Some aspects of the taxonomy, genetics, carotenogenesis and chemical composition of a red yeast Rhodotorula rubra TP1

Acheampong, Edward Asafo-Adjei (2000) Some aspects of the taxonomy, genetics, carotenogenesis and chemical composition of a red yeast Rhodotorula rubra TP1. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

A new strain of yeast was isolated from yogurt and tentatively identified as Rhodoturula ruhra TP1 It was reported that the yeast produced structures resembling ascospores and, hence, was the first report of a sexual stage in Rhodotorula rubra. Preliminary studies in fish feeding trials indicate that the yeast may be an attractive candidate for use in the aquaculture industry for coloring fish muscle. To exploit the potentials of the new yeast isolate, a comprehensive study was undertaken to: I) confirm the phylogenetic affinity of the new isolate using molecular, biochemical and physiological techniques; 2) identify and quantify the pigments produced by the yeast; 3) isolate mutants with increased pigment production; 4) examine the cell wall for industrially useful polysaccharides; 5) isolate and characterize the carotenogenic enzymes. Studies on the sexuality of the new isolate could not confirm the production of ascospores or any structures resembling spores. To determine the exact phylogenetic relationship of the new isolate, various biochemical and physiological tests were carried out. These tests included assimilation of various carbon and nitrogen sources, isozyme electrophoresis and analysis of cellular long-chain fatty acids. Based on the comparison of the electrophoretic mobilities (IJ) of 8 isozymes in the new isolate and 8 other yeast isolates (Rhodotontla nthra ATCC 9449, Saccharomyces cerevisiae, Phaffia rhodozyma, Rhodotontla glutinis, Rhodotontla graminis. Rhodotorula minuta, Rhodosporidium toruloides and Cryptoccocus macerans), it was concluded that the new isolate could not be distinguished from R. ruhra and should therefore be considered a variant strain. The study also suggested that cellulose acetate electrophoresis could be an invaluable taxonomic tool for the identification of isolates of yeast. Similarly, on the basis of the numerical analysis of the cellular long-chain fatty acid composition 9 isolates, assimilation patterns of various carbon sources, Diazonium blue (DBB), urease and nitrate tests, the new isolate should be confirmed as a variant strain of R. ruhra. To determine whether the phylogeny of the new isolate could be determined on the basis of other identification protocols aside from the biochemical and physiological tests, a ponion of the ribosomal DNA (rONA) and internal transcribed spacer was amplified by the polymerase chain reaction and then sequenced. Comparison ofthe DNA sequences ofthe small subunit ribosomal RNA (ISS rRNA) coding regions and the internal transcribed spacer (ITS) of the new isolate with those of other yeast isolates revealed that the new yeast isolate may be a variant strain of R. ruhra. The new isolate had 93 and lOO% sequence similarity with R. rubra ATCC 9449 tbr the ISS rRNA and ITS sequences, respectively. Furthermore, the evolutionary distances estimated from the 18S rRNA genes and the ITS sequences of the two organisms were 0.015 and 0.000, respectively. Using gas liquid chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy, it was determined that the cell wall polysaccharides of the new isolate consisted of mainly mannans with 13-( 1 ~3) and ~-(I ~4) mannopyranosyl units. The major monosaccharides of the cell wall were determined to be mannose (50.53%), glucose (25.53%), galactose (12.27%), fucose (8.6%) and rhamnose (3.2%). Characterization of pigments produced by the new isolate involved the use of column and thin layer chromatography, high performance liquid chromatography and light spectroscopy. The new isolate was found to produce P-carotene, torulene, torularhodin, phytoene and phytotluene with percentage compositions of 39.85, 30.65, 24.5, 2.2 and 3.1%, respectively. Studies on the genetics of the new isolate included mutagenesis with nitrosoguanadine. Several mutants with increased production of pigments were isolated. Some of these mutants produced the same types of pigments produced by the parental strain while others were found to be blocked at the hydrogenation step and, therefore, produced only ~- carotene. The preliminary studies on the isolation and characterization of the carotenogenic enzymes involved the solubilization and polyethylene glycol precipitation of a cell-free 40,000 x: g supernatant fraction that converted [14C]MVA to phytoene, P-carotene, torulene and torularhodin. The effects of three non-ionic detergents, Tweens 40, 60 and 80 over a 0.5 to 3 % (w/v) concentration range on enzyme activity and protein release were investigated. Enzymatic activity was retained with all three detergents, however, I% Tween 60 was found to be the least inhibitory. The efficacy of the new isolate to color the flesh of rainbow trout was demonstrated in a 16 week feeding triaL Even though a commercial canthaxanthin containing diet induced better pigmentation than the test yeast supplemented diet, the level of pigmentation obtained with the test yeast was comparable to the level reported tor rainbow trout as sufficient for adequate visual color impression. The highest growth rate was obtained with fish fed a diet containing no pigment (negative control group) and the lowest growth rate was observed in fish fed a diet supplemented with test yeast. While the proximate analysis of the flesh showed significant increases in the total protein content of fish in all groups at the end of the feeding trial, the levels were found to be lower than those reported for rainbow trout by other workers and were found to be associated with increased moisture content. Finally, it was observed that the test yeast fed fish had increased polyunsaturated fatty acids content whereas the fatty acid profile remaining relatively unchanged in all other groups.

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