Isolation and characterization of a Δ-9 fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the Δ-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned Δ-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the Δ-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.     

Melanization of flavonoids by fungal and bacterial laccases

Laccase activity in plants results in the formation of a number of brown pigments, often referred to as tannins. Laccase-dependent pigment production is also catalogued in numerous fungal and bacterial species. The laccase of the haploid yeast Cryptococcus neoformans forms melanin-like pigmentation outside the cell wall in the presence of exogenous substrates. While this process is a contributing factor to its virulence in humans, the evolutionary intent for the laccase function remains a mystery. We show here that C. neoformans and Bacillus subtilis have the ability to create melanin-like pigments from a variety of flavonoid molecules across a range of conformations, preferring those with 3',4'-dihydroxylations. Since flavonoids are ubiquitous plant molecules and often-considered antimicrobial agents, we postulate that they are the intended natural targets of laccase activity and result in the formation of a defensive melanin-like coat. These results suggests a new mechanism by which flavonoid-melanin formation may occur, using not only A- and C-ring linkages, but also monomer links through the B-ring of the flavonoid structure. We also show that resveratrol and other non- and mono-hydroxylated polyphenol substrates have the ability to restrict pigment formation and may be potent inhibitors of laccase activity.     

Environmental isolation of Cryptococcus neoformans from an endemic region of HIV-associated cryptococcosis in Thailand

We successfully isolated Cryptococcus neoformans from chicken faeces in suburban areas of Thailand. C. neoformans was isolated from 36/150 houses (24.0%) in the dry season and 6/150 (4.0%) in the rainy season. All environmental isolates were of serotype A. The high isolation rate of 24% from chicken faeces has never been reported previously. Our environmental study could probably explain the high incidence of cryptococcal meningitis in HIV patients in Thailand.     

Gas-flushed packaging contributes to calcium lactate crystals in Cheddar cheese

Gas-flushed packaging is commonly used for cheese shreds and cubes to prevent aggregation and loss of individual identity. Appearance of a white haze on cubed cheese is unappealing to consumers, who may refrain from buying, resulting in lost revenue to manufacturers. The objective of this study was to determine whether gas flushing of Cheddar cheese contributes to the occurrence of calcium lactate crystals (CLC). Cheddar cheese was manufactured using standard methods, with addition of starter culture, annatto, and chymosin. Two different cheese milk compositions were used: standard (lactose:protein = 1.47, protein:fat = 0.90, lactose = 4.8%) and ultrafiltered (UF; lactose:protein = 1.23, protein:fat = 0.84, lactose = 4.8%), with or without adjunct Lactobacillus curvatus. Curds were milled when whey reached 0.45% titratable acidity, and pressed for 16 h. After aging at 7.2°C for 6 mo, cheeses were cubed (1 × 1 × 4 cm) and either vacuum-packaged or gas-flushed with carbon dioxide, nitrogen, or a 50:50 mixture of carbon dioxide and nitrogen, then aged for an additional 3 mo. Heavy crystals were observed on surfaces of all cubed cheeses that were gas-flushed, but not on cheeses that were vacuum-packaged. Cheeses without Lb. curvatus exhibited l(+)-CLC on surfaces, whereas cheeses with Lb. curvatus exhibited racemic mixtures of l(+)/d(−)-CLC throughout the cheese matrices. The results show that gas flushing (regardless of gas composition), milk composition, and presence of nonstarter lactic acid bacteria, can contribute to the development of CLC on cheese surfaces. These findings stress the importance of packaging to cheese quality.     

Heavy metal tolerance in the psychrotolerant Cryptococcus sp. isolated from deep‐sea sediments of the Central Indian Basin

A deep‐sea isolate of the psychrotolerant yeast Cryptococcus sp. (NIOCC#PY13) obtained from polymetallic nodule‐bearing sediments of the Central Indian Basin was examined for its capacity to grow in the presence of various concentrations of the heavy metal salts i.e., ZnSO₄, CuSO₄, Pb(CH₃COO)₂ and CdCl₂. It demonstrated considerable growth in the presence of 100 mg/l concentrations of the above‐mentioned four heavy metal salts both at 30°C and 15°C. This strain tolerated comparatively higher levels of these four metal salts than other deep‐sea and terrestrial yeast isolates belonging to Cryptococcus, Rhodotorula, Rhodosporidium and Sporidiobolus spp. Optimum pH for growth of this isolate was in the range of 6–8 in the presence of heavy metal salts at these two temperatures. Scanning electron microscopic (SEM) studies exhibited altered cell surface morphology of the cells under the influence of heavy metals compared to that with control. The adsorption of heavy metals to the cells was demonstrated by FTIR and EDAX analysis. As evidenced by atomic absorption spectrophotometric (AAS) analysis, about 30–90% of the heavy metals were removed from the culture supernatant after 4 days of growth at 30°C. This deep‐sea yeast isolate appears to be a potential candidate for bioremediation of metal‐contaminated sites. Moreover, its metal tolerance properties provide a significant insight into its ecological role and adaptations to growth in such extreme conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of Acetic Acid on Growth and Lipid Production by Cryptococcus albidus

The cell growth and lipid accumulation process of Cryptococcus albidus were investigated using acetic acid as the sole carbon source at different concentrations. C. albidus showed high tolerance to acetic acid at a high concentration of 30 g L^?1. The highest lipid content (32.69 ± 0.50 %) and lipid yield (0.96 ± 0.05 g L^?1) were both obtained in the medium with an initial acetic acid concentration of 30 g L^?1 on day five. Interestingly, the maximum lipid content and lipid yield was obtained on a different day in a medium with different acetic acid concentration. The fatty acid composition of the lipids accumulated by C. albidus was 16–23 % palmitic acid (C16:0), 3–5 % linolenic acid (C18:3), 42–51 % linoleic acid (C18:2) and 23–27 % oleic acid (C18:1), which was similar to that of soybean oil; thus, this microbial oil has great potential value as a renewable biodiesel feedstock. This work also provides valuable information for further research to use cheap substrates containing a high concentration of acetic acid (such as lignocellulosic hydrolysates), which is an economical and environmentally friendly form of microbial oil production.     

Utilisation of a yeast pectinase in olive oil extraction and red wine making processes

The possibility of using an endo-polygalacturonase produced by the yeast Cryptococcus albidus var albidus (yeast pectinase) in the mechanical olive oil extraction process and in the production of red wine was investigated. Compared with the control and olive pastes treated with a commercial enzyme preparation, an increase in oil yield was achieved by treatment with the yeast pectic preparation. Also, the finished oil quality (turbidity, oxidation induction time, chlorophyll, and the content of polyphenols and aromatic compounds) was generally better. Treatment of red musts with yeast pectinase resulted in an improved aromatic profile in the wines, even in the absence of an increase in colour intensity. Moreover, the use of the yeast pectinase did not cause any particular increase in the methanol content of the finished wines.     

Purification, characterization and functional cloning of inositol oxygenase from Cryptococcus

The enzyme inositol oxygenase (myo-inositol : oxygen oxidoreductase; E.C. 1.13.99.1) is a monooxygenase that converts inositol into glucuronic acid in the presence of molecular oxygen. This enzyme is integrated into a pathway leading to either degradation and energy production or the biosynthesis of precursors for polysaccharides. The enzyme was purified from the yeast Cryptococcus lactativorus by a five-step chromatography procedure. The purified enzyme shows a molecular mass of 37 kDa on SDS-PAGE, similar to the estimation of the size of the native enzyme determined by size exclusion chromatography. Peptides of the inositol oxygenase protein derived from a tryptic digest were sequenced de novo by nanoelectrospray tandem mass spectrometry. Using degenerate oligonucleotides, the corresponding gene was cloned from first strand cDNA. The open reading frame encodes a 315 amino acid polypeptide with a predicted molecular mass of 36.9 kDa. Inositol oxygenase is a single copy gene in C. lactativorus. It has close homologues in other fungi such as Cryptococcus neoformans and Neurospora crassa. Biochemical characterization of the enzyme showed a pH optimum of 6-6.5 and a temperature optimum of 30 degrees C. Myo-inositol is the only accepted substrate with a Km of ca. 5 mM. The enzyme contains a Fe-centre but the enzyme activity is resistant to KCN.