Biosynthesis of soluble carnitine acetyltransferases from the yeast Candida tropicalis.
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ABSTRACT:
SUBMITTER: Kozulic B
PROVIDER: S-EPMC1149380 | biostudies-other | 1988 Aug
REPOSITORIES: biostudies-other
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Project description:Backgroundα-Humulene is a plant-derived monocyclic sesquiterpenoid with multiple pharmacological activities, and far-reaching potential for the development of new drugs. Currently, the production of α-humulene is typically achieved via plant extraction, which is not sustainable and limited by low yields. The oleaginous yeast Candida tropicalis has recently emerged as a valuable host for producing high-value-added chemicals. However, the potential of C. tropicalis for terpenoid production has not been exploited.ResultsIn this study, C. tropicalis was engineered for de novo synthesis of α-humulene from glucose. To improve α-humulene production, the codon-optimised α-humulene synthase gene and the entire endogenous farnesyl diphosphate synthesis pathway were co-overexpressed. Furthermore, bottlenecks in the α-humulene synthase pathway were identified and relieved by overexpressing α-humulene synthase, acetoacetyl-CoA thiolase and NADH-dependent HMG-CoA reductase. Combined with fermentation medium optimisation, the engineered strain produced 195.31 mg/L of α-humulene in shake flasks and 4115.42 mg/L in a bioreactor through fed-batch fermentation, a 253- and 5345-fold increase over the initial production, respectively.ConclusionsThis study demonstrates the potential of C. tropicalis for α-humulene production, and presents a platform for the biosynthesis of other terpenoids.
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Project description:The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species--Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast.
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Project description:Candida spp. yeast-like fungi are opportunistic pathogens in humans and have been recently found to release extracellular vesicles (EVs) that are involved in many vital biological processes in fungal cells. These include communication between microorganisms and host-pathogen interactions during infection. The production of EVs and their content have been significantly characterized in the most common candidal species Candida albicans, including the identification of numerous virulence factors and cytoplasmic proteins in the EV cargo. We have here conducted the isolation and proteomic characterization of EVs produced by the clinically important non-albicans Candida species C. glabrata, C. tropicalis and C. parapsilosis. With the use of ultracentrifugation of the cell-free culture supernatant, the candidal EVs were collected and found to be a heterogeneous population of particles for each species with sizes ranging from 60-280 nm. The proteinaceous contents of these vesicles were analyzed using LC-MS/MS, with particular attention paid to surface-expressed proteins that would come into immediate and direct contact with host cells. We thereby identified 42 extracellular and surface-connected proteins from C. glabrata, 33 from C. parapsilosis, and 34 from C. tropicalis, including membrane-associated transporters, glycoproteins and enzymes involved in the organization of the fungal cell wall, as well as several cytoplasmic proteins, including alcohol dehydrogenase, enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and pyruvate kinase, for which the vesicular transport is a possible mechanism underlying their non-classical secretion.
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Project description:Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2-6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36-49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.
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Project description:Candida tropicalis is considered as the leading pathogen in nosocomial fungemia and hepatosplenic fungal infections in patients with cancer, particularly in leukemia. The yeast-filament transition is required for virulent infection by Candida. Several studies have explored the genome-wide transcription profile of Candida, however, no report on the transcriptional profile of C. tropicalis under yeast-filament transition has been published. In this study, the transcriptomes of three C. tropicalis isolates with different adhesion and biofilm formation abilities, identified in our previous studies, were analyzed in both the yeast and filament states using RNA-Seq. Differentially expressed genes were found for each isolate during the transition. A total of 115 genes were up- or down- regulated in the two hyphal-producing isolates (ZRCT 4 and ZRCT 45). Among these differentially expressed genes, only two were down-regulated during the yeast-filament transition. Furthermore, six filament-associated genes were up-regulated in the hyphae-producing isolates. According to Candida Hypha Growth Database established in this study, 331 hyphae- related genes were discovered in C. tropicalis. ALS1 and ALS3 were down-regulated and up-regulated, respectively, during filamentous growth of C. tropicalis. These findings proved a better understanding of gene expression dynamics during the yeast-filament transition in C. tropicalis.
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Project description:Candida tropicalis is a globally distributed human pathogenic yeast, especially prevalent in tropical and sub-tropical regions. Over the last several decades, a large number of studies have been published on the genetic diversity and molecular epidemiology of C. tropicalis from different parts of the world. However, the global pattern of genetic variation remains largely unknown. Here we analyzed the published multilocus sequence data at six loci for 876 isolates from 16 countries representing five continents. Our results showed that 280 of the 2677 (10.5%) analyzed nucleotides were polymorphic, resulting in a mean of 82 (a range of 38-150) genotypes per locus and a total of 633 combined diploid sequence types (DSTs). Among these, 93 combined DSTs were shared by 336 strains, including 10 by strains from different continents. Analysis of Molecular Variance (AMOVA) showed that 89% of the observed genetic variations were found within regional and national populations while < 10% was due to among-country separations. Pairwise geographic population analyses showed overall low but statistically significant genetic differentiation between most geographic populations, with the Singaporean and Indian populations being the most distinct from other populations. However, the Mantel test showed no significant correlation between genetic distance and geographic distance among the geographic populations. Consistent with high genetic variation within and limited variations among geographic populations, results from STRUCTURE analyses showed that the 876 isolates could be grouped into 15 genetic clusters, with each cluster having a broad geographic distribution. Together, our results suggest frequent gene flows among certain regional, national, and continental populations of C. tropicalis, resulting in abundant regional and national genetic diversities of this important human fungal pathogen.
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Project description:The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1a delta/t1bdelta null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a delta/t3b delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All thiolase activities in this yeast disappeared with the ct-t1a delta/t1bdelta and ct-t3a delta/t3bdelta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bdelta null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the beta-oxidation of a C4 substrate butyrate.
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Project description:Medically important yeasts of the genus Candida secrete aspartic proteinases (Saps), which are of particular interest as virulence factors. Like Candida albicans, Candida tropicalis secretes in vitro one dominant Sap (Sapt1p) in a medium containing bovine serum albumin (BSA) as the sole source of nitrogen. Using the gene SAPT1 as a probe and under low-stringency hybridization conditions, three new closely related gene sequences, SAPT2 to SAPT4, encoding secreted proteinases were cloned from a C. tropicalis lambdaEMBL3 genomic library. All bands identified by Southern blotting of EcoRI-digested C. tropicalis genomic DNA with SAPT1 could be assigned to a specific SAP gene. Therefore, the SAPT gene family of C. tropicalis is likely to contain only four members. Interestingly, the SAPT2 and SAPT3 gene products, Sapt2p and Sapt3p, which have not yet been detected in C. tropicalis cultures in vitro, were produced as active recombinant enzymes with the methylotrophic yeast Pichia pastoris as an expression system. As expected, reverse transcriptase PCR experiments revealed a strong SAPT1 signal with RNA extracted from cells grown in BSA medium. However, a weak signal was obtained with all other SAPT genes under several conditions tested, showing that these SAPT genes could be expressed at a basic level. Together, these experiments suggest that the gene products Sapt2p, Sapt3p, and Sapt4p could be produced under conditions yet to be described in vitro or during infection.
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Project description:Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.
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Project description:Biofilm is the fortitude of Candida species infections which eventually causes candidiasis in human. C. tropicalis is one of the predominant Candida species commonly found in systemic infections, next to C. albicans. In Candida species, biofilm maturity initiates irreversible surface attachment of cells and barricades the penetration of conventional antifungals. Hence, the current study investigated the antifungal and antivirulence potency of palmitic acid (PA) against C. tropicalis mature biofilm and its associated virulence factors. In vitro results revealed an effective inhibition of biofilm in PA-treated C. tropicalis, compared to C. albicans and C. glabrata. Also, PA reduced C. tropicalis mature biofilm at various time points. Further, PA treatment triggered apoptosis in C. tropicalis through ROS mediated mitochondrial dysfunction as demonstrated by confocal microscopic observation of PI, DAPI and DCFDA staining. PA regulated other virulence factors such as cell surface hydrophobicity, ergosterol biosynthesis, protease and lipase after 48 h of treatment. Downregulation of ERG11 (Lanosterol 14-alpha demethylase) was contributed to the reduction of ergosterol in PA-treated C. tropicalis. However, enhanced hyphal growth was observed in PA-treated C. tropicalis through upregulation HWP1 (Hyphal wall protein) and EFG1 (Enhanced filamentous growth). This study highlighted the antibiofilm and antivirulence potency of PA against C. tropicalis. Hence, PA could be applied synergistically with other antifungal agents to increase the efficacy for regulating NCAC infections.
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