Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

佐夫色绿藻高产虾青素突变体的筛选及虾青素合成机理研究

为了提高佐夫色绿藻(Chromochloris zofingiensis)细胞内虾青素含量,研究通过甲基磺酸乙酯诱变构建了含有20000个单克隆的突变体库,并筛选出一株高产虾青素的突变体12C10。在异养正常培养条件下,当葡萄糖耗完时, 12C10虾青素含量比野生型提高74%;缺氮诱导第4天时,虾青素含量比野生型高25%。利用广泛靶向代谢组学分析在正常培养条件下12C10与野生型在代谢物水平上的差异。与野生型相比, 12C10中除谷氨酸外的氨基酸及脂肪酸的合成普遍下降,但是谷氨酸的水平显著提高。氨基酸和脂肪酸合成减少为虾青素合成提供更多的碳骨架、NADPH和ATP。谷氨酸的积累可能一方面刺激了磷酸戊糖途径中6-磷酸葡萄糖脱氢酶的活性促进NADPH的产生,另一方面导致氧自由基产生促进虾青素合成。代谢物组分析结果还表明12C10中虾青素合成的增强可能与乙烯合成的增强有关。研究为进一步通过代谢调控提高C. zofingiensis虾青素含量奠定了基础,对指导C. zofingiensis虾青素积累新工艺的开发具有重要意义。     

Two-step process for ketocarotenoid production by a green alga, Chlorococcum sp. strain MA-1

The production of ketocarotenoids (KCs) from Chlorococcum sp. strain MA-1 was investigated by a two-step process. In the first step, 18 g biomass l(-1) was achieved by feeding glucose to the heterotrophic cultures; in the second step, the high-density cultures were treated with light illumination or chemical stress in dark, respectively, to induce KC synthesis. Light-treated cultures could produce 103 mg total KCs l(-1) and 32 mg astaxanthin l(-1), three times higher than those from chemical-treated cultures, in the 10 days of induction. The percentages of individual KCs (hydroxyechinenone, canthaxanthin, adonirubin and astaxanthin) in the total KCs were not markedly influenced by the different stress conditions. The developed two-step process provides a feasible strategy for commercial production of ketocarotenoids by the green microalga, Chlorococcum sp. strain MA-1.     

Regulation of reductase formation in Proteus mirabilis

Summary The levels of several redox enzymes in a chlorate-resistant mutant of Proteus mirabilis, which is partially affected in the formation of formate hydrogenlyase, thiosulfate reductase and tetrathionate reductase, were compared with those of the wild type. The composition of the electron transport system of both strains was almost the same in cells grown aerobically, but very different in cells grown anaerobically. In the mutant, the cytochrome content increased twofold, whereas the level of the anaerobic enzymes is strongly diminished. The anaerobic formation of electron transport components in the mutant was, in contrast to that of the wild type, not influenced significantly by azide. During anaerobic growth with nitrate low levels of a functional nitrate reductase system were formed in the mutant. Under these conditions the formation of formate dehydrogenase, formate hydrogenlyase, formate oxidase, thiosulfate reductase, tetrathionate reductase, cytochrome b_563,5 and partly that of cytochrome a₂, was repressed. The repressive effect of nitrate, however, was completely abolished by azide. Therefore, it seems likely that a functional nitrate reductase system, rather than nitrate, controls the formation of the enzymes repressible by nitrate.     

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.     

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 col-umn was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus plu-vialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The re-sults showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingien-sis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a re-markably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).     

Improvement of astaxanthin production by Phaffia rhodozyma through mutation and optimization of culture conditions

Phaffia rhodozyma strains were treated with the mutagenic agent NTG several times and plated onto yeast-malt agar containing β-ionone as a selective medium. One of the NTG-treated strains (NCHU-FS301) produced considerably more astaxanthin than the parent CBS-6938 (strain NCHU-FS301 produced 1515.63 μg/g and CBS-6938 565.08 μg/l). When the kinetic parameters of the specific growth rate (μ) and specific astaxanthin productivity (q_p) were used to judge the association between growth behavior and product formation, NCHU-FS301 was shown to be a more positive growth-associated fermentation type than the parent strain. A study of the effects of the carbon source on red pigment formation revealed that glucose could support the highest total astaxanthin production (7809.3 μg/l). Yeast extract was the best nitrogen source in supporting the highest total astaxanthin formation (8637.5 μg/l). When mixed nitrogen sources were used, a mixture of yeast extract, beef extract, and potassium nitrate (1:1:1) supported more pigmentation (8052.6 μg/l) than the other mixtures tested. Astaxanthin-overproducing mutants could be useful in providing a natural source of astaxanthin for the aquacultural industry.     

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O₂-resistant photosynthesis. The apparent K_m for H₂O₂ was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.     

Effects of oxidative stress on the production of carotenoid pigments byPhaffia rhodozyma (Xanthophyllomyces dendrorhous)

The resistance to killing by free radicals of two mutants ofPhaffia rhodozyma was determined. Mutant 5–7 did not produce astaxanthin but produced β-carotene, while mutant 3–4 did not produce any carotenoid pigments. The resistance of mutant 5–7 was the same as that of the wild type but mutant 3–4 was rapidly killed. Carotenoid pigments increased the resistance to killing by free radicals. We investigated the effects of free radicals, generated by H₂O₂ and Fe²⁺ added to the medium, on wild-type cells and mutants ofP. rhodozyma. Unpigmented mutants of basidiomycetous yeasts (Rhodotorula spp. and others) are more susceptible to killing by UV-irradiation than the pigmented, wild-type strains. Therefore, we investigated the effect of free radicals on a similar basidiomycetous yeast,P. rhodozyma, a species of economic importance, in the biological production of astaxanthin.     

Factors affecting expression of enhanced catalase activity in a tobacco mutant with o(2)-resistant photosynthesis

Tobacco (Nicotiana tabacum) mutants with 40 to 50% more catalase activity than wild type show O₂-resistant photosynthesis under conditions of high photorespiration. More than 90% of the population of mutant plants of an M₇ and M₈ generation had enhanced catalase activity, and nearly 40% had activities >3 standard deviations above the mean of wild type. Superoxide dismutase activity was the same in mutant and wild-type leaves. The greater photosynthetic rate of mutant leaves previously observed in the laboratory was confirmed with field-grown plants that showed significantly higher rates (8%) than wild type during 8 days of measurements during a 19-day period of active growth. The tip region of expanding mutant leaves had higher catalase activity than the base of the lamina, and photosynthesis was O₂ resistant in 42% O₂ in the tip compared with the base, thus further supporting the hypothesis that there is a biochemical linkage between these traits. Plants grown in high light (270 micromole photons per square meter per second) had greater catalase activity and an activity ratio of mutant to wild type of 1.45 compared with 1.22 for those grown in low light (130 micromole photons per square meter per second). After acclimation for 3 weeks, plants transferred from low to high light showed increasing activities, and after 5 days the activity ratio of mutant to wild type was the same as in plants acclimated in higher light. The role of enhanced catalase activity in reducing photorespiratory CO₂ is discussed.     

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%). Supported by the Frontier Research Grant of the SCSIO, the Hundred Talents program of Chinese Academy of Sciences, and National Natural Sciences of China projects (Grant No. 40776087)     

耐辐射球菌基因DRB0099缺失突变株的构建及逆境分析

耐辐射球菌(Deinococcus radiodurans R1)有着极强的辐射抗性.研究其抗辐射的机理对于处理放射性废料有着潜在的应用价值.在耐辐射球菌的基因组中,许多序列的功能未知.其中DRB0099尤为引人注意.将DRB0099缺失突变构建该基因的突变株.对野生型和突变体进行比较后发现,在正常生长条件下的前期阶段(0～16 h),突变体生长速度比野生型慢.16 h以后,野生型逐渐进入稳定生长期.这时,突变株的生长速度高于野生型.但是,野生型的浓度一直高于突变株.表明在DRB0099被删除后,耐辐射球菌的生长可能受到了阻滞.在紫外线照射的条件下,尽管野生型随着照射剂量的增加,存活率越来越低,但是要比突变体高许多.野生型具有比突变体更强的修复DNA双链断裂的能力.DRB0099可能直接参与了对DNA的修复.突变体对H2O2的敏感程度高于野生型,表明野生型耐辐射球菌在对抗活性氧保护其蛋白质、DNA或者DNA修复方面具有比突变体更强的功能.在低浓度H2O2处理条件下,尽管野生型和突变体的存活率都出现下降趋势,但二者的差值并不大.随着H2O2剂量的增加,二者的差值越来越大.表明随着活性氧浓度的增加,蛋白质和DNA损伤的数量增加,失去DRB0099基因功能的突变体比野生型更容易受到损伤.在紫外线照射处理或者H2O2处理条件下,DRB0099能够保护蛋白质和DNA.     

Root morphology and cadmium uptake kinetics of the cadmium-sensitive rice mutant

To understand the physiological mechanism that confers Cd sensitivity, root morphology and Cd uptake kinetics of the Cd-sensitive mutant and wild type rice were investigated. The root length, root surface area, and root number of mutant rice decreased more significantly with increasing Cd concentration in growth media compared with the wild type rice. The uptake kinetics for ¹⁰⁹Cd²⁺ in roots of both the mutant and wild type rice were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period. Concentration-dependent Cd²⁺ influx in both species could be characterized by the Michaelis-Menten equation, with similar apparent K_m values for mutant and wild type rice (2.54 and 2.37 μM, respectively). However, the V_max for Cd²⁺ influx in mutant root cells was nearly 2-fold higher than that for wild type rice, indicating that enhanced absorption into the root is one of the mechanisms involved in Cd sensitivity in mutant rice.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Induction and Characterization of UV Resistant Calothrix braunii

A UV resistant mutant of Calothrix braunii has been isolated after repeated exposure to UV-C (254 nm) radiation. LD₅₀ for wild type against UV-B was 1.74 hand 100% lethality was achieved after 3.5 h. Whereas, UV resistant mutant showed LD₅₀ at 3.33 h and loss of complete survival after 5 h exposure. The growth rate of mutant was about 29 per cent greater than that of the wild type. The photosynthetic pigments, chlorophyll a and phycoerythrin were stimulated by 36.2 and 41.2 per cent, respectively over wild type. There were no differences in nitrogenase activity nitrate reductase activity, extracellular ammonia production and of plasmid DNA.     

协同诱变法选育虾青素高产优良酵母菌株

以红法夫酵母(Phaffia rhodozyma)野生型菌株GH1为研究对象,选育虾青素高产优良酵母菌株,并初步研究其生长特性。对野生型菌株GH1用紫外线(UV)和亚硝基胍(NTG)协同诱变,用抗霉素A筛选,选育出虾青素高产突变菌株GH1-7.1。突变菌株GH1-7.1的虾青素最高产量达1200μg/gDCW,比野生菌株GH1的虾青素最高产量271μg/gDCW高出4倍,并且生长加快,达到最高生物量和最高虾青素产量的时间比野生菌株缩短了24h;野生菌株GH1的最适生长温度为20℃,高于28℃则不生长,而突变菌株GH1-7.1的最适生长温度为28～30℃,比野生菌株GH1提高8～10℃。首次采用协同诱变的方法成功选育了虾青素高产酵母菌株,并且生长周期短,最适的生长温度提高,是适宜南方高温环境发酵工程应用的优良菌株。     

Increased astaxanthin production by a Phaffia rhodozyma mutant grown on date juice from Yucca fillifera

The wild strain and the astaxanthin-overproducing mutant strain 25–2 of Phaffia rhodozyma were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium composed of g L⁻¹: KH₂PO₄ 2.0; MgSO₄ 0.5; CaCl₂ 0.1; urea 1.0 and supplemented with date juice of Yucca fillifera as a carbon source (yuca medium). The highest astaxanthin production (6170 μg L⁻¹) was obtained at 22.5 g L⁻¹ of reducing sugars. The addition of yeast extract to the yuca medium at concentrations of 0.5–3.0 g L⁻¹ inhibited astaxanthin synthesis. The yuca medium supported a higher production of astaxanthin, 2.5-fold more than that observed in the YM medium. Journal of Industrial Microbiology & Biotechnology (2000) 24, 187–190. Received 14 July 1999/ Accepted in revised form 02 December 1999     

Kinetics of NH(4) Assimilation in Zea mays: Preliminary Studies with a Glutamate Dehydrogenase (GDH1) Null Mutant

In higher plants it is now generally considered that glutamate dehydrogenase (GDH) plays only a small or negligible role in ammonia assimilation. To test this specific point, comparative studies of ¹⁵NH₄⁺ assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDH1-positive wild type from which this mutant was derived. The kinetics of ¹⁵NH₄⁺ assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% (¹⁵NH₄)₂SO₄ via the aerated root medium in hydroponic culture over a 24-h period. The GDH1-null mutant, with a 10- to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of ¹⁵NH₄⁺ assimilation into total reduced nitrogen. Observed rates of root ammonium assimilation were 5.9 and 3.1 micromoles per hour per gram fresh weight for the wild type and mutant, respectively. The lower rate of ¹⁵NH₄⁺ assimilation in the mutant was associated with lower rates of labeling of several free amino acids (including glutamate, glutamine-amino N, aspartate, asparagine-amino N, and alanine) in both roots and shoots of the mutant in comparison to the wild type. Qualitatively, these labeling kinetics appear consistent with a reduced flux of ¹⁵N via glutamate in the GDH1-null mutant. However, the responses of the two genotypes to the potent inhibitor of glutamine synthetase, methionine sulfoximine, and differences in morphology of the two genotypes (particularly a lower shoot:root ratio in the GDH1-null mutant) urge caution in concluding that GDH1 is solely responsible for these differences in ammonia assimilation rate.