Metabolic Engineering of Candida tropicalis for the De Novo Synthesis of ß-Ionone.

ß-ionone, a norisoprenoid, is a natural aromatic compound derived from plants, which displays various biological activities including anticancer, antioxidant and deworming properties. Due to its large biomass and strong environmental tolerance, the nonconventional oleaginous yeast Candida tropicalis was selected to efficiently synthesize ß-ionone. We initially investigated the capacity of the cytoplasm and subcellular compartments to synthesize ß-ionone independently. Subsequently, through adaptive screening of enzymes, functional identification of subcellular localization signal peptides and subcellular compartment combination strategies, a titer of 152.4 mg/L of ß-ionone was achieved. Finally, directed evolution of rate-limiting enzyme and overexpression of key enzymes were performed to enhance ß-ionone production. The resulting titer was 400.5 mg/L in shake flasks and 730 mg/L in a bioreactor. This study demonstrates the first de novo synthesis of ß-ionone in C. tropicalis, providing a novel cellular chassis for terpenoid fragrances with considerable industrial potential.

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis.

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Unlocking Cd(II) biosorption potential of Candida tropicalis XTA 1874 for sustainable wastewater treatment.

Cd(II) is a potentially toxic heavy metal having carcinogenic activity. It is becoming widespread in the soil and groundwater by various natural and anthropological activities. This is inviting its immediate removal. The present study is aimed at developing a Cd(II) resistant strain isolated from contaminated water body and testing its potency in biological remediation of Cd(II) from aqueous environment. The developed resistant strain was characterized by SEM, FESEM, TEM, EDAX, FT-IR, Raman Spectral, XRD and XPS analysis. The results depict considerable morphological changes had taken place on the cell surface and interaction of Cd(II) with the surface exposed functional groups along with intracellular accumulation. Molecular contribution of critical cell wall component has been evaluated. The developed resistant strain had undergone Cd(II) biosorption study by employing adsorption isotherms and kinetic modeling. Langmuir model best fitted the Cd(II) biosorption data compared to the Freundlich one. Cd(II) biosorption by the strain followed a pseudo second order kinetics. The physical parameters affecting biosorption were also optimized by employing response surface methodology using central composite design. The results depict remarkable removal capacity 75.682 ± 0.002% of Cd(II) by the developed resistant strain from contaminated aqueous medium using 500 ppm of Cd(II). Quantitatively, biosorption for Cd(II) by the newly developed resistant strain has been increased significantly (p < 0.0001) from 4.36 ppm (non-resistant strain) to 378.41 ppm (resistant strain). It has also shown quite effective desorption capacity 87.527 ± 0.023% at the first desorption cycle and can be reused effectively as a successful Cd(II) desorbent up to five cycles. The results suggest that the strain has considerable withstanding capacity of Cd(II) stress and can be employed effectively in the Cd(II) bioremediation from wastewater.

Variations in candidalysin amino acid sequence influence toxicity and host responses.

Candida albicans causes millions of mucosal infections in humans annually. Hyphal overgrowth on mucosal surfaces is frequently associated with tissue damage caused by candidalysin, a secreted peptide toxin that destabilizes the plasma membrane of host cells thereby promoting disease and immunopathology. Candidalysin was first identified in C. albicans strain SC5314, but recent investigations have revealed candidalysin "variants" of differing amino acid sequence in isolates of C. albicans, and the related species C. dubliniensis, and C tropicalis, suggesting that sequence variation among candidalysins may be widespread in natural populations of these Candida species. Here, we analyzed ECE1 gene sequences from 182 C. albicans isolates, 10 C. dubliniensis isolates, and 78 C. tropicalis isolates and identified 10, 3, and 2 candidalysin variants in these species, respectively. Application of candidalysin variants to epithelial cells revealed differences in the ability to cause cellular damage, changes in metabolic activity, calcium influx, MAPK signalling, and cytokine secretion, while biophysical analyses indicated that variants exhibited differences in their ability to interact with and permeabilize a membrane. This study identifies candidalysin variants with differences in biological activity that are present in medically relevant Candida species. IMPORTANCE: Fungal infections are a significant burden to health. Candidalysin is a toxin produced by Candida albicans that damages host tissues, facilitating infection. Previously, we demonstrated that candidalysins exist in the related species C. dubliniensis and C. tropicalis, thereby identifying these molecules as a toxin family. Recent genomic analyses have highlighted the presence of a small number of candidalysin "variant" toxins, which have different amino acid sequences to those originally identified. Here, we screened genome sequences of isolates of C. albicans, C. dubliniensis, and C. tropicalis and identified candidalysin variants in all three species. When applied to epithelial cells, candidalysin variants differed in their ability to cause damage, activate intracellular signaling pathways, and induce innate immune responses, while biophysical analysis revealed differences in the ability of candidalysin variants to interact with lipid bilayers. These findings suggest that intraspecies variation in candidalysin amino acid sequence may influence fungal pathogenicity.

Multifunctional in vitro, in silico and DFT analyses on antimicrobial BagremycinA biosynthesized by Micromonospora chokoriensis CR3 from Hieracium canadense.

Among the actinomycetes in the rare genera, Micromonospora is of great interest since it has been shown to produce novel therapeutic compounds. Particular emphasis is now on its isolation from plants since its population from soil has been extensively explored. The strain CR3 was isolated as an endophyte from the roots of Hieracium canadense, and it was identified as Micromonospora chokoriensis through 16S gene sequencing and phylogenetic analysis. The in-vitro analysis of its extract revealed it to be active against the clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Candida tropicalis (15 mm). No bioactivity was observed against Gram-negative bacteria, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 706003. The Micromonospora chokoriensis CR3 extract was also analyzed through the HPLC-DAD-UV-VIS resident database, and it gave a maximum match factor of 997.334 with the specialized metabolite BagremycinA (BagA). The in-silico analysis indicated that BagA strongly interacted with the active site residues of the sterol 14-α demethylase and thymidylate kinase enzymes, with the lowest binding energies of - 9.7 and - 8.3 kcal/mol, respectively. Furthermore, the normal mode analysis indicated that the interaction between these proteins and BagA was stable. The DFT quantum chemical properties depicted BagA to be reasonably reactive with a HOMO-LUMO gap of (ΔE) of 4.390 eV. BagA also passed the drug-likeness test with a synthetic accessibility score of 2.06, whereas Protox-II classified it as a class V toxicity compound with high LD50 of 2644 mg/kg. The current study reports an endophytic actinomycete, M. chokoriensis, associated with H. canadense producing the bioactive metabolite BagA with promising antimicrobial activity, which can be further modified and developed into a safe antimicrobial drug.

Metabolic engineering of Candida tropicalis for efficient 1,2,4-butanetriol production.

1,2,4-Butanetriol serves as a precursor in the manufacture of diverse pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. The study involved further modifications to an engineered Candida tropicalis strain, aimed at improving the production efficiency of 1,2,4-butanetriol. Faced with the issue of xylonate accumulation due to the low activity of heterologous xylonate dehydratase, we modulated iron metabolism at the transcriptional level to boost intracellular iron ion availability, thus enhancing the enzyme activity by 2.2-fold. Addressing the NADPH shortfall encountered during 1,2,4-butanetriol biosynthesis, we overexpressed pivotal genes in the NADPH regeneration pathway, achieving a 1,2,4-butanetriol yield of 3.2 g/L. The introduction of calcium carbonate to maintain pH balance led to an increased yield of 4 g/L, marking a 111% improvement over the baseline strain. Finally, the use of corncob hydrolysate as a substrate culminated in 1,2,4-butanetriol production of 3.42 g/L, thereby identifying a novel host for the conversion of corncob hydrolysate to 1,2,4-butanetriol.

Isolation and characterization of Candida tropicalis B: a promising yeast strain for biodegradation of petroleum oil in marine environments.

The increasing interest in environmental protection laws has compelled companies to regulate the disposal of waste organic materials. Despite efforts to explore alternative energy sources, the world remains heavily dependent on crude petroleum oil and its derivatives. The expansion of the petroleum industry has significant implications for human and environmental well-being. Bioremediation, employing living microorganisms, presents a promising approach to mitigate the harmful effects of organic hydrocarbons derived from petroleum. This study aimed to isolate and purify local yeast strains from oil-contaminated marine water samples capable of aerobically degrading crude petroleum oils and utilizing them as sole carbon and energy sources. One yeast strain (isolate B) identified as Candida tropicalis demonstrated high potential for biodegrading petroleum oil in seawater. Physiological characterization revealed the strain's ability to thrive across a wide pH range (4-11) with optimal growth at pH 4, as well as tolerate salt concentrations ranging from 1 to 12%. The presence of glucose and yeast extract in the growth medium significantly enhanced the strain's biomass formation and biodegradation capacity. Scanning electron microscopy indicated that the yeast cell diameter varied based on the medium composition, further emphasizing the importance of organic nitrogenous sources for initial growth. Furthermore, the yeast strain exhibited remarkable capabilities in degrading various aliphatic and aromatic hydrocarbons, with a notable preference for naphthalene and phenol at 500 and 1000 mg/l, naphthalene removal reached 97.4% and 98.6%, and phenol removal reached 79.48% and 52.79%, respectively. Optimization experiments using multi-factorial sequential designs highlighted the influential role of oil concentration on the bioremediation efficiency of Candida tropicalis strain B. Moreover, immobilized yeast cells on thin wood chips demonstrated enhanced crude oil degradation compared to thick wood chips, likely due to increased surface area for cell attachment. These findings contribute to our understanding of the potential of Candida tropicalis for petroleum oil bioremediation in marine environments, paving the way for sustainable approaches to address oil pollution.

Bioinspired peptides induce different cell death mechanisms against opportunistic yeasts.

The management of fungal diseases imposes an urgent need for the development of effective antifungal drugs. Among new drug candidates are the antimicrobial peptides, and especially their derivatives. Here, we investigated the molecular mechanism of action of three bioinspired peptides against the opportunistic yeasts Candida tropicalis and Candida albicans. We assessed morphological changes, mitochondrial functionality, chromatin condensation, ROS production, activation of metacaspases, and the occurrence of cell death. Our results indicated that the peptides induced sharply contrasting death kinetics, of 6 h for RR and 3 h for D-RR to C. tropicalis and 1 h for WR to C. albicans. Both peptide-treated yeasts exhibited increased ROS levels, mitochondrial hyperpolarization, cell size reduction, and chromatin condensation. RR and WR induced necrosis in C. tropicalis and C. albicans, but not D-RR in C. tropicalis. The antioxidant ascorbic acid reverted the toxic effect of RR and D-RR, but not WR, suggesting that instead of ROS there is a second signal triggered that leads to yeast death. Our data suggest that RR induced a regulated accidental cell death in C. tropicalis, D-RR induced a programmed cell death metacaspase-independent in C. tropicalis, while WR induced an accidental cell death in C. albicans. Our results were obtained with the LD100 and within the time that the peptides induce the yeast death. Within this temporal frame, our results allow us to gain clarity on the events triggered by the peptide-cell interaction and their temporal order, providing a better understanding of the death process induced by them.

Optimization of xylanase production by Pichia kudriavzevii and Candida tropicalis isolated from the wood product workshop.

Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50 °C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50 °C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50 °C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.

Rice Big Grain1 enhances biomass and plant growth-promoting traits in rhizospheric yeast Candida tropicalis.

The Big Grain1 (BG1) gene of rice (Oryza sativa L.) is reported to increase the yield of rice crops; however, its molecular mechanism is largely concealed. To explore its functional prospects, we have taken a structure-function-based approach. In silico analyses suggest OsBG1 is a DNA- and phytohormone-binding protein. Heterologous expression of OsBG1 with galactose-inducible promoter GAL1p in the rhizospheric yeast Candida tropicalis SY005 revealed 7.9- and 1.5-fold higher expression of the gene at 12 and 24 h, respectively, compared to the expression at 36 h post-galactose induction. Functional activity of the induced OsBG1 in engineered yeast increased cell density, specific growth rate, and biomass by 28.5%, 29.8%, and 14.1%, respectively, and decreased the generation time by 21.25%. Flow cytometry-based cell cycle analysis of OsBG1-expressing yeast cells exhibited an increase in the cells of the G2/M population by 15.8% after 12 h of post-galactose induction. The gene expression study of yeast transformants disclosed that OsBG1 regulates cell division by upregulating the expression of the endogenous gene cyclin B1 (CtCYB1) by 1.3- and 1.9-folds at 10 and 12 h, respectively, compared to the control, and is positively influenced by the phytohormone indole acetic acid (IAA). Further, the study revealed that OsBG1 significantly increases biofilm formation, stress tolerance, and IAA production in C. tropicalis SY005, implying its prospective role in enhancing plant growth-promoting traits in microbes. OsBG1-expressing rhizospheric yeast cells significantly improved the germination and growth parameters of the bio-inoculated rice seeds. Altogether, this study suggests OsBG1 can be employed to genetically improve suitable bio-inoculants for their plant growth-promoting traits to augment crop productivity. KEY POINTS: • In silico analyses suggested OsBG1 is a phytohormone-binding transcription factor. • OsBG1 enhanced growth in rhizospheric Candida tropicalis by upregulating CtCYB1. • OsBG1 improved plant growth-promoting traits of the rhizospheric yeast C. tropicalis.

Current advances in Candida tropicalis: Yeast overview and biotechnological applications.

Candida tropicalis is a nonconventional yeast with medical and industrial significance, belonging to the CTG clade. Recent advancements in whole-genome sequencing and genetic analysis revealed its close relation to other unconventional yeasts of biotechnological importance. C. tropicalis is known for its immense potential in synthesizing various valuable biomolecules such as ethanol, xylitol, biosurfactants, lipids, enzymes, α,ω-dicarboxylic acids, single-cell proteins, and more, making it an attractive target for biotechnological applications. This review provides an update on C. tropicalis biological characteristics and its efficiency in producing a diverse range of biomolecules with industrial significance from various feedstocks. The information presented in this review contributes to a better understanding of C. tropicalis and highlights its potential for biotechnological applications and market viability.

Analysis of CDR1 and MDR1 Gene Expression and ERG11 Substitutions in Clinical Candida tropicalis Isolates from Alexandria, Egypt.

INTRODUCTION: Candida tropicalis is a common non-albicans Candida (NAC) species that causes numerous fungal infections. Increasing antifungal resistance to azoles in NAC is becoming a major health problem worldwide; however, in Egypt, almost no data is available regarding fluconazole resistance mechanisms in C. tropicalis. The current study aims to investigate two possible important molecular mechanisms involved in fluconazole resistance in C. tropicalis isolates. MATERIALS: Fifty-four clinical C. tropicalis isolates were included. Identification and antifungal susceptibility profiles of the isolates were carried out using the VITEK 2 compact system. The molecular investigation of fluconazole resistance included the expression of the CDR1 and MDR1 genes by quantitative real-time RT-PCR as well as the sequence analysis of the ERG11 gene. RESULTS: Antifungal susceptibility testing identified 30 fluconazole-non-susceptible isolates. Statistically, CDR1 gene expression in fluconazole-non-susceptible isolates was significantly higher than that in fluconazole-susceptible isolates, with MDR1 gene expression levels that were similar in both non-susceptible and susceptible isolates. Sequence analysis of the ERG11 gene of 26 fluconazole-resistant isolates identified two missense mutations: A395T (Y132F) and G1390A (G464S). CONCLUSIONS: This study has highlighted the role of overexpression of the CDR1 gene and ERG11 gene mutations in fluconazole non-susceptibility. Further studies in Egypt are required to investigate other possible molecular mechanisms involved in azole resistance.

Candida tropicalis as a novel dietary additive to reduce methane emissions and nitrogen excretion in sheep.

The goal of this study was to investigate Candida tropicalis as a kind of environmentally friendly dietary additive to manipulate ruminal fermentation patterns, reduce methane emissions and nitrogen excretion, and to screen the appropriate dose for sheep. Twenty-four Dorper × thin-tailed Han crossbred ewes (51.12 kg ± 2.23 kg BW) were selected and randomly divided into four groups which were fed Candida tropicalis at dose of 0 (control), 4 × 108 (low dose), 4 × 109 (medium dose), and 4 × 1010 (high dose) colony-forming units (CFU)/d per head, respectively. The experiment lasted 33 days with 21 days for adaptation and 12 days for nutrient digestibility trial and respiratory gases sampling. The results showed that nutrients intake was not affected by Candida tropicalis supplementation (P > 0.05), whereas apparent digestibility of nutrients significantly increased compared with the control group (P < 0.05). Nitrogen and energy utilization increased with Candida tropicalis supplementation (P < 0.05). Compared with the ewes of the control group, rumen fluid pH and NH3-N concentration were not affected (P > 0.05), whereas total volatile fatty acid concentration and molar proportion of propionate were greater (P < 0.05), and molar proportion of acetate and the ratio of acetate to propionate were less (P < 0.05) when the ewes were fed Candida tropicalis. Daily total CH4 production (L/d) and CH4 emissions yield (L/d of CH4 per kg of dry matter intake, metabolic weight, or digestibility dry matter intake) were decreased at the low dose group (P < 0.05). The abundance of total bacteria, methanogen, and protozoa in rumen fluid was significantly higher at medium dose and high dose of Candida tropicalis supplementation (P < 0.05) compared with low dose and the control group. In summary, Candida tropicalis supplementation has a potential to reduce CH4 emissions and nitrogen excretion, and the optimal dose should be 4 × 108 CFU/d per head.

Dimorphism of Candida tropicalis and its effect on nitrogen and phosphorus removal and sludge settleability.

Candida tropicalis PNY, a novel dimorphic strain with the capacity of simultaneous carbon, nitrogen and phosphorus removal in anaerobic and aerobic conditions, was isolated from activated sludge. Dimorphism of C. tropicalis PNY had effect on removing nitrogen and phosphorous and slightly affected COD removal under aerobic condition. Sample with high hypha formation rate (40 ± 5%) had more removal efficiencies of NH4+-N (50 mg/L) and PO43--P (10 mg/L), which could achieve 82.19% and 97.53%, respectively. High hypha cells dosage exhibited good settleability and filamentous overgrowth was not observed. According to label-free quantitative proteomics assays. Up-regulated proteins involved in the mitogen-activated protein kinase (MAPK) pathway indicated the active growth and metabolism process of sample with high hypha formation rate (40 ± 5%). And proteins concerning about glutamate synthetase and SPX domain-contain protein explain for the nutrient removal mechanism including assimilation of ammonia and polyphosphates synthesis.

Candida tropicalis oligopeptide transporters assist in the transmembrane transport of the antimicrobial peptide CGA-N9.

Candida tropicalis is often reported as the second or third most common pathogen causing fungal infections. Antimicrobial peptides (AMPs) have attracted increasing attention for their broad-spectrum antimicrobial properties and low cytotoxicity. Our previous studies have shown that CGA-N9, a non-membrane-rupturing AMP, crosses the cell membrane to exert anticandidal activity. We speculate that there are some related transporters that assist in the transmembrane transport of CGA-N9. In this study, the relationship between CGA-N9 lethality kinetics and its real-time transmembrane amount in C. tropicalis cells was investigated. The results demonstrated that there was a positive correlation between its candicidal activity and transmembrane amount. A total of 12 oligopeptide transporter (OPT) coding sequences (CDSs) were cloned from C. tropicalis by using the conservative OPT gene sequences of Candida spp. to design primers and were named C. tropicalis OPTs (CtOPTs). The results of RT‒qPCR demonstrated that the expression levels of CtOPT1, CtOPT9 and CtOPT12 were correlated with the CGA-N9 transmembrane amount in a time-dependent manner. The results of molecular docking demonstrated that CtOPT1, CtOPT9 and CtOPT12 interact strongly with CGA-N9. Therefore, CtOPT1, CtOPT9 and CtOPT12 were predicted to assist in the transmembrane transport of the AMP CGA-N9.

Valorization of rice straw, sugarcane bagasse and sweet sorghum bagasse for the production of bioethanol and phenylacetylcarbinol.

Open burning of agricultural residues causes numerous complications including particulate matter pollution in the air, soil degradation, global warming and many more. Since they possess bio-conversion potential, agro-industrial residues including sugarcane bagasse (SCB), rice straw (RS), corncob (CC) and sweet sorghum bagasse (SSB) were chosen for the study. Yeast strains, Candida tropicalis, C. shehatae, Saccharomyces cerevisiae, and Kluyveromyces marxianus var. marxianus were compared for their production potential of bioethanol and phenylacetylcarbinol (PAC), an intermediate in the manufacture of crucial pharmaceuticals, namely, ephedrine, and pseudoephedrine. Among the substrates and yeasts evaluated, RS cultivated with C. tropicalis produced significantly (p ≤ 0.05) higher ethanol concentration at 15.3 g L-1 after 24 h cultivation. The product per substrate yield (Yeth/s) was 0.38 g g-1 with the volumetric productivity (Qp) of 0.64 g L-1 h-1 and fermentation efficiency of 73.6% based on a theoretical yield of 0.51 g ethanol/g glucose. C. tropicalis grown in RS medium produced 0.303 U mL-1 pyruvate decarboxylase (PDC), a key enzyme that catalyzes the production of PAC, with a specific activity of 0.400 U mg-1 protein after 24 h cultivation. This present study also compared the whole cells biomass of C. tropicalis with its partially purified PDC preparation for PAC biotransformation. The whole cells C. tropicalis PDC at 1.29 U mL-1 produced an overall concentration of 62.3 mM PAC, which was 68.4% higher when compared to partially purified enzyme preparation. The results suggest that the valorization of lignocellulosic residues into bioethanol and PAC will not only aid in mitigating the environmental challenge posed by their surroundings but also has the potential to improve the bioeconomy.

Biodegradation of phenol by Candida tropicalis sp.: Kinetics, identification of putative genes and reconstruction of catabolic pathways by genomic and transcriptomic characteristics.

Candida tropicalis sp. was isolated with predominant biodegradation capability to phenol compounds, even with high concentration or in acid environment. The biodegradation of phenol was evaluated at the following concentrations 10-1750 mg L-1, the strain exhibited well biodegradation efficiency. The maximum specific growth rate was 0.660 h-1 and the specific biodegradation rates was 0.47 mg (phenol) [(mg (VSS) h]-1. Differentially expressed genes were screened out, and results revealed a complete process of energy and carbon metabolism. The genes' arrangements and phylogenetic information showed the unique genetic characteristics of the strain. Catabolic pathways were reconstructed and some key phenol-degrading genes were obviously upregulated, including pheA, catA, OXCT and fadA. A notable detail that CMBL encoding carboxymethylenebutenolidase was speculated to be involved in a shortened pathway of phenol biodegradation, thereby contributing to the reconstruction of the novel phenol catabolic pathway through the hydrolases of dienelactone. Finally, key enzymes were verified by the analysis of specific activity.

Inactivation of the Complement Lectin Pathway by Candida tropicalis Secreted Aspartyl Protease-1.

Candida tropicalisis an opportunistic fungal pathogen and is one of the most frequently isolated non-albicans species. It can cause localised as well as invasive systemic infections particularly in immunocompromised patients. Increased resistance to common anti-fungal drugs is an emerging problem. In order to establish disseminated infections, Candida has evolved several strategies to escape the host immune system. A detailed understanding of how C. tropicalis escapes the host immune attack is needed as it can help develop novel anti-fungal therapies. Secreted aspartyl proteinases (Saps) of C. albicans have been shown to be determinants of virulence and immune evasion. However, the immune evasion properties of C. tropicalis Saps have been poorly characterised. This study investigated the immune evasion properties of C. tropicalis secreted aspartic protease 1 (Sapt1).Sapt1 was recombinantly produced using a Kluyveromyces lactis yeast expression system. A range of complement proteins and immunogloublins were screened to test if Sapt1 had any proteolytic activity. Sapt1 efficiently cleaved human mannose-binding lectin (MBL) and collectin-11, which are the initiating molecules of the lectin pathway of the complement system, but not l-ficolin. In addition, Sapt1 cleaved DC-SIGN, the receptor on antigen presenting dendritic cells. Proteolysis was prominent in acidic condition (pH 5.2), a characteristic of aspartyl protease. No proteolytic activity was detected against complement proteins C1q, C3, C3b, IgG and IgA. In view of the ability of Sapt1 to cleave MBL and collectin-11, we found that Sapt1 could prevent activation of the complement lectin pathway. RT-qPCR analysis using three different C. tropicalis clinical isolates (oral, blood and peritoneal dialysis fluid) revealed relatively higher levels of mRNA expression of Sapt1 gene when compared to a reference strain; Sapt1 protein was found to be secreted by all the tested strains. Lectin pathway and its initiating components are crucial to provide front line defence against Candida infections. For the first time, we have shown that a Candida protease can proteolytically degrade the key initiating components of lectin pathway and inhibit complement activation. Findings from this study highlight the importance of exploring Sapt1 as a potential therapeutic target. We conclude that C. tropicalis secretes Sapt1 to target the complement lectin pathway, a key pattern recognition and clearance mechanism, for its survival and pathogenesis.

Integrated bioinformatics, modelling, and gene expression analysis of the putative pentose transporter from Candida tropicalis during xylose fermentation with and without glucose addition.

The transport of substrates across the cell membrane plays an essential role in nutrient assimilation by yeasts. The establishment of an efficient microbial cell factory, based on the maximum use of available carbon sources, can generate new technologies that allow the full use of lignocellulosic constituents. These technologies are of interest because they could promote the formation of added-value products with economic feasibility. In silico analyses were performed to investigate gene sequences capable of encoding xylose transporter proteins in the Candida tropicalis genome. The current study identified 11 putative transport proteins that have not yet been functionally characterized. A phylogenetic tree highlighted the potential C. tropicalis xylose-transporter proteins CtXUT1, CtXUT4, CtSTL1, CtSTL2, and CtGXT2, which were homologous to previously characterized and reported xylose transporters. Their expression was quantified through real-time qPCR at defined times, determined through a kinetic analysis of the microbial growth curve in the absence/presence of glucose supplemented with xylose as the main carbon source. The results indicated different mRNA expression levels for each gene. CtXUT1 mRNA expression was only found in the absence of glucose in the medium. Maximum CtXUT1 expression was observed in intervals of the highest xylose consumption (21 to 36 h) that corresponded to consumption rates of 1.02 and 0.82 g/L/h in the formulated media, with xylose as the only carbon source and with glucose addition. These observations indicate that CtXUT1 is an important xylose transporter in C. tropicalis. KEY POINTS: • Putative xylose transporter proteins were identified in Candida tropicalis; • The glucose concentration in the cultivation medium plays a key role in xylose transporter regulation; • The transporter gene CtXUT1 has an important role in xylose consumption by Candida tropicalis.

Candida tropicalis prompted effectively simultaneous removal of carbon, nitrogen and phosphorus in activated sludge reactor: Microbial community succession and functional characteristics.

A new Candida tropicalis that simultaneously remove nitrogen and phosphorus, and degrade organic matters was isolated. Three continuous stirred tank reactors inoculated with C. tropicalis, activated sludge, and their co-existing system in aerobic condition were operated for 150 days. Results demonstrated that the inoculation of C. tropicalis in the co-existing system remarkably improved the carbon, nitrogen, and phosphorus removal efficiencies. The co-existing system had increased carbon, nitrogen, and phosphorus removal efficiencies (92%, 73%, and 63%, respectively); decreased biomass (reduced from 1200 mg/L to 500 mg/L); and C. tropicalis as the dominant strain. The relative abundance of traditional nitrogen- and phosphorus-removing microorganisms, such as Mycobacterium, Flavonifactor, and Devsia, increased in the co-existing system. Metagenomic analysis showed that the presence of the PCYT2, EPT1, and phnPP genes and more complexed metabolism pathways in the co-existing system might be responsible for the more activated metabolism process.