Sphingolipid diversity in Candida auris: unraveling interclade and drug resistance fingerprints.

In this study, we explored the sphingolipid (SL) landscape in Candida auris, which plays pivotal roles in fungal biology and drug susceptibility. The composition of SLs exhibited substantial variations at both the SL class and molecular species levels among clade isolates. Utilizing principal component analysis, we successfully differentiated the five clades based on their SL class composition. While phytoceramide (PCer) was uniformly the most abundant SL class in all the isolates, other classes showed significant variations. These variations were not limited to SL class level only as the proportion of different molecular species containing variable number of carbons in fatty acid chains also differed between the isolates. Also a comparative analysis revealed abundance of ceramides and glucosylceramides in fluconazole susceptible isolates. Furthermore, by comparing drug-resistant and susceptible isolates within clade IV, we uncovered significant intraclade differences in key SL classes such as high PCer and low long chain base (LCB) content in resistant strains, underscoring the impact of SL heterogeneity on drug resistance development in C. auris. These findings shed light on the multifaceted interplay between genomic diversity, SLs, and drug resistance in this emerging fungal pathogen.

BEM2, a RHO GTPase Activating Protein That Regulates Morphogenesis in S. cerevisiae, Is a Downstream Effector of Fungicidal Action of Fludioxonil.

Fludioxonil belongs to the phenylpyrrole group of fungicides with a broad antifungal spectrum that has been widely used in agricultural practices for the past thirty years. Although fludioxonil is known to exert its fungicidal action through group III hybrid histidine kinases, the downstream effector of its cytotoxicity is poorly understood. In this study, we utilized a S. cerevisiae model to decipher the cytotoxic effect of fludioxonil. Through genome wide transposon mutagenesis, we have identified Bem2, a Rho GTPase activating protein, which is involved in this process. The deletion of BEM2 resulted in fludioxonil resistance. Our results showed that both the GAP and morphogenesis checkpoint activities of Bem2 were important for this. We also provided the genetic evidence that the role of Bem2 in the cell wall integrity (CWI) pathway and cell cycle regulation could contribute to the fludioxonil resistance phenotype.

Unmasking of CgYor1-Dependent Azole Resistance Mediated by Target of Rapamycin (TOR) and Calcineurin Signaling in Candida glabrata.

In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter KOs were analyzed for their susceptibility toward antimycotic drugs. Although CgYOR1 has previously been reported to be upregulated in various azole-resistant clinical isolates of C. glabrata, deletion of this gene did not change the susceptibility to any of the tested azoles. Additionally, Cgyor1Δ showed no change in susceptibility toward oligomycin, which is otherwise a well-known substrate of Yor1 in other yeasts. The role of CgYor1 in azole susceptibility only became evident when the major transporter CgCDR1 gene was deleted. However, under nitrogen-depleted conditions, Cgyor1Δ demonstrated an azole-susceptible phenotype, independent of CgCdr1. Notably, Cgyor1Δ cells also showed increased susceptibility to target of rapamycin (TOR) and calcineurin inhibitors. Moreover, increased phytoceramide levels in Cgyor1Δ and the deletions of regulators downstream of TOR and the calcineurin signaling cascade (Cgypk1Δ, Cgypk2Δ, Cgckb1Δ, and Cgckb2Δ) in the Cgyor1Δ background and their associated fluconazole (FLC) susceptibility phenotypes confirmed their involvement. Collectively, our findings show that TOR and calcineurin signaling govern CgYor1-mediated azole susceptibility in C. glabrata. IMPORTANCE The increasing incidence of Candida glabrata infections in the last 40 years is a serious concern worldwide. These infections are usually associated with intrinsic azole resistance and increasing echinocandin resistance. Efflux pumps, especially ABC transporter upregulation, are one of the prominent mechanisms of azole resistance; however, only a few of them are characterized. In this study, we analyzed the mechanisms of azole resistance due to a multidrug resistance-associated protein (MRP) subfamily ABC transporter, CgYor1. We demonstrate for the first time that CgYor1 does not transport oligomycin but is involved in azole resistance. Under normal growing conditions its function is masked by major transporter CgCdr1; however, under nitrogen-depleted conditions, it displays its azole resistance function independently. Moreover, we propose that the azole susceptibility due to removal of CgYor1 is not due to its transport function but involves modulation of TOR and calcineurin cascades.

Distinct role of HAMP and HAMP-like linker domains in regulating the activity of Hik1p, a hybrid histidine kinase 3 from Magnaporthe oryzae.

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.

Sphingolipidomics of drug resistant Candida auris clinical isolates reveal distinct sphingolipid species signatures.

Independent studies from our group and others have provided evidence that sphingolipids (SLs) influence the antimycotic susceptibility of Candida species. We analyzed the molecular SL signatures of drug-resistant clinical isolates of Candida auris, which have emerged as a global threat over the last decade. This included Indian hospital isolates of C. auris, which were either resistant to fluconazole (FLCR) or amphotericin B (AmBR) or both drugs. Relative to Candida glabrata and Candida albicans strains, these C. auris isolates were susceptible to SL pathway inhibitors such as myriocin and aureobasidin A, suggesting that SL content may influence azole and AmB susceptibilities. Our analysis of SLs confirmed the presence of 140 SL species within nine major SL classes, namely the sphingoid bases, Cer, αOH-Cer, dhCer, PCer, αOH-PCer, αOH-GlcCer, GlcCer, and IPC. Other than for αOH-GlcCer, most of the SLs were found at higher concentrations in FLCR isolates as compared to the AmBR isolates. SLs were at intermediate levels in FLCR + AmBR isolates. The observed diversity of molecular species of SL classes based on fatty acyl composition was further reflected in their distinct specific imprint, suggesting their influence in drug resistance. Together, the presented data improves our understanding of the dynamics of SL structures, their synthesis, and link to the drug resistance in C. auris.

Assessment of antifungal resistance and associated molecular mechanism in Candida albicans isolates from different cohorts of patients in North Indian state of Haryana.

The present study examines the trend in distribution of Candida species and their antifungal resistance patterns in hospitals across Haryana, a North Indian state with poorly addressed epidemiology of fungal infections. In our collection of 228 Candida isolates, Candida albicans dominated in both high vaginal swab (HVS) and urine samples while Candida glabrata and Candida tropicalis were the second-highest non-albicans Candida species (NAC), respectively. Of note, in blood samples, C. tropicalis and C. albicans were present in equal numbers. All 228 isolates were subjected to antifungal susceptibility tests, whereby 51% of C. albicans recovered from HVS samples displayed fluconazole resistance. To understand its mechanistic basis, expression profiling of efflux pump genes CDR1, CDR2, MDR1 and azole drug target, ERG11 was performed in 20 randomly selected resistant isolates, wherein many isolates elicited higher expression. Further, ERG11 gene sequencing suggested that most of the isolates harbored mutations, which are not reported with azole resistance. However, one isolate, RPCA9 (MIC 64 µg/mL) harbored triple mutation (Y132C, F145L, A114V), wherein Y132 and F145 sites were previously implicated in azole resistance. Interestingly, one isolate, (RPCA61) having MIC > 128 µg/mL harbored a novel mutation, G129R. Of note, HVS isolates RPCA 21, RPCA 22, and RPCA 44 (MICs 64 to > 128 µg/mL) did not show any change in alteration in ERG11 or overexpression of efflux pump genes. Together, this study presents a first report of Candida infections in selected hospitals of Haryana State.

ABC Transporter Genes Show Upregulated Expression in Drug-Resistant Clinical Isolates of Candida auris: A Genome-Wide Characterization of ATP-Binding Cassette (ABC) Transporter Genes.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including Candida species. Candida auris, a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of C. auris. Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the C. auris type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the C. auris drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant C. auris isolates suggested their importance in the drug resistance phenotype of this pathogen. CAUR_02725 orthologous to CDR1, a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of C. auris. Homologs of other ABC transporter genes, such as CDR4, CDR6, and SNQ2, also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in C. auris.

Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast.

Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast Saccharomyces cerevisiae but also in the pathogenic yeast Candida albicans We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of S. cerevisiae, Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in C. albicans, Mlt1p, could also fulfill this function. Evidence that the process is energy dependent comes from the finding that a Mlt1p mutant version made by converting a critical lysine residue in the Walker A motif of nucleotide-binding domain 1 (required for ATP hydrolysis) to alanine (K710A) was not able to transport azoles. Additionally, we have shown that, as for other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at position 765 (F765Δ) results in mislocalization of the Mlt1 protein; this mislocalized protein was devoid of the azole-resistant attribute. This finding suggests that the presence of this protein on vacuolar membranes is an important factor in azole resistance. Further, we report the importance of conserved residues, because conversion of two serines (positions 973 and 976, in the regulatory domain and in the casein kinase I [CKI] consensus sequence, respectively) to alanine severely affected the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by the ABC transporter Ybt1 and its homologue Mlt1 as an alternative strategy to circumvent drug toxicity among pathogenic and nonpathogenic yeasts.

Overexpression of the CORVET complex alleviates the fungicidal effects of fludioxonil on the yeast Saccharomyces cerevisiae expressing hybrid histidine kinase 3.

The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogen-activated protein kinase (MAPK). In addition to its role in osmoadaptation, HHK3 is involved in hyphal morphogenesis, conidiation, virulence, and cellular adaptation to oxidative stress. However, the molecular mechanisms by which it controls these processes remain obscure. Moreover, HHK3 is a molecular target for antifungal agents such as fludioxonil, which thereby interferes with the HOG/p38 pathway, leading to the abnormal accumulation of glycerol and subsequent cell lysis. Here, we used a chemical genomics approach with the yeast Saccharomyces cerevisiae to better understand the fungicidal action of fludioxonil and the role of HHK3 in fungal growth and physiology. Our results indicated that the abnormal accumulation of glycerol is not the primary cause of fludioxonil toxicity. Fludioxonil appears to impair endosomal trafficking in the fungal cells. We found that the components of class C core vacuole/endosome tethering (CORVET) complex are essential for yeast viability in the presence of a subthreshold dose of fludioxonil and that their overexpression alleviates fludioxonil toxicity. We also noted that by impeding secretory vesicle trafficking, fludioxonil inhibits hyphal growth in the opportunistic fungal pathogen Candida albicans Our results suggest that HHK3 regulates fungal hyphal growth by affecting vesicle trafficking. Together, our results reveal an important role of CORVET complex in the fungicidal action of fludioxonil downstream of HHK3.

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance.

The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans.

Ncb2, the ß subunit of NC2 complex, a heterodimeric regulator of transcription was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant isolate (AR) of Candida albicans. This study examines its genome-wide role by profiling Ncb2 occupancy between genetically matched pair of azole sensitive (AS) and AR clinical isolates. A comparison of Ncb2 recruitment between the two isolates displayed that 29 genes had higher promoter occupancy of Ncb2 in the AR isolate. Additionally, a host of genes exhibited exclusive occupancy of Ncb2 at promoters of either AR or AS isolate. The analysis also divulged new actors of multi-drug resistance, whose transcription was activated owing to the differential occupancy of Ncb2. The conditional, sequence-specific positional escape of Ncb2 from the core promoter in AS isolate and its preferential recruitment to the core promoter of certain genes in AR isolates was most noteworthy means of transcription regulation. Together, we show that positional rearrangement of Ncb2 resulting in either activation or repression of gene expression in response to drug-induced stress, represents a novel regulatory mechanism that opens new opportunities for therapeutic intervention to prevent development of drug tolerance in C. albicans cells.

The serine/threonine phosphatase DhSIT4 modulates cell cycle, salt tolerance and cell wall integrity in halo tolerant yeast Debaryomyces hansenii.

The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li+, Na+ and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii.

Functional dissection of HAMP domains in NIK1 ortholog from pathogenic yeast Candida lusitaniae.

Nik1 orthologs or group III hybrid histidine kinases (HHK) are ubiquitous signaling molecules in fungal pathogens. Besides osmosensing, they are also involved in hyphal morphogenesis, virulence, and conidiation. They are important molecular targets for antifungal agents. Nik1 orthologs contain a varying number of HAMP domain repeats (poly-HAMP) in the N-terminal region. Poly-HAMP plays a crucial role in their function. So far, the role of HAMP domains in their function has been studied only in a few Nik1 orthologs. In this paper, we describe the functional characterization of a Nik1 ortholog (ClNik1p) from Candida lusitaniae, an emerging and important fungal pathogen. We show that ClNik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG pathway in Saccharomyces cerevisiae. Our data suggests a differential role of the HAMP domains in the functionality of ClNik1p. The HAMP domains H1, H2, H3 and H5 are essential for kinase activity, and H4 domain has a regulatory role. Among the HAMP like linker domains, only H4b was crucial for the activity of ClNik1p.

Plasma Membrane Proteolipid 3 Protein Modulates Amphotericin B Resistance through Sphingolipid Biosynthetic Pathway.

Invasive opportunistic fungal infections of humans are common among those suffering from impaired immunity, and are difficult to treat resulting in high mortality. Amphotericin B (AmB) is one of the few antifungals available to treat such infections. The AmB resistance mechanisms reported so far mainly involve decrease in ergosterol content or alterations in cell wall. In contrast, depletion of sphingolipids sensitizes cells to AmB. Recently, overexpression of PMP3 gene, encoding plasma membrane proteolipid 3 protein, was shown to increase and its deletion to decrease, AmB resistance. Here we have explored the mechanistic basis of PMP3 effect on AmB resistance. It was found that ergosterol content and cell wall integrity are not related to modulation of AmB resistance by PMP3. A few prominent phenotypes of PMP3 delete strain, namely, defective actin polarity, impaired salt tolerance, and reduced rate of endocytosis are also not related to its AmB-sensitivity. However, PMP3 overexpression mediated increase in AmB resistance requires a functional sphingolipid pathway. Moreover, AmB sensitivity of strains deleted in PMP3 can be suppressed by the addition of phytosphingosine, a sphingolipid pathway intermediate, confirming the importance of this pathway in modulation of AmB resistance by PMP3.

Hybrid histidine kinases in pathogenic fungi.

Histidine kinases (HK) sense and transduce via phosphorylation events many intra- and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N-terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape-function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.

Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1­5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1­5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1­5 protein inside experimental data-based models showed how two chains of HAMP1­5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1­5 structure mediate its function.

DrugMint: a webserver for predicting and designing of drug-like molecules.

BACKGROUND: Identification of drug-like molecules is one of the major challenges in the field of drug discovery. Existing approach like Lipinski rule of 5 (Ro5), Operea have their own limitations. Thus, there is a need to develop computational method that can predict drug-likeness of a molecule with precision. In addition, there is a need to develop algorithm for screening chemical library for their drug-like properties. RESULTS: In this study, we have used 1347 approved and 3206 experimental drugs for developing a knowledge-based computational model for predicting drug-likeness of a molecule. We have used freely available PaDEL software for computing molecular fingerprints/descriptors of the molecules for developing prediction models. Weka software has been used for feature selection in order to identify the best fingerprints. We have developed various classification models using different types of fingerprints like Estate, PubChem, Extended, FingerPrinter, MACCS keys, GraphsOnlyFP, SubstructureFP, Substructure FPCount, Klekota-RothFP, Klekota-Roth FPCount. It was observed that the models developed using MACCS keys based fingerprints, discriminated approved and experimental drugs with higher precision. Our model based on one hundred fifty nine MACCS keys predicted drug-likeness of the molecules with 89.96% accuracy along with 0.77 MCC. Our analysis indicated that MACCS keys (ISIS keys) 112, 122, 144, and 150 were highly prevalent in the approved drugs. The screening of ZINC (drug-like) and ChEMBL databases showed that around 78.33% and 72.43% of the compounds present in these databases had drug-like potential. CONCLUSION: It was apparent from above study that the binary fingerprints could be used to discriminate approved and experimental drugs with high accuracy. In order to facilitate researchers working in the field of drug discovery, we have developed a webserver for predicting, designing, and screening novel drug-like molecules (http://crdd.osdd.net/oscadd/drugmint/).

Studies on xylitol production by metabolic pathway engineered Debaryomyces hansenii.

Debaryomyces hansenii is one of the most promising natural xylitol producers. As the conversion of xylitol to xylulose mediated by NAD(+) cofactor dependent xylitol dehydrogenase (XDH) reduces its xylitol yield, xylitol dehydrogenase gene (DhXDH)-disrupted mutant of D. hansenii having potential for xylose assimilating pathway stopping at xylitol, was used to study the effects of co-substrates, xylose and oxygen availability on xylitol production. Compared to low cell growth and xylitol production in cultivation medium containing xylose as the only substrate, XDH disrupted mutants grown on glycerol as co-substrate accumulated 2.5-fold increased xylitol concentration over those cells grown on glucose as co-substrate. The oxygen availability, in terms of volumetric oxygen transfer coefficient, kLa (23.86-87.96 h(-1)), affected both xylitol productivity and yield, though the effect is more pronounced on the former. The addition of extra xylose at different phases of xylitol fermentation did not enhance xylitol productivity under experimental conditions.

The sixth HAMP domain negatively regulates the activity of the group III HHK containing seven HAMP domains.

In fungi, the group III hybrid histidine kinases (HHK) act as important sensors to regulate osmoadaptation, hyphal growth, morphogenesis, conidia formation and virulence. They are molecular targets for antifungal agent fludioxonil. They typically have HAMP domain repeats at the NH2-terminus that are important for their activity. Interestingly, the numbers of HAMP domain vary among the orthologs from different genera. The orthologs from basidiomycetes harbor seven HAMP domains whereas those from yeast contain five HAMP domains. In order to understand the functioning of a seven-HAMP module, we have constructed a yeast-like chimera DhNik1-Tco1 containing seven HAMP domains. The functional characterization of this chimera in yeast Saccharomyces cerevisiae showed that the sixth HAMP domain played important regulatory role. Our results indicated that the negative regulation of histidine kinase activity by the penultimate HAMP domain could possibly be an evolutionarily conserved theme in the group III HHK containing different lengths of poly HAMP module.

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii.

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.