To realize the economical production of ethanol and other bio-based chemicals from lignocellulosic biomass by consolidated bioprocessing (CBP), various cellulases from different sources were tested to improve the level of cellulase secretion in the yeast Saccharomyces cerevisiae by screening an optimal translational fusion partner (TFP) as both a secretion signal and fusion partner. Among them, four indispensable cellulases for cellulose hydrolysis, including Chaetomium thermophilum cellobiohydrolase (CtCBH1), Chrysosporium lucknowense cellobiohydrolase (ClCBH2), Trichoderma reesei endoglucanase (TrEGL2), and Saccharomycopsis fibuligera β-glucosidase (SfBGL1), were identified to be highly secreted in active form in yeast. Despite variability in the enzyme levels produced, each recombinant yeast could secrete approximately 0.6–2.0 g/L of cellulases into the fermentation broth. The synergistic effect of the mixed culture of the four strains expressing the essential cellulases with the insoluble substrate Avicel and several types of cellulosic biomass was demonstrated to be effective. Co-fermentation of these yeast strains produced approximately 14 g/L ethanol from the pre-treated rice straw containing 35 g/L glucan with 3-fold higher productivity than that of wild type yeast using a reduced amount of commercial cellulases. This process will contribute to the cost-effective production of bioenergy such as bioethanol and biochemicals from cellulosic biomass.

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 ...

The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121.

Understanding the characteristics and regulation mechanisms of cell wall integrity (CWI) in yeast is important not only for basic research but also in biotechnological applications. We found significantly different CWIs in two representative strains of the thermotolerant methylotrophic yeast Hansenula polymorpha. Compared to the A16 strain (classified as Ogataea polymorpha), the DL1-L strain (classified as Ogataea parapolymorpha) has a thinner cell wall that was found to be more fragile following long-term cultivation and more sensitive to zymolyase. To gain a deeper insight into this difference, we compared the characteristics of the Mpk1pmediated CWI signaling pathway in the two strains. While a DL1-L mutant deficient in Mpk1p (mpk1Δ) showed severe growth retardation at both normal and high growth temperatures and in the presence of cell-wall disrupting agents, the A16 mpk1Δ mutant displayed only a mild defect in cell growth. Sorbitol effect on rescuing growth retardation was different in the two mpk1Δ strains, which could partly be ascribed to subtle differences in the activation of HOG pathway. Among the cell wall disruptors evaluated, only caffeine clearly increased phosphorylation of Mpk1p in DL1-L, but not in A16. A transcriptome analysis of the DL1-L strain revealed that caffeine significantly increased the expression of a subset of cell-wall related genes in an Mpk1p-dependent manner, but not the expected Rlm1-target genes. Taken together, our data support an essential role for Mpk1p in maintaining CWI in H. polymorpha, although the requirement for Mpk1p and its regulation under diverse stress conditions varies depending on the strain background.