

10.1111/pbi.12175 ◽

2014 ◽

Vol 12 (6) ◽

pp. 718-727 ◽

Cited By ~ 22

Author(s):

Yulia A. Meshcheriakova ◽

Pooja Saxena ◽

George P. Lomonossoff

Keyword(s):

Gene Expression ◽

Transient Expression ◽

Heterologous Gene Expression ◽

Expression System ◽

Fine Tuning ◽

Heterologous Gene ◽

Untranslated Regions ◽

Transient Expression System

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Bestowing Inducibility on the Cloned Methanol Dehydrogenase Promoter (PmxaF) of Methylobacterium extorquens by Applying Regulatory Elements of Pseudomonas putida F1

Applied and Environmental Microbiology ◽

10.1128/aem.02002-06 ◽

2006 ◽

Vol 72 (12) ◽

pp. 7723-7729 ◽

Cited By ~ 22

Author(s):

Young J. Choi ◽

Lyne Morel ◽

Denis Bourque ◽

Alaka Mullick ◽

Bernard Massie ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Putida ◽

Heterologous Gene Expression ◽

Expression System ◽

Regulatory Elements ◽

Heterologous Gene ◽

Expression Vectors ◽

Transcriptional Level ◽

Broad Host Range ◽

Methylobacterium Extorquens

ABSTRACT P mxaF is a strong methanol-inducible promoter in Methylobacterium extorquens. When this promoter is cloned in expression vectors and used to drive heterologous gene expression, methanol inducibility is either greatly reduced or entirely lost. In order to bestow inducibility upon the cloned P mxaF promoter in expression vectors, we adopted combinational methods (regulatory elements of the Pseudomonas putida F1 cym and cmt operons and Tn7 transposon system) to control reporter gene expression at the transcriptional level in M. extorquens. An operator fragment (26 nucleotides) of the cmt operon was inserted downstream of the cloned P mxaF promoter in the broad-host-range expression vector (pCHOI3). The repressor gene (cymR) located upstream of the cym operon in P. putida F1 was amplified by PCR. To avoid cellular toxicity for M. extorquens caused by the overexpression of CymR, single and/or double copies of cymR were integrated into the chromosome of M. extorquens using the mini-Tn7 transposon system. Cultures containing the chromosomally integrated cymR gene were subsequently transformed with pCHOI3 containing modified P mxaF (i.e., P mxaF plus operator). In this construct, inducibility is afforded by cumate (p-isopropylbenzoate). In this report, we describe the inducible and tightly regulated expression of heterologous genes (bgl [for β-galactosidase], est [for esterase], and gfp [for green fluorescent protein]) in M. extorquens. This is the first documented example of an inducible/regulated heterologous gene expression system in M. extorquens.

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