scholarly journals Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

2015 ◽  
Vol 14 (3) ◽  
pp. 278-285 ◽  
Author(s):  
Saori Oda ◽  
Hiroya Yurimoto ◽  
Nobuhisa Nitta ◽  
Yu Sasano ◽  
Yasuyoshi Sakai
Keyword(s):  
Gene Expression ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Promoter Regions ◽  
Inducible Gene Expression ◽  
Content Type ◽  
Inducible Promoters ◽  
Hap Complex ◽  
Inducible Gene

ABSTRACT We identified genes encoding components of the Hap complex, CbHAP2 , CbHAP3 , and CbHAP5 , as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii . We found that the Cbhap2 Δ, Cbhap3 Δ, and Cbhap5 Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae , the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii . Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

scholarly journals Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

2013 ◽  
Vol 79 (21) ◽  
pp. 6795-6802 ◽  
Author(s):  
Andreas Kaczmarczyk ◽  
Julia A. Vorholt ◽  
Anne Francez-Charlot
Keyword(s):  
Gene Expression ◽  
Caulobacter Crescentus ◽  
Paracoccus Denitrificans ◽  
Expression System ◽  
Model Organisms ◽  
Inducible Gene Expression ◽  
Gene Expression System ◽  
Content Type ◽  
Wide Range ◽  
Inducible Gene

ABSTRACTTunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of thePseudomonas putidaF1cym/cmtsystem. The resulting promoter, termed PQ5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in otherAlphaproteobacteria, such as the model organismsCaulobacter crescentus,Paracoccus denitrificans, andMethylobacterium extorquens. In the noninduced state, expression from PQ5is low enough to allow gene depletion analysis, as demonstrated with the essential genephyPofSphingomonassp. strain Fr1. A set of PQ5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

scholarly journals Comprehensive Set of Integrative Plasmid Vectors for Copper-Inducible Gene Expression in Myxococcus xanthus

2012 ◽  
Vol 78 (8) ◽  
pp. 2515-2521 ◽  
Author(s):  
Nuria Gómez-Santos ◽  
Anke Treuner-Lange ◽  
Aurelio Moraleda-Muñoz ◽  
Elena García-Bravo ◽  
Raquel García-Hernández ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myxococcus Xanthus ◽  
Gliding Motility ◽  
Expression Vectors ◽  
Plasmid Vectors ◽  
Inducible Gene Expression ◽  
Content Type ◽  
Marker Selection ◽  
Effect Of Copper ◽  
Inducible Gene

ABSTRACTMyxococcus xanthusis widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover,M. xanthusis a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression inM. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters inM. xanthusreported so far, the multicopper oxidasecuoApromoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in theM. xanthuschromosome. The vectors have been tested and gene expression quantified using thelacZgene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of thepilBgene. These versatile vectors are likely to deepen our understanding of the biology ofM. xanthusand may also have biotechnological applications.

scholarly journals Development of a Hyperosmotic Stress Inducible Gene Expression System by Engineering the MtrA/MtrB-Dependent NCgl1418 Promoter in Corynebacterium glutamicum

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingwen Huang ◽  
Jiuzhou Chen ◽  
Yu Wang ◽  
Tuo Shi ◽  
Xiaomeng Ni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Corynebacterium Glutamicum ◽  
Expression System ◽  
Regulation Of Gene Expression ◽  
Hyperosmotic Stress ◽  
Inducible Gene Expression ◽  
Gene Expression System ◽  
Inducible Promoters ◽  
Inducible Gene

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.

scholarly journals Development and Characterization of a Xylose-Inducible Gene Expression System for Clostridium perfringens

2011 ◽  
Vol 77 (23) ◽  
pp. 8439-8441 ◽  
Author(s):  
Hirofumi Nariya ◽  
Shigeru Miyata ◽  
Tomomi Kuwahara ◽  
Akinobu Okabe
Keyword(s):  
Gene Expression ◽  
Clostridium Perfringens ◽  
Expression System ◽  
Xylose Isomerase ◽  
Inducible Gene Expression ◽  
Content Type ◽  
Regulated Expression ◽  
Operator Sequence ◽  
Inducible Gene

ABSTRACTA xylose-inducible gene expression vector forClostridium perfringenswas developed. Plasmid pXCH contains a chromosomal region fromClostridium difficile(xylR-PxylB):xylR, encoding the xylose repressor,xylO, thexyloperator sequence, and PxylB, the divergent promoter upstream ofxylBAencoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study ofC. perfringens.

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