烷化剂NTG诱变虾青素产生菌红法夫酵母的研究

虾青素是一种很有效的生物抗氧化剂和某些生物的天然着色剂,应用前景广阔。红法夫酵母是 生产虾青素的一个来源,优点颇多。天然菌株虾青素产量较少,缺少实用价值。实验采用烷化剂ＮＴＧ 诱变红法夫酵母,筛选出类胡萝卜素产量高的诱变株。用薄层层析对红法夫酵母产生的色素及其皂 化产物进行分析,并对各个成分的扫描光谱进行了比较。认为红法夫酵母产生的类胡萝卜素成分主 要是虾青素及虾青素二酯,还有一部分β－胡萝卜素。同时,还对虾青素产生的时相和ＢＨＴ对虾青素 光分解的保护作用进行了初步研究。     

Complementation analysis with new genetic markers in Phaffia rhodozyma

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade^- and met^- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.     

Metabolic flux analysis of the two astaxanthin-producing microorganisms Haematococcus pluvialis and Phaffia rhodozyma in the pure and mixed cultures

The two major astaxanthin-producing microorganisms Phaffia rhodozyma and Haematococcus pluvialis exhibited elevated astaxanthin yields under the mixed culture regime, and the changes in flux distribution were investigated by means of metabolic flux analysis (MFA). In the mixed culture of the two strains, the carbon flux towards astaxanthin formation in P. rhodozyma increased by 20%, which may be due to the enriched oxygen evolved through the photosynthesis of H. pluvialis. On the other hand, the uptake of pyruvate and CO(2) excreted by P. rhodozyma also facilitated astaxanthin synthesis in H. pluvialis, which reduced 33% of the carbon flux exported from Calvin cycle to the catabolic pathway, and in turn raised the carbon flux to glyceraldehyde-3-phosphate by 25%. As a result, the carbon flux diverted to astaxanthin synthesis increased 2.8-fold in comparison with that in the pure culture.     

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast Phaffia rhodozyma and its sexual form, Xanthophyllomyces dendrorhous

Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous) is a basidiomycetous yeast that has been found in tree exudates in the Northern Hemisphere at high altitudes and latitudes. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We isolated X. dendrorhous from the Southern Hemisphere (Patagonia, Argentina), where it was associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus trees. We compared internal transcribed spacer (ITS)-based phylogenies of P. rhodozyma and its tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae) and found them to be generally concordant, suggesting that different yeast lineages colonize different trees and providing an explanation for the phylogenetic distance observed between the type strains of P. rhodozyma and X. dendrorhous. We hypothesize that the association of Xanthophyllomyces with Cyttaria derives from a previous association of the yeast with Nothofagus, and the sister relationship between Nothofagaceae and Betulaceae plus Fagaceae correlates with the phylogeny of X. dendrorhous strains originating from these three plant families. The two most basal strains of X. dendrorhous are those isolated from Cornus, an ancestral genus in the phylogenetic analysis of the host trees. Thus, we question previous conclusions that P. rhodozyma and X. dendrorhous represent different species since the polymorphisms detected in the ITS and intergenic spacer sequences can be attributed to intraspecific variation associated with host specificity. Our study provides a deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny. Such knowledge is essential for the comprehension of many aspects of the biology of this organism and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.     

法夫酵母 JMU-MVP14菌体中类胡萝卜素成分分析

结合薄层色谱、柱色谱、以及高效液相色谱对虾青素高产菌株-法夫酵母JMU-MVP14中的类胡萝卜素成分进行初步研究。研究结果表明,硅胶柱层析和氧化镁柱层析相结合的方法对法夫酵母JMU-MVP14菌体中的类胡萝卜素成分有很好的分离效果。经过柱层析分离纯化后,各组分中类胡萝卜素的种类单一,有利于进一步通过各种波谱技术对其进行定性。此方法弥补了单纯依靠高效液相色谱（ODS 柱）对法夫酵母 JMU-MVP14菌体中类胡萝卜素分离效果不佳,可供选择的商业化类胡萝卜素标准品少,液相保留时间漂移等因素给法夫酵母JMU-MVP14菌体中类胡萝卜素定性带来的不足。     

Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma

O kagbue , R.N. & L ewis , M.J. 1985. Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma. Journal of Applied Bacteriology 59 , 243–255.
In mixed culture Bacillus circulans WL-12 hydrolysed cell walls of Phaffia rhodozyma and rendered astaxanthin extractable from the yeast. pH control was critical to survival and lytic activity of the bacillus; the optimum range was 6.2–6.8. The optimum range of temperature was 20–24C. Glucose (1–2%) was efficient in minimizing catabolite repression of the lytic enzyme complex of the bacillus. Slow-feeding of glucose improved ultimate yields of lytic enzyme but did not acclerate yeast cell wall modification. A relatively high inoculum level of B. circulans accelerated modification of P. rhodozyma in the mixed culture: when the bacterial inoculum was four times that of the yeast, over 80% of total astaxanthin was extractable in 48 h. High bacterial inoculum size also stimulated yeast autolysis and necessitated early harvest of the mixed culture. Results obtained in shake flasks were duplicated in 5-litre fermentors and suggest that the mixed culture has potential industrial value for producing a biomass containing biologically-available astaxanthin. Extractability of astaxanthin was also achieved when mixed culture filtrate was incubated with pure cultured Phaffia cells. When suitably fortified with nutrients, the filtrate also supported simultaneous yeast growth and modification of the yeast cell walls. A scheme incorporating mixed culture with B. circulans WL-12 and re-use of culture filtrate has been proposed for enzymatic processing of Phaffia rhodozyma for inclusion in animal diets.     

红发夫酵母积累虾青素的代谢调控机理研究进展

由红发夫酵母合成的虾青素是一种非常有商业价值的类胡萝卜素.综述了近年来国内外在红发夫酵母中虾青素的生物合成途径、合成代谢调控机理等领域的相关研究进展,并提出了国内应开展的相关创新性研究.     

红发夫酵母积累虾青素的代谢调控机理研究进展

由红发夫酵母合成的虾青素是一种非常有商业价值的类胡萝卜素。综述了近年来国内外在红发夫酵母中虾青素的生物合成途径、合成代谢调控机理等领域的相关研究进展, 并提出了国内应开展的相关创新性研究。     

Molecular diversity and intragenomic variability in the yeast genus Xanthophyllomyces: the origin of Phaffia rhodozyma?

The teleomorphic basidiomycetous yeast Xanthophyllomyces dendrorhous is important as a commercial source of astaxanthin, which is a component of feeds for mariculture. Phaffia rhodozyma is the anamorphic state of Xanthophyllomyces; however, there are conflicting reports in the literature concerning the presence of a sexual cycle in P. rhodozyma. The current study attempted to explain this enigma. Strains were obtained from the Phaff Yeast Culture Collection (University of California, Davis) and other sources in the northern hemisphere. Molecular sequences of three nuclear rDNA regions were examined: the internal transcribed spacers (ITS), intergenic spacer (IGS1) and the D1D2 region at the 5' end of the 26S gene. Different levels of genetic variability were observed in the three regions. The D1D2 differentiated major groups of strains, while an increased variability in the ITS suggested that the ITS region could be employed as an ecological marker. The greatest variability was in the IGS1 region, where strains can be defined by the presence and location of indels. Intragenomic sequence heterogeneity in the ITS and IGS1 regions led to the hypothesis that the type strain of P. rhodozyma (CBS 5905(T), UCD 67-210(T)) was derived as a mating-deficient basidiospore from the parent teleomorphic strain CBS 9090.     

Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

法夫红酵母高产虾青素菌株的研究概况

虾青素是一种天然类胡萝卜素,具有较强的抗氧化作用。法夫红酵母是一种能合成虾青素的真菌。本文主要概述法夫红酵母高产虾青素菌株的育种、高产株选择、培养条件和虾青素的提取检测方法     

STUDIES ON DIPLOID STRAINS OF DICTYOSTELIUM DISCOIDEUM

Ross, Ian K. (Yale U., New Haven, Conn.) Studies on diploid strains of Dictyostelium discoideum. Amer. Jour. Bot. 47 (1) : 54—59. Illus. 1960.–Three strains of Dictyostelium discoideum having the diploid number of chromosomes (14) at all stages of their life cycle were examined. No evidence of sexuality as shown by syngamy or meiosis was found in the diploid strains. Two of the diploid strains were unstable and reverted to the haploid type with 7 chromosomes. These haploid strains had a sexual phase in which syngamy and meiosis were observed. The nuclear behavior of both the diploid and haploid strains differed from that reported in previous papers.     

高温湿热酸法破壁提取法夫酵母胞内虾青素

法夫酵母是一种能积累虾青素的红酵母, 对其进行破壁是当前虾青素工业化提取生产的瓶颈工艺。研究在高温湿热条件下,低浓度盐酸对法夫酵母破壁而提取其胞内虾青素的工艺。探讨了不同破壁温度、盐酸浓度、液料比与破壁处理时间等因素对法夫酵母破壁后虾青素及类胡萝卜素提取率的影响, 确定了高温湿热酸法破壁提取虾青素的最佳条件为: 饱和蒸汽压力 0.1 MPa (121°C), 盐酸浓度0.5 mol/L, 液料比 (V/W)30 mL/g, 破壁时间2 min。在最佳条件下进行中试放大实验, 可得到虾青素与类胡萝卜素的提取率分别为: (84.8±3.2)%, (93.3±2)%。经优化后的新破壁工艺安全高效, 不会有毒性残留, 具有良好的工业应用前景。     

稀土元素对红酵母的生长及类胡萝卜素合成的影响

探索了稀土元素镧Ｌａ^３＋、铈Ｃｅ^３＋和钕Ｎｄ^３＋对红酵母ｐｈａｆｆｉａ　ｒｈｏｄｏｚｙｍａ的生长,类胡萝卜素合成及菌体氨基酸组成的影响。三种稀土元素在所测试的浓度范围内对红酵母细胞生物量仅有轻度的抑制作用,但对类胡萝卜素的合成有促进作用。当培养基中分别加入２０ｍｇ／Ｌ的ＬａＣｌ_３、５ｍｇ／Ｌ的ＣｅＣｌ_３,和５０ｍｇ／ＬＮｄＣｌ_３时,刺激类胡萝卜素合成作用最强,产     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods

The growth and carotenoid production of Phaffia rhodozyma in fed-batch cultures with different feeding methods and grown at specific growth rates similar to the batch culture was compared. With constant feeding, exponential feeding, DO-stat and pH-stat fed-batch cultures of Phaffia rhodozyma, the highest biomass (17.4 g/l) and lowest carotenoid content (307 g/g cell) of Phaffia rhodozyma was from the DO-stat fed-batch culture. The lowest biomass (14.7 g/l) and highest carotenoid content (412 g/g cell) was from the exponential, fed-batch culture.     

Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.     

Simple method for the isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma.

A method is described for the quantitative and, possibly, large-scale extraction of astaxanthin from the yeast Phaffia rhodozyma. The method utilizes extracellular enzymes produced by the bacterium Bacillus circulans WL-12, which partially digests the yeast cell wall and renders the carotenoid pigments extractable by acetone or ethanol. Complete recovery of astaxanthin from heat-killed P. rhodozyma cells was obtained after growing B. circulans WL-12 on these yeast cells for 26 h and then extracting the yeast-bacterium mixture with acetone. A bacteria-free lytic system, which gave quantitative extraction of astaxanthin from P. rhodozyma, was obtained by concentrating the culture broth from the growth of B. circulans WL-12 on P. rhodozyma cells. Hydrolytic enzyme activities detected in this concentrate included beta-(1 leads to 3)-glucanase, beta-(1 leads to 6)-glucanase, alpha-(1 leads to 3)-glucanase, xylanase, and chitinase. The lytic system was found to work most efficiently at pH 6.5 and with low concentrations of yeast.     

Totally chlorine-free bleaching of flax pulp

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression. Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release. In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro. Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin. The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S. cerevisiae.