Hemolymph metabolism of black soldier fly (Diptera: Stratiomyidae), response to different supplemental fungi.

The black soldier fly, Hermetia illucens L. (Diptera: Stratiomyidae), is commonly used for organic waste recycling and animal feed production. However, the often inadequate nutrients in organic waste necessitate nutritional enhancement of black soldier fly larvae, e.g., by fungal supplementation of its diet. We investigated the amino acid composition of two fungi, Candida tropicalis (Castell.) Berkhout (Saccharomycetales: Saccharomycetaceae) and Pichia kudriavzevii Boidin, Pignal & Besson (Saccharomycetales: Pichiaceae), from the black soldier fly gut, and commercial baker's yeast, Saccharomyces cerevisiae Meyen ex E.C. Hansen (Saccharomycetales: Saccharomycetaceae), and their effects on larval growth and hemolymph metabolites in fifth-instar black soldier fly larvae. Liquid chromatography-mass spectrometry was used to study the effect of fungal metabolites on black soldier fly larval metabolism. Amino acid analysis revealed significant variation among the fungi. Fungal supplementation led to increased larval body mass and differential metabolite accumulation. The three fungal species caused distinct metabolic changes, with each over-accumulating and down-accumulating various metabolites. We identified significant alteration of histidine metabolism, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism in BSF larvae treated with C. tropicalis. Treatment with P. kudriavzevii affected histidine metabolism and citrate cycle metabolites, while both P. kudriavzevii and S. cerevisiae treatments impacted tyrosine metabolism. Treatment with S. cerevisiae resulted in down-accumulation of metabolites related to glycine, serine, and threonine metabolism. This study suggests that adding fungi to the larval diet significantly affects black soldier fly larval metabolomics. Further research is needed to understand how individual amino acids and their metabolites contributed by fungi affect black soldier fly larval physiology, growth, and development, to elucidate the interaction between fungal nutrients and black soldier fly physiology.

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli.

5-Methyltetrahydrofolate (5-MTHF) is the sole active form of folate functioning in the human body and is widely used as a nutraceutical. Unlike the pollution from chemical synthesis, microbial synthesis enables green production of 5-MTHF. In this study, Escherichia coli BL21 (DE3) was selected as the host. Initially, by deleting 6-phosphofructokinase 1 and overexpressing glucose-6-phosphate 1-dehydrogenase and 6-phosphogluconate dehydrogenase, the glycolysis pathway flux decreased, while the pentose phosphate pathway flux enhanced. The ratios of NADH/NAD+ and NADPH/NADP+ increased, indicating elevated NAD(P)H supply. This led to more folate being reduced and the successful accumulation of 5-MTHF to 44.57 µg/L. Subsequently, formate dehydrogenases from Candida boidinii and Candida dubliniensis were expressed, which were capable of catalyzing the reaction of sodium formate oxidation for NAD(P)H regeneration. This further increased the NAD(P)H supply, leading to a rise in 5-MTHF production to 247.36 µg/L. Moreover, to maintain the balance between NADH and NADPH, pntAB and sthA, encoding transhydrogenase, were overexpressed. Finally, by overexpressing six key enzymes in the folate to 5-MTHF pathway and employing fed-batch cultivation in a 3 L fermenter, strain Z13 attained a peak 5-MTHF titer of 3009.03 µg/L, the highest level reported in E. coli so far. This research is a significant step toward industrial-scale microbial 5-MTHF production.

Analysis of ester-producing performance in high-yield ethyl hexanoate yeast and the effect on metabolites in bio- enhanced Daqu, the starter for Baijiu and other traditional fermented foods.

AIMS: Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied. METHODS AND RESULTS: Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56 mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds. CONCLUSION: In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Enhanced triolein and ethyl ferulate interesterification performance by CRL-AuNPs.

This study established a Candida rugosa lipase (CRL) system to catalyze triolein and ethyl ferulate interesterification. The products were identified, and the binding mode between the substrates and CRL was predicted through molecular docking. Three methods for preparing CRL-AuNPs were proposed and characterized. It was found that the addition of 40 mL of 15 nm gold nanoparticles increased the CRL activity from 3.05 U/mg to 4.75 U/mg, but the hybridization efficiency was only 32.7 %. By using 4 mL of 0.1 mg/mL chloroauric acid, the hybridization efficiency was improved to 50.7 %, but the enzyme activity was sharply decreased. However, when the molar ratio of Mb to HAuCl4 was 0.2, the hybridization efficiency increased to 71.8 %, and the CRL activity was also enhanced to 5.98 U/mg. Under optimal conditions, the enzyme activity of CRL-AuNPs③ was maintained at 95 % after 6 repetitions and 85.6 % after 30 days at room temperature.

Divergent mitochondrial responses and metabolic signal pathways secure the azole resistance in Crabtree-positive and negative Candida species.

Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat to human health. Several reports have shown that mitochondria are associated with the virulence of pathogenic fungi. However, there are few studies on the mechanisms of mitochondria-mediated azoles resistance. Here, we first performed mitochondrial proteomic analysis on multiple Candida species (Candida albicans, Nakaseomyces glabrata, Pichia kudriavzevii, and Candida auris) and analyzed the differentially expressed mitochondrial proteins (DEMPs) between azole-sensitive and azole-resistant Candida species. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology analysis, and protein-protein interaction network analysis of DEMPs. Our results showed that a total of 417, 165, and 25 DEMPs were identified in resistant C. albicans, N. glabrata, and C. auris, respectively. These DEMPs were enriched in ribosomal biogenesis at cytosol and mitochondria, tricarboxylic acid cycle, glycolysis, transporters, ergosterol, and cell wall mannan biosynthesis. The high activations of these cellular activities, found in C. albicans and C. auris (at low scale), were mostly opposite to those observed in two fermenter species-N. glabrata and P. kudriavzevii. Several transcription factors including Rtg3 were highly produced in resistant C. albicans that experienced a complex I activation of mitochondrial electron transport chain (ETC). The reduction of mitochondrial-related activities and complex IV/V of ETC in N. glabrata and P. kudriavzevii was companying with the reduced proteins of Tor1, Hog1, and Snf1/Snf4.IMPORTANCECandida spp. are common organisms that cause a variety of invasive diseases. However, Candida spp. are resistant to azoles, which hinders antifungal therapy. Exploring the drug-resistance mechanism of pathogenic Candida spp. will help improve the prevention and control strategy and discover new targets. Mitochondria, as an important organelle in eukaryotic cells, are closely related to a variety of cellular activities. However, the role of mitochondrial proteins in mediating azole resistance in Candida spp. has not been elucidated. Here, we analyzed the mitochondrial proteins and signaling pathways that mediate azole resistance in Candida spp. to provide ideas and references for solving the problem of azole resistance. Our work may offer new insights into the connection between mitochondria and azoles resistance in pathogenic fungi and highlight the potential clinical value of mitochondrial proteins in the treatment of invasive fungal infections.

Improved lipase performance by covalent immobilization of Candida antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support.

Development of immobilized lipase with excellent catalytic performance and low cost is the major challenge for large-scale industrial applications. In this study, green renewable microcrystalline cellulose (MCC) that was hydrophobically modified with D-alanine (Ala) or L-lysine (Lys) was used for immobilizing Candida antarctica lipase B (CALB). The improved catalytic properties were investigated by experimental and computational methods. CALB immobilized on MCC-Ala with higher hydrophobicity showed better catalytic activity than CALB@MCC-Lys because the increased flexibility of the lid region of CALB@MCC-Ala favored the formation of open conformation. Additionally, the low root mean square deviation and the high ß-sheet and α-helix contents of CALB@MCC-Ala indicated that the structure became more stable, leading to a significantly enhanced stability (54.80% and 90.90% relative activity at 70 °C and pH 9.0, respectively) and good reusability (48.92% activity after 5 cycles). This study provides a promising avenue to develop immobilized lipase with high catalytic properties for industry applications.

Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans.

Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal ammonia-generating component. We show that alkalization is dependent on the SPS-sensor-regulated transcription factor STP2 and the proline-responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2-/- strain. Alkalization is dependent on mitochondrial activity and in wild-type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co-culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal-induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal-associated processes are more critical in promoting dysbiosis and virulent fungal growth.

Glycerol Production from Undetoxified Lignocellulose Hydrolysate by a Multiresistant Engineered Candida glycerinogenes.

Glycerol is an important platform compound with multidisciplinary applications, and glycerol production using low-cost sugar cane bagasse hydrolysate is promising. Candida glycerinogenes, an industrial yeast strain known for its high glycerol production capability, has been found to thrive in bagasse hydrolysate obtained through a simple treatment without detoxification. The engineered C. glycerinogenes exhibited significant resistance to furfural, acetic acid, and 3,4-dimethylbenzaldehyde within undetoxified hydrolysates. To further enhance glycerol production, genetic modifications were made to Candida glycerinogenes to enhance the utilization of xylose. Fermentation of undetoxified bagasse hydrolysate by CgS45 resulted in a glycerol titer of 40.3 g/L and a yield of 40.4%. This process required only 1 kg of bagasse to produce 93.5 g of glycerol. This is the first report of glycerol production using lignocellulose, which presents a new way for environmentally friendly industrial production of glycerol.

Role of the extracellular matrix in Candida biofilm antifungal resistance.

Clinical infection due to Candida species frequently involve growth in biofilm communities. Recalcitrance despite antifungal therapy leads to disease persistence associated with high morbidity and mortality. Candida possesses several tools allowing evasion of antifungal effects. Among these, protection of biofilm cells via encasement by the extracellular matrix is responsible for a majority drug resistance phenotype. The Candida matrix composition is complex and includes a mannan-glucan complex linked to antifungal drug sequestration. This mechanism of resistance is conserved across the Candida genus and impacts each of the available antifungal drug classes. The exosome pathway is responsible for delivery and assembly of much of the Candida extracellular matrix as functional vesicle protein and polysaccharide cargo. Investigations demonstrate the vesicle matrix delivery pathway is a useful fungal biofilm drug target. Further elucidation of the vesicle pathway, as well as understanding the roles of biofilm driven cargo may provide additional targets to aid the diagnosis, prevention, and treatment of Candida biofilms.

The Adr1 transcription factor directs regulation of the ergosterol pathway and azole resistance in Candida albicans.

IMPORTANCE: Research often relies on well-studied orthologs within related species, with researchers using a well-studied gene or protein to allow prediction of the function of the ortholog. In the opportunistic pathogen Candida albicans, orthologs are usually compared with Saccharomyces cerevisiae, and this approach has been very fruitful. Many transcription factors (TFs) do similar jobs in the two species, but many do not, and typically changes in function are driven not by modifications in the structures of the TFs themselves but in the connections between the transcription factors and their regulated genes. This strategy of changing TF function has been termed transcription factor rewiring. In this study, we specifically looked for rewired transcription factors, or Candida-specific TFs, that might play a role in drug resistance. We investigated 30 transcription factors that were potentially rewired or were specific to the Candida clade. We found that the Adr1 transcription factor conferred resistance to drugs like fluconazole, amphotericin B, and terbinafine when activated. Adr1 is known for fatty acid and glycerol utilization in Saccharomyces, but our study reveals that it has been rewired and is connected to ergosterol biosynthesis in Candida albicans.

Untargeted metabolomics and phenotype data indicate the therapeutic and prophylactic potential of Lysimachia candida Lindl. towards high-fat high-fructose-induced metabolic syndrome in rats.

The present study evaluated the therapeutic potential of the medicinal plant Lysimachia candida Lindl. against metabolic syndrome in male SD rats fed with a high-fat high-fructose (HFHF) diet. Methanolic extract of Lysimachia candida Lindl. (250 mg kg-1 body weight p.o.) was administrated to the HFHF-fed rats daily for 20 weeks. Blood samples were collected, and blood glucose levels and relevant biochemical parameters were analysed and used for the assessment of metabolic disease phenotypes. In this study, Lysimachia candida decreased HFHF diet-induced phenotypes of metabolic syndrome, i.e., obesity, blood glucose level, hepatic triglycerides, free fatty acids, and insulin resistance. Liquid chromatography-mass spectrometry-based metabolomics was done to study the dynamics of metabolic changes in the serum during disease progression in the presence and absence of the treatment. Furthermore, multivariate data analysis approaches have been employed to identify metabolites responsible for disease progression. Lysimachia candida Lindl. plant extract restored the metabolites that are involved in the biosynthesis and degradation of amino acids, fatty acid metabolism and vitamin metabolism. Interestingly, the results depicted that the treatment with the plant extract restored the levels of acetylated amino acids and their derivatives, which are involved in the regulation of beta cell function, glucose homeostasis, insulin secretion, and metabolic syndrome phenotypes. Furthermore, we observed restoration in the levels of indole derivatives and N-acetylgalactosamine with the treatment, which indicates a cross-talk between the gut microbiome and the metabolic syndrome. Therefore, the present study revealed the potential mechanism of Lysimachia candida Lindl. extract to prevent metabolic syndrome in rats.

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.

DVL, lectin from Dioclea violacea seeds, has multiples mechanisms of action against Candida spp via carbohydrate recognition domain.

Lectins are proteins of non-immunological origin with the ability to bind to carbohydrates reversibly. They emerge as an alternative to conventional antifungals, given the ability to interact with carbohydrates in the fungal cell wall inhibiting fungal growth. The lectin from D. violacea (DVL) already has its activity described as anti-candida in some species. Here, we observed the anti-candida effect of DVL on C. albicans, C. krusei and C. parapsilosis and its multiple mechanisms of action toward the yeasts. Additionally, it was observed that DVL induces membrane and cell wall damage and ROS overproduction. DVL was also able to cause an imbalance in the redox system of the cells, interact with ergosterol, inhibit ergosterol biosynthesis, and induce cytochrome c release from the mitochondrial membrane. These results endorse the potential application of DVL in developing a new antifungal drug to fight back against fungal resistance.

Study on the synthesis of pine sterol esters in solvent-free systems catalyzed by Candida rugosa lipase immobilized on hydrophobic macroporous resin.

BACKGROUND: Pine sterol ester is a type of novel food source nutrient with great advantages in lowering blood cholesterol levels, inhibiting tumors, preventing prostate enlargement, and regulating immunity. Macroporous resins with large specific surface area, stable structures, and various functional groups (epoxy, amino, and octadecyl groups) have been selected for immobilization of Candida rugosa lipase (CRL) to improve its stability and efficiency in the synthesis of pine sterol esters. A solvent-free strategy using oleic acid (substrate) as an esterification reaction medium is an important alternative for avoiding the use of organic solvents. RESULTS: The immobilization conditions of CRL immobilized on several types of commercial macroporous resins were optimized. Fortunately, by adsorption (hydrophobic interaction), a high immobilization efficiency of CRL was obtained using macroporous resins with hydrophobic octadecyl groups with an immobilization efficiency of 86.5%, enzyme loading of 138.5 mg g-1 and enzyme activity of 34.7 U g-1 . The results showed that a 95.1% yield could be obtained with a molar ratio of oleic acid to pine sterol of 5:1, an enzyme amount of 6.0 U g-1 (relative to pine sterol mass) at 50 °C for 48 h. CONCLUSION: The hydrophobic macroporous resin (ECR8806M) with a large specific surface area and abundant functional groups was used to achieve efficient immobilization of CRL. CRL@ECR8806M is an efficient catalyst for the synthesis of phytosterol esters and has the potential for further large-scale applications. Therefore, this simple, green, and low-cost strategy for lipase immobilization provides new possibilities for the high-efficiency production of pine sterol esters and other food source nutrients. © 2023 Society of Chemical Industry.

Candida parapsilosis isolates carrying mutations outside FKS1 hotspot regions confer high echinocandin tolerance and facilitate the development of echinocandin resistance.

Candida parapsilosis is a significant cause of candidemia worldwide. Echinocandin-resistant (ECR) and echinocandin-tolerant (ECT) C. parapsilosis isolates have been reported in various countries but are rare. Resistance and tolerance are predominantly caused by mutations related to the hotspot (HS) regions of the FKS1 gene. A relatively high proportion of clinical C. parapsilosis isolates carrying mutations outside the HS regions has been noted in some studies, but an association with echinocandin (EC) resistance or tolerance was not explored. Herein, CRISPR-Cas9 was used and the association between amino acid substitution in FKS1 outside HS 1/2 (V595I, S745L, M1328I, F1386S, and A1422G) with EC susceptibility profile was delineated. None of the mutations conferred EC resistance, but they resulted in a significantly higher level of EC tolerance than the parental isolate, ATCC 22019. When incubated on agar plates containing ECs, specifically caspofungin and micafungin, ECR colonies were exclusively observed among ECT isolates, particularly mutants carrying V595I, S745L, and F1386S. Additionally, mutants had significantly better growth rates in yeast extract peptone dextrose (YPD) and YPD containing agents inducing membrane and oxidative stresses. The mutants had a trivial fitness cost in the Galleria mellonella model relative to ATCC 22019. Collectively, this study supports epidemiological studies to catalog mutations occurring outside the HS regions of FKS1, even if they do not confer EC resistance. These mutations are important as they potentially confer a higher level of EC tolerance and a higher propensity to develop EC resistance, therefore unveiling a novel mechanism of EC tolerance in C. parapsilosis. The identification of EC tolerance in C. parapsilosis may have direct clinical benefit in patient management.

Rational genome and metabolic engineering of Candida viswanathii by split CRISPR to produce hundred grams of dodecanedioic acid.

Candida viswanathii is a promising cell factory for producing dodecanedioic acid (DDA) and other long chain dicarboxylic acids. However, metabolic engineering of C. viswanathii is difficult partly due to the lack of synthetic biology toolkits. Here we developed CRISPR-based approaches for rational genome and metabolic engineering of C. viswanathii. We first optimized the CRISPR system and protocol to promote the homozygous gene integration efficiency to >60%. We also designed a split CRISPR system for one-step integration of multiple genes into C. viswanathii. We uncovered that co-expression of CYP52A19, CPRb and FAO2 that catalyze different steps in the biotransformation enhances DDA production and abolishes accumulation of intermediates. We also unveiled that co-expression of additional enzyme POS5 further promotes DDA production and augments cell growth. We harnessed the split CRISPR system to co-integrate these 4 genes (13.6 kb) into C. viswanathii and generated a stable strain that doubles the DDA titer (224 g/L), molar conversion (83%) and productivity (1.87 g/L/h) when compared with the parent strain. This study altogether identifies appropriate enzymes/promoters to augment dodecane conversion to DDA and implicates the potential of split CRISPR system for metabolic engineering of C. viswanathii.

Characterization of selected species of Pichia and Candida for their growth capacity in apple and grape must and their biofilm parameters.

Pichia and Candida species include biofilm-forming yeasts able to spoil foods and beverages. Strains belonging to 10 Pichia and Candida species isolated from apples, grape musts, and wines were analysed. They were subjected to molecular typing and characterized for their ability to grow and ferment must for cider and wine production, and for their biofilm properties. All strains grew similarly in apple and grape must. Glucose-fermenting strains displayed differentiated fermentation performances. Great variation in SO2 and ethanol sensitivity was observed among the strains. Pichia manshurica strains showed high tolerance to both molecules. Eleven and five surface-spreading biofilm (MAT) phenotypes were identified in solid and liquid media, respectively. Strains produced biofilms with variable thicknesses and widths in culture tubes. Cell adherence and aqueous-hydrocarbon biphasic hydrophobicity assays were carried out. Some Pichia manshurica and P. membranifaciens strains exhibited a high capacity to form a thick biofilm and had high cell adherence and hydrophobicity values. These strains could be more likely to colonize the internal surfaces of tanks. This study evidenced that some Pichia and Candida strains can proliferate during apple and grape must fermentation and may be detrimental the beverage quality, due to their specific biofilm properties.

Identifying Potential Molecular Targets in Fungi Based on (Dis)Similarities in Binding Site Architecture with Proteins of the Human Pharmacolome.

Invasive fungal infections represent a public health problem that worsens over the years with the increasing resistance to current antimycotic agents. Therefore, there is a compelling medical need of widening the antifungal drug repertoire, following different methods such as drug repositioning, identification and validation of new molecular targets and developing new inhibitors against these targets. In this work we developed a structure-based strategy for drug repositioning and new drug design, which can be applied to infectious fungi and other pathogens. Instead of applying the commonly accepted off-target criterion to discard fungal proteins with close homologues in humans, the core of our approach consists in identifying fungal proteins with active sites that are structurally similar, but preferably not identical to binding sites of proteins from the so-called "human pharmacolome". Using structural information from thousands of human protein target-inhibitor complexes, we identified dozens of proteins in fungal species of the genera Histoplasma, Candida, Cryptococcus, Aspergillus and Fusarium, which might be exploited for drug repositioning and, more importantly, also for the design of new fungus-specific inhibitors. As a case study, we present the in vitro experiments performed with a set of selected inhibitors of the human mitogen-activated protein kinases 1/2 (MEK1/2), several of which showed a marked cytotoxic activity in different fungal species.

Ammonia nitrogen recovery from biogas slurry by SCP production using Candida utilis.

The safe and robust yeast Candida utilis was employed for nitrogen recovery as single cell protein from biogas slurry. The maximum biomass of 6.2 g/L with protein content of 53.5% was produced in batch cultivation with glucose as the carbon source, C/N ratio of 3:1, NH4+-N concentration of 3000 mg/L, initial pH of 8.0, and the addition of 0.35% (w/v) Na2HPO4. It was speculated that C. utilis can grow well with free ammonia below 197 mg/L. In fed-batch fermentation, a biomass of 14.8 g/L was obtained, and the maintenance of aerobic conditions was critical to improving the production of single cell protein. The sterilized and non-sterilized biogas slurry can be used as an effective pH regulator. The obtained single cell protein was a nutritious, safe, and reliable protein source. This study provides novel insights into nitrogen recovery via C. utilis as a single cell protein from biogas slurry.