Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH ^# ), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.     

Chemostat study of xylitol production by Candida guilliermondii

The mechanism of production of xylitol from xylose by Candida guilliermondii was studied using chemostat cultures and enzymatic assays. The maximum dilution rate in aerobic conditions was 0.34 1/h. No xylitol was produced. Under oxygen-limited conditions xylose uptake was impaired and glycerol accumulated but no xylitol was detected. Under transient oxygen limitation, caused by a gradual decrease in the agitation rate, onset of xylitol, acetate and residual xylose accumulation occurred simultaneously when q _O2 dropped below 25 mmol/C-mmol cell dry weight (CDW) per hour. Ethanol and glycerol started to accumulate when q _O2 dropped below 20 mmol/C-mmol CDW per hour. The highest in vitro enzyme activities were found at the lowest dilution rate studied (0.091/h) under aerobic conditions. The amount of active enzymes or cofactor availability did not limit the rate of xylose consumption. Our results confirm that a surplus of NADH during transient oxygen limitation inhibited the activity of xylitol dehydrogenase which resulted in xylitol accumulation. Phosphoglucoisomerase (E.C. 5.3.1.9.) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) activities suggest re-shuttling of the metabolites into the pentose phosphate pathway. Received: 7 March 2000 / Received revision: 9 June 2000 / Accepted: 18 June 2000     

Alcohol oxidation by Candida guilliermondii yeasts grown on hexadecanol

A number of enzyme systems involved in the first steps of hexadecane oxidation can be induced by hexadecanol, an intermediate product of hexadecane degradation. It has also been found that, in Candida guilliermondii cells and in their mitochondrial fraction, an oxidase system is induced when the cells are grown on hexadecanol. This system is similar to that in cells grown on hexadecane; it oxidises higher alcohols at a high rate and is not inhibited by the inhibitors of the man phosphorylating respiration chain. The membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase activities resistant to pyrazole, an inhibitor of cytosol ethanol dehydrogenase, are induced together with the oxidase activity when the cells are grown on hexadecanol as well as on hexadecane. The oxidation of higher alcohols by whole cells is entirely inhibited by azide although their oxidation by mitochondria is resistant to the action of azide; apparently, azide inhibits the transport of alcohols into the cell.     

Xylitol production from aspenwood hemicellulose hydrolysate by Candida guilliermondii

The production of xylitol by the yeast Candida guilliermondii was investigated in batch fermentations with aspenwood hemicellulose hydrolysate and compared with results obtained in semi-defined media with a mixture of glucose and xylose. The hemicellulose hydrolysate had to be supplemented by yeast extract and the maximum xylitol yield (0.8 g g^–1) and productivity (0.6 g l^–1 h^–1) were reached by controlling oxygen input.     

Anabolic and catabolic enzymes of urea metabolism in a carbohydrate-utilizing strain of Candida guilliermondii]

The yeast "H" of the genus Candida guilliermondii can grow on hydrocarbons as the only source for carbon. Urea can serve as a nitrogen source for this yeast which lacks detectable urease activity. During urea metabolism ammonia has never been accumulated in the culture medium. However, transferring the yeast from complete urea-medium into an urea containing phophate-buffer, the degradation of urea continues and ammonia is accumulated as well as CO2 evolved. In cell-free extracts of the yeast urea amidolyase activity was detected in the presence of ATP, biotin and specific cations. Obviously, the synthesis of urea amidolyase is induced by urea and arginine and repressed by the catabolite ammonia. Similarly the synthesis of arginase is regulated by arginine and ammonia. The analytical data of the arginase action differ significantly in relation to the carbon source of the culture medium. Both the level of arginase and ornithine carbamyl-transferase change in a characteristic way during the batch-culture. From the lower level of arginase in relation to ornithine carbamyltransferase it can be concluded that especially in alkane-metabolizing yeast the arginine catabolism is not very intensive.     

Enzymatic degradation of nitriles by a Candida guilliermondii UFMG-Y65

Candida guilliermondii UFMG-Y65, isolated from a gold mine, was able to utilize different nitriles and the corresponding amides as sole source of nitrogen, at concentrations up to 2 M. Resting cells cultivated on YCB-acetonitrile medium showed nitrile hydrolyzing enzyme activities against acrylonitrile and benzonitrile. These enzymes were inducible and intracellular; the optimum pH was 7.0-8.0, and the optimum temperature 25 degrees C-30 degrees C. Liquid chromatographic analysis indicated that C. guilliermondii UFMG-Y65 metabolized 12 mM benzonitrile to 11 mM benzoic acid and 10 mM acrylonitrile to 7.9 mM acrylic acid. The results suggest that C. guilliermondii UFMG-Y65 may be useful for the bioproduction of amides and acids, and for the bioremediation of environments contaminated with nitriles.     

季也蒙假丝酵母对采后桃果实软腐病的抑制效果

The biocontrol capability of Candida guilliermondii (Cast) Langeroret Guerra against Rhizopus stolonifer (Ehrenb.ex Fr.) Vuill. postharvest rot of peach fruits was studied in vitro and in vivo under different storage temperatures. C. guilliermondii at 5.0×108 CFU (colony-forming unit)/mL of washed cells provided complete control of 5×104 spores /mL of R. stolonifer during storage at 25 ℃ for 4 d, 15 ℃ for 7 d and 3℃ for 30 d. Temperature had no significant effect on the biocontrol efficacy. Antagonist population increased 45.6, 34.4 and 33.1 times per site at 25℃, 15℃ and 3℃ for 3, 3 and 7 d,respectively, then the population stabilized for the remaining storage period. Cell-free culture filtrate of C. guilliermondii was not effective in preventing decay and resulted in even greater lesion diameter than those of sterile distilled water at 3 ℃.These results showed that competition for nutrient, not antibiotic production, plays a major role in the biocontrol capability of C. guilliermondii against rhizopus rot of peach fruits. As the interval between wounding and inoculation with the pathogen increased from 0 to 72 h, susceptibility of wounds to decay by R. stolonifer decreased from 100% of 0 h to 5% of 4 h and 0% of 24 h, then increased to 10% of 48 h and 40% of 72 h.     

Production of L-arabitol from L-arabinose by Candida entomaea and Pichia guilliermondii

 Lignocellulosic biomass, particularly corn fiber, represents a renewable resource that is available in sufficient quantities from the corn wet milling industry to serve as a low cost feedstock for production of fuel alcohol and valuable coproducts. Several enzymatic and chemical processes have potential for the conversion of cellulose and hemicellulose to fermentable sugars. The hydrolyzates are generally rich in pentoses (D-xylose and L-arabinose) and D-glucose. Yeasts produce a variety of polyalcohols from pentose and hexose sugars. Many of these sugar alcohols have food applications as low-calorie bulking agents. During the screening of 49 yeast strains capable of growing on L-arabinose, we observed that two strains were superior secretors of L-arabitol as a major extracellular product of L-arabinose. Candida entomaea NRRL Y-7785 and Pichia guilliermondii NRRL Y-2075 produced L-arabitol (0.70 g/g) from L-arabinose (50 g/l) at 34°C and pH 5.0 and 4.0, respectively. Both yeasts produced ethanol (0.32–0.33 g/g) from D-glucose (50 g/l) and only xylitol (0.43–0.51 g/g) from D-xylose (50 g/l). Both strains preferentially utilized D-glucose>D-xylose>L-arabinose from mixed substrate (D-glucose, D-xylose and L-arabinose, 1:1:1, 50 g/l, total) and produced ethanol (0.36–0.38 g/g D-glucose), xylitol (0.02–0.08 g/g D-xylose) and L-arabitol (0.70–0.81 g/g L-arabinose). The yeasts co-utilized D-xylose (6.2–6.5 g/l) and L-arabinose (4.9–5.0 g/l) from corn fiber acid hydrolyzate simultaneously and produced xylitol (0.10 g/g D-xylose) and L-arabitol (0.53–0.54 g/g L-arabinose). Received: 24 April 1995/Received revision: 9 August 1995/Accepted: 7 September 1995     

Formation of xylitol in Candida guilliermondii 2581 culture

The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Biosynthesis of beta-phenethyl alcohol in Candida guilliermondii.

$\text{Candida guilliermondii}$ produced β-phenethyl alcohol and β-phenyllactic acid when grown in a synthetic medium containing L-phenylalanine as sole source of nitrogen. The cell-free preparations from these cells showed the following enzymes: phenylalanine aminotransferase, phenylpyruvate decarboxylase, phenylpyruvate reductase and phenylacetaldehyde reductase. The cell-free preparations of $\text{C. guilliermondii}$ grown in medium with ammonium sulfate, lacked these enzyme activities, indicating the inducible nature of these enzymes. The results indicate the role of β-phenylpyruvate as a key intermediate in the pathway of biosynthesis of β-phenethyl alcohol and β-phenyllactic acid from L-phenylalanine.     

Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii

A detoxification method using activated charcoal with concentrated rice straw hemicellulosic hydrolysate improved the conversion of xylose to xylitol by the yeast Candida guilliermondii by 22%. This was achieved when the hydrolysate:charcoal ratio was 40 g g^–1, resulting in removal of 27% of phenolic compounds. Under this condition, the xylitol yield factor (0.72 g g^–1) and volumetric productivity (0.61 g l^–1 h^–1) were close to those attained in a semi-defined medium simulating hydrolysate sugars.     

Biodegradation of pyrene by Candida sp. S1 under high salinity conditions

Polycyclic aromatic hydrocarbon is a toxic recalcitrant environmental pollutant and its removal from the environment is very essential. In this study, a novel S1 strain isolated from the tropical rain forest was identified as Candida species based on 18S rRNA. The pyrene biodegradation was performed by Candida sp. S1. Pyrene was 35% degraded in 15 days. The percentage of pyrene biodegradation increased up to 75% with 24 g L⁻¹ of sodium chloride and decreased along with increasing salinity. Under the acidic condition, the biodegradation was increased up to 60% at pH 5. It was also found that the increasing glucose concentration of more than 10 g L⁻¹ had no significant effect on pyrene biodegradation, while agitation proved to have greater influence. There was a positive relationship between biomass growth and biodegradation rate of pyrene. One pyrene metabolite was identified from the extract solution and analyzed by a thin-layer chromatography, UV–visible absorption and gas chromatography–mass spectrometry. The metabolite found in the pyrene degradation was benzoic acid. Suitable conditions must be found to promote a successful microbial augmentation in liquid culture.

     

Development of a URA5 integrative cassette for gene disruption in the Candida guilliermondii ATCC 6260 strain

We designed an efficient transformation system for Candida guilliermondii based on a ura5 ATCC 6260 derived recipient strain and a URA5 recyclable selection marker. This “URA5 blaster” disruption system represents a powerful tool to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.