Selection of Astaxanthin-Overproducing Mutants of Phaffia rhodozyma with β-Ionone

β-Ionone, an end ring analog of β-carotene, inhibits astaxanthin production in the red yeast Phaffia rhodozyma. Astaxanthin-overproducing mutants of this yeast are easily spotted on β-ionone-containing yeast malt agar plates. β-Ionone appears to block astaxanthin synthesis at the β-carotene level.     

Isolation of Phaffia rhodozyma Mutants with Increased Astaxanthin Content

Plating of the astaxanthin-producing yeast Phaffia rhodozyma onto yeast-malt agar containing 50 μM antimycin A gave rise to colonies of unusual morphology, characterized by a nonpigmented lower smooth surface that developed highly pigmented vertical papillae after 1 to 2 months. Isolation and purification of the pigmented papillae, followed by testing for pigment production in shake flasks, demonstrated that several antimycin isolates were increased two- to fivefold in astaxanthin content compared with the parental natural isolate (UCD-FST 67-385). One of the antimycin strains (ant-1) and a nitrosoguanidine derivative of ant-1 (ant-1-4) produced considerably more astaxanthin than the parent (ant-1 had 800 to 900 μg/g; ant-1-4 had 900 to 1,300 μg/g; and 67-385 had 300 to 450 μg/g). The mutant strains were compared physiologically with the parent. The antimycin mutants grew slower on ammonia, glutamate, or glutamine as nitrogen sources compared with the natural isolate and also had lower cell yields on several carbon sources. Although isolated on antimycin plates, they were found to be more susceptible to antimycin A, apparently owing to the spatial separation of the papillae from the agar. They were also more susceptible than the parent to the respiratory inhibitor thenoyltrifluoroacetone and were slightly more susceptible to cyanide, but did not differ from the natural isolate in susceptibility to azide. The antimycin-derived strains were also killed faster than the parent by hydrogen peroxide. The carotenoid compositions of the parent and the antimycin-derived strains were similar to those previously determined in the type strain (UCD-FST 67-210) except that two carotenoids not previously found in the type strain were present in increased quantities in the antimycin mutants and phoenicoxanthin was a minor component. The chemical properties of the unknown carotenoids suggested that the strains isolated on antimycin agar tended to oxygenate and desaturate carotene precursors to a greater extent than the parent. The physiology of the antimycin isolates and the known specificity of antimycin for cytochrome b in the respiratory chain suggests that alteration of cytochrome b or cytochrome P-450 components involved in oxygenation and desaturation of carotenes in mitochondria are affected, which results in increased astaxanthin production. These astaxanthin-overproducing mutants and more highly pigmented derivative strains could be useful in providing a natural source of astaxanthin for the pen-reared-salmon industry or for other farmed animals that contain astaxanthin as their principal carotenoid.     

Selection and evaluation of astaxanthin-overproducing mutants of Phaffia rhodozyma

Mutagenesis of Phaffia rhodozyma with NTG yielded a mutant with an astaxanthin content of 1688 g (g dry biomass)^-1, a cell yield coefficient of 0.47 on glucose and a maximum specific growth rate of 0.12 h^-1. Re-mutation of the mutant decreased the cell yield and maximum specific growth rate but increased the astaxanthin content. The use of mannitol or succinate as carbon sources enhanced pigmentation, yielding astaxanthin contents of 1973 g g^-1 and 1926 g g^-1, respectively. The use of valine as sole nitrogen source also increased astaxanthin production, but severely decreased the maximum specific growth rate and cell yield coefficient. The optimum pH for growth of P. rhodozyma was between pH 4.5 and 5.5, whereas the astaxanthin content remained constant above pH 3.     

Phaffia rhodozyma is polyploid

The ploidy of the red yeast Phaffia rhodozyma was evaluated using flow cytometric analyses of propidium iodide- stained cells and mutagenic inactivation kinetics. Our findings suggest that Phaffia rhodozyma is not haploid. Auxotrophic strains were generated at a high frequency following treatment of mutagenized cells with a combination of benomyl and ethyl acetate. Studies of an auxotrophic mutant using flow cytometry and UV inactivation indicated possible chromosome loss to an aneuploid state. Received 21 June 1998/ Accepted in revised form 25 September 1998     

虾青素高产突变株的选育

阐述了法夫酵母中从葡萄糖到虾青素的整条生物合成途径。用亚硝基胍（NTG）作为诱变剂,2－脱氧－D－葡萄糖（2－DG）作为“筛子”,通过三轮诱变和一次自然分离,得到高产虾青素的菌株4－26,其类胡萝卜素总量提高了371％,为7．07μg／ml,虾青素的比率从75％提高到79％,并且其稳定性良好。对2－DG和二苯胺作为筛子的作用机理也作了初步探讨。     

红发夫酵母的生物学特性

红发夫酵母以单细胞为主,有时能形成假菌丝。菌落因菌体产生虾青素等类胡萝卜素而呈红色,类胡萝卜素均匀地分布于细胞脂质中。红发夫酵母为专性好氧菌,细胞产生类胡萝卜素需要大量氧气。葡萄糖和蔗糖为最佳碳源,酵母膏是最佳氮源。红发夫酵母具克拉布特里效应（Crabtree)。红发夫酵母生长温度范围为4－27℃,属于兼性嗜冷的低温型微生物。最适生长pH为6．0,色素形成的最适pH为5．0。     

Transformation of Phaffia rhodozyma by electroporation

Summary The transformation of Phaffia rhodozyma by electroporation was better than the one by the lithium chloride (LiCl) or spheroplast transformation methods. The influence of several parameters on transformation efficiency was studied. Electroporation conditions of 1200 Volts, 50 Farads and 2000 Ohms were proved successful. A maximum of 1.5 × 10³ transformants per 100 ng of DNA was reached.     

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.     

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.     

Transformation of the astaxanthin-producing yeast Phaffia rhodozyma

Summary This paper describes the genetic transformation of the astaxanthin-producing yeast Phaffia rhodozyma with the cloning vector pGH-1. The plasmid replicates autonomously in this yeast, and the selection of transformants was possible by using both, the URA3 marker from Saccharomyces cerevisiae, and the kanamycin resistance (Km^R) determinant from the bacterial transposon Tn903.     

Isolation of astaxanthin-overproducing mutants of Phaffia rhodozyma

We isolated mutants of Phaffia rhodozyma strain NRRL Y-17269 that overproduced astaxanthin when grown on corn-based fuel ethanol stillage (thin stillage, TS, or fuel ethanol byproducts). Ten ml cultures of mutant strain JB2 produced 1.54 ± 0.21 mg carotenoid/mg dry weight when grown on 70% thin stillage at pH 5.2, compared with 0.38 ± 0.04 g/mg produced by the parental strain. Furthermore, JB2 produced similar astaxanthin concentrations when grown in either thin stillage or yeast malt broth. By comparison, previously described astaxanthin overproducing strain NRRL Y-17811 yielded 1.08 ± 0.07 g/mg in yeast malt broth but only 0.67 ± 0.03 g/mg in thin stillage. Five liter fermentation experiments using JB2 grown on 70% thin stillage at pH 5.2 yielded 2.01 ± 0.17 g/mg astaxanthin. Thus, JB2 is uniquely suited for astaxanthin production from low cost thin stillage.     

红色酵母原生质体形成和再生条件的研究

了不同渗透压稳定剂、酶浓度、酶解时间,温度等对红色酵母原生质体的形成与再生的影响。实验结果表明：使用对数生长期早期的细胞,蜗牛酶浓度１％,３０℃处理５０－６ｍｉｎ,山梨醇为渗透压稳定剂,有利于原生质体制备和再生。     

虾青素高产菌的选育

红发夫酵母（Phaffia rhodozyma)是微生物发酵法生产虾青素的优良菌株,作者采用Cs^137-γ射线重复辐照,并进行亚硝基胍（NTG）诱变处理,选育得到一株高产虾青素的红发夫酵母YB-20-28突变株,该菌株摇瓶发酵的生物量达老人家酵母YB-20-28突变株,该菌株摇瓶发酵的生物量达36.3g/L,总色素含量为1216.0μg/g,较出发菌株提高308%,虾青素产量达30.9μg/mL,是一株颇具开发潜力的虾青素高产菌株。     

Ethanol increases carotenoid production in Phaffia rhodozyma

Addition of ethanol (0.2%) to cultures of the yeast Phaffia rhodozyma increased the specific rate of carotenoid production [(carotenoid)(cell mass)⁻¹(time)⁻¹]. The incremental increase in carotenoid synthesis with ethanol was highest in carotenoid-hyperproducing strains. Ethanol increased carotenoid production when it was added at various points during the lag and active growth phases. Ethanol increased alcohol dehydrogenase and hydroxy-methyl-glutaryl-CoA (HMG-CoA) reductase activities. Our results indicate that increased carotenoid production by ethanol is associated with induction of HMG-CoA reductase and possibly activation of oxidative metabolism. Received 24 December 1996/ Accepted in revised form 27 May 1997     

利用亚硝酸钠选育法夫酵母虾青素高产菌株

以亚硝酸钠作为筛选剂选择性分离法夫酵母虾青素高产菌株。实验研究表明,在亚硝酸钠存在的情况下,法夫酵母的生长和虾青素合成量均会减少;当亚硝酸钠浓度为5000μmol/L时,法夫酵母的致死率为100%。挑取200株经过甲基磺酸乙酯（EMS）诱变后的法夫酵母,以5000μmol/L的亚硝酸钠为筛选剂摇瓶发酵后测得虾青素体积产率为正突变的菌株有87株,正突变率为43.5%。挑取其中8株进行复筛,编号为N030的菌株比出发菌株的虾青素体积产率和细胞产率分别提高了39.3%和89.3%。结果说明,亚硝酸钠可作为法夫酵母虾青素高产菌株的筛选剂,用于提高菌种的筛选效率。     

虾青素摇瓶发酵条件的研究

通过对P .rhodozyma 4 - 2 6的虾青素摇瓶发酵条件包括接种比、通气量和温度等 ,以及培养基主要组分的优化 ,得到最优培养基及培养条件。在此条件下发酵 72h ,虾青素量可达 13 4 5μg/ml或 146 5μg/gCDW ,较优化前提高 1 4 8倍。     

红法夫酵母产特种胡萝卜素酮的研究

红法夫酵母(Phaffia rhodozyma)可产3-羟-3’,4-二脱氢-β,φ－胡萝卜素－4－酮(HDCO)．经多次诱变和HDCO生成条件研究．HDCO产率由原来的30μg,g(细胞干重)提高至510μg／g(细胞干重)．HDC0占P．Rhodozyma所产总类胡萝卜素含量由8%提高到45%。还对促进和抑制HDCO生成因子及可能的机理进行初步探讨。酵母产HDCO的研究在国内外尚属首次报道。     

Extraction and quantitation of astaxanthin from Phaffia rhodozyma

Summary The rapid, quantitative release of astaxanthin and other carotenoids from the yeast Phaffia rhodozyma is described. Hashed cells are ruptured with dimethylsulfoxide (DMSO) and carotenoids extracted into an organic solvent. Extraction and spectrophotometric quantitation of total carotenoids is rapid, reproducible and only small volumes (0.1–2 ml) of culture are required. HPLC analysis in normal phase silica gel column indicates that astaxanthin comprises 65–95% of the total pigmented carotenoids of P. rhodozyma.     

高产虾青素红法夫酵母的紫外线诱变

虾青素(3,3'-二羟基-β,β'-胡萝卜素-4,4'-二酮)是一种类胡萝卜、素类物质,具有巨大的开发价值。本文通过单一紫外线、紫外线-氯化锂复合处理,结合选择性平板的筛选作用,对菌株G26进行诱变育种。实验结果表明:紫外线照射3～9min,法夫酵母的死亡率在70%～95%之间;当死亡率为78.57%时,正变率达到最大值71.43%。经多次单独紫外线处理,以及紫外线-氯化锂复合诱变,获得稳定高产突变株G49,虾青素产量达到10 321μg/L,含量为713.8μg/g DCW,较出发菌株G26分别提高31.48%和20.47%。