Identification of the Cadmium-Inducible Hansenula polymorpha SEO1 Gene Promoter by Transcriptome Analysis and Its Application to Whole-Cell Heavy-Metal Detection Systems

Abstract

The genomewide gene expression profiling of the methylotrophic yeast Hansenula polymorpha exposed to cadmium (Cd) allowed us to identify novel genes responsive to Cd treatment. To select genes whose promoters can be useful for construction of a cellular Cd biosensor, we further analyzed a set of H. polymorpha genes that exhibited >6-fold induction upon treatment with 300 M Cd for 2 h. The putative promoters, about 1,000-bp upstream fragments, of these genes were fused with the yeast-enhanced green fluorescence protein (GFP) gene. The resultant reporter cassettes were introduced into H. polymorpha to evaluate promoter strength and specificity. The promoter derived from the H. polymorpha SEO1 gene (HpSEO1) was shown to drive most strongly the expression of GFP upon Cd treatment among the tested promoters. The Cd-inducible activity was retained in the 500-bp deletion fragment of the HpSEO1 promoter but was abolished in the further truncated 250-bp fragment. The 500-bp HpSEO1 promoter directed specific expression of GFP upon exposure to Cd in a dose-dependent manner, with Cd detection ranging from 1 to 900 M. Comparative analysis of the Saccharomyces cerevisiae SEO1 (ScSEO1) promoter revealed that the ScSEO1 promoter has a broader specificity for heavy metals and is responsive to arsenic and mercury in addition to Cd. Our data demonstrate the potential use of the HpSEO1 promoter as a bioelement in whole-cell biosensors to monitor heavy metal contamination, particularly Cd.Industrial activities lead to large-scale environmental contamination with toxic heavy metals, such as cadmium (Cd) and mercury (Hg). These metals are toxic even at low levels and tend to accumulate in the body over an extended period of time, which can eventually cause serious health problems in humans. Therefore, the development of monitoring systems for these metal ions in the environment has become increasingly important in order to prevent chronic exposure to these pollutants (2). As a response to this need, numerous biological systems and nonbiological sensors based on the emerging nanotechnology have been developed to monitor heavy metal contamination (38, 43). In particular, biosensors that use unicellular microorganisms as analytical tools to monitor heavy metals have drawn attention because of several practical advantages (1): the large population size, rapid growth rate, low cost, and easy maintenance. Moreover, the feasibility of genetic manipulation makes microbial cells an attractive choice as environmental bioreporters.Many current microbial whole-cell sensors are based on genetically modified microorganisms (43,28). In general, microbial biosensors comprise the molecular fusion of two linked genetic elements: a sensing bioelement and a reporter gene. In most cases, the sensing element is a promoter that specifically responds to the presence or absence of the target molecule, and the reporter gene, which is fused to the sensing element, encodes a quantifiable molecule such as a bioluminescent or fluorescent protein (13). In creating ...

A pilot study for construction of a new cadmium-sensing yeast strain carrying a reporter plasmid with the <i>JLP1</i> promoter

“…These results revealed that the JLP1 promoter is more appropriate than the SEO1 promoter that was used in the reporter assay strain by Park et al (2007). The greater Cd-induced transcription of lacZ from the JLP1 promoter than the SEO1 promoter in this study is supported by the findings of a yeast microarray study (Momose and Iwahashi, 2001).…”

“…The promoter sequences of the CUP1, SEO1, and UFO1 genes have been used to detect heavy metals. Yeast strains possessing CUP1 reporter plasmids respond to copper (Cu) and cadmium (Cd) (Tohoyama et al, 1992), those possessing the SEO1 reporter respond to Cd, arsenic (As), and mercury (Hg) (Park et al, 2007), and those possessing the UFO1 reporter respond to arsenate and ultraviolet light B (UVB) (Bakhrat et al, 2011).…”

Development of a Conditional Gene Expression System Using a Zearalenone-Inducible Promoter for the Ascomycete Fungus Gibberella zeae

“…These systems have been used as tools for studying the molecular regulation of target gene expression and protein production of genes in industrial fungi (20,39). Further, recombinant bacterial and fungal strains that possess a metal-inducible promoter have been used as biosensors for monitoring heavy metal contamination (25,33). A woundinducible promoter was also recently used to generate transgenic plants that can recognize fungal infections (36).…”

“…Regulation of gene expression by conditional promoters, including copper-, cadmium-, thiamine-, and alcohol-responsive promoters, has been developed in various organisms (23,25,30,(34)(35). These systems have been used as tools for studying the molecular regulation of target gene expression and protein production of genes in industrial fungi (20,39).…”

Targeting Genes of Cd Induced Oxidative Stress Response in Yeasts

“…Until recently, it was considered that the sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae (with glutathione being the main cd intracellular chelator), but different from Schizosaccharomys pombe and Candida glabrata (27). however, several papers report identification of cadmium-ion tolerant Hansenula species (31,44), suggesting that much profounder investigations should be made on the cadmium tolerance of these yeast microorganisms.…”