Media optimization for the production of beta-carotene by Blakeslea trispora: a statistical approach

Blakeslea trispora (+) MTCC, Blakeslea trispora NRRL 2895 (+), Blakeslea trispora NRRL 2896 (-) as well as intraspecific mating of both the strain types have been studied for optimum production of beta-carotene. Intraspecific mating of both the strain types increased the yield of beta-carotene to a considerable level (98+/-2mg/l) as compared to wild strains. Effect of different media components such as carbon, nitrogen, and sulphates, and that of process variables such as pH and inoculum size on beta-carotene production by Blakeslea trispora in shake flask culture was investigated. One factor at-a-time method was employed for the optimization of media components. Response surface methodology (RSM) was further used to determine the optimum values of process variables for maximum beta-carotene production. The fit of the quadratic model was found to be significant. A significant increase in beta-carotene production (139+/-1mg/l) was achieved using RSM.     

Effect of high pressure on the carotenoid content of Blakeslea trispora Thaxter mycelium]

High hydrostatic pressure was shown to decrease the content of beta- and lambda-carotenes in the mycelium of Blakeslea trispora, but to increase the production of lycopene, neurosporene and phytoene.     

Assimilation of 1-14-C-octadecane by the (-)4 strain of Blakeslea trispora]

The assimilation of 1-14C-octadecane by the (-) 4 strain of Blakeslea trispora was studied, i.e. the kinetics of 14CO2 evolution and the distribution of the label in the biomass, proteins, lipids, and water-soluble products of the fungus. B1. trispora oxidized octadecane even in the presence of glucose in the medium. The distribution of the label between the protein and lipid fractions was different when the fungus was grown on octadecane or on a mixture of octadecane with glucose. After 6 hours of the incubation the content of the label in proteins (the experiment with 1-14C-octadecane as a carbon source) was 33 percent, and 60 percent in lipids. In the experiment with a mixture of glucose and hydrocarbon, the content of the label was 8 percent in proteins and 95 percent in lipids. These results show the participation of n-alkane and the products of its oxidation in the metabolism of Blakeslea trispora.     

三孢布拉霉生产番茄红素研究进展

番茄红素是一种能够预防某些癌症,心血管疾病等慢性病的重要类胡萝卜素。三孢布拉霉是产生番茄红素的主要微生物之一。对番茄红素的理化性质,三孢布拉霉的生物学特性,高产菌株的选育,培养基及工艺的优化,番茄红素的提取,市场状况及开发前景等进行了综述。     

Superoxide dismutase and catalase activities in carotenoid-synthesizing fungi Blakeslea trispora and Neurospora crassa under the oxidative stress

The addition of menadione into the medium during cultivation of Neurospora crassa in the dark activated its constitutive superoxide dismutase. Exposure to light not only activated superoxide dismutase and catalase, but also increased the content of neurosporaxanthin. Superoxide dismutase activity in the mixed (+/-) mycelium of Blakeslea trispora synthesizing beta-carotene in the dark was much lower than that in Neurospora crassa. The superoxide dismutase activity further decreased in oxidative stress. The catalase activity decreased with an increase in the content of beta-carotene. Our results indicate that neurosporaxanthin possesses photoprotective properties in Neurospora crassa. In Blakeslea trispora (+/-) fungi, this compound acts as a major antioxidant during inactivation of enzymes that detoxify reactive oxygen species.     

Light induction of the carotenoid biosynthesis pathway in Blakeslea trispora

A gene of Blakeslea trispora has been cloned by heterologous hybridization with the Mucor circinelloides crgA gene, a repressor of light-inducible carotenogenesis. This gene is the ortholog of the M. circinelloides crgA, since it was able to restore the wild-type phenotype of a null crgA mutant of M. circinelloides. The expression of B. trispora crgA gene is light-induced and photoadapted, as occurs for M. circinelloides crgA. Light induction and photoadaptation of B. trispora crgA was also observed in M. circinelloides, which suggests that the mechanisms involved in light regulation are basically conserved between these filamentous fungi. Conservation of the regulatory pathway that controls carotene biosynthesis was supported by the light-induced and photoadapted expression of all structural carotenogenic genes of B. trispora. Consequently, the beta-carotene content of dark grown mycelia of B. trispora increased upon illumination with white light.     

卡那霉素抗性基因在三孢布拉霉菌种中的表达

利用一个带有自主复制子,但缺失自身启动子的源于转座子Tn-5的卡那霉素抗性基因的质粒PVB32,在大肠杆菌中克隆丝状真菌三孢布拉霉DNA中有启动子功能的DNA片段;通过原生质体转化,获得了三孢布拉霉对卡那霉素抗性的表达,且抗性表现稳定,可通过孢子无性繁殖稳定遗传下去。     

Cloning,characterization and heterologous expression of the Blakeslea trispora gene encoding orotidine-5'-monophosphate decarboxylase

The pyrG gene of the fungus Blakeslea trispora, encoding orotidine-5'-monophosphate decarboxylase (OMPD) enzyme, was cloned by heterologous hybridization of a genomic library with the Mucor circinelloides pyrG gene. The deduced amino acid sequence of the B. trispora pyrG gene is highly similar to the OMPD from other organisms. Hybridization analyses revealed that the only copy of this gene present in the genome of B. trispora is constitutively expressed. Heterologous complementation of a mutant of M. circinelloides deficient in OMPD activity with the B. trispora pyrG gene and promoter sequence confirmed the function of this gene. This functional complementation demonstrates that heterologous expression in M. circinelloides might be used to investigate the function of genes of B. trispora.     

Zygote formation in Blakeslea trispora: morphological peculiarities and relationship with carotenoid synthesis

Changes associated with zygospore formation in the mucorous fungus Blakeslea trispora were studied. Zygospores are dormant cells with thickened cell walls and large central lipid vacuoles containing large amounts of lycopene. We established for the first time that B. trispora gametangia of different sexes differ in their carotenoid content and revealed that zygote formation involves a novel structure that consists of densely intertwined hyphae. Using inhibitory analysis (blocking beta-carotene synthesis with diphenylamine and 2-amino-6-methylpyridine), we showed that suppression of carotene production results in the inhibition of zygote formation. Hence, we established a manifest dependence of zygote formation on beta-carotene synthesis.     

三孢布拉霉发酵生产番茄红素的提取工艺及残留溶剂去除

用超声波皂化法从三孢布拉霉(Blakeslea trispora)发酵菌丝中提取番茄红素,对皂化条件、超声处理时间、提取温度和时间进行优化,得到番茄红素含量大于0.5%的番茄红素油树脂,总提取率达92.9%。提出溶剂提取与SC-CO2去除溶剂的技术路线,使番茄红素粗提物的溶剂残留符合标准。     

Effect of beta-carotene accumulation on spectral parameters of microscopic fungi Blakeslea trispora biomass

Parameters of reflectance spectra in visible region and colorimetry for Blakeslea trispora biomass with different content of beta-carotene were determined. Correlation level of pigment accumulation and spectral characteristics were revealed. It is established, that objective control of color characteristics for biomass is possible using measurement of colorimetric parameters.     

三孢布拉氏霉生物合成β-胡萝卜素的研究──Ⅰ.三孢布拉氏霉负菌优良菌株SCB201的选育

采用紫外照射、亚硝基胍（NTG）和离子束综合诱变处理,获得一株三孢布拉氏霉菌负亩优良菌株SCB201。在优化了的培养条件下,它与三孢布拉氏霉菌正菌SCB200接合培养产β－胡萝卜素达到2g／1,较其亲株0．2g／1的水平提高了10倍。     

Initial stages of trisporic acid synthesis in Blakeslea trispora

Biotransformation of beta-carotene with enzyme preparations isolated from the mycelium of Blakeslea trispora resulted in the formation of its hydroxylated metabolite and apocarotenals, products of oxidative degradation of this compound. By spectral, chromatographic, and chemical properties, the beta-carotene derivative was identified as 4-hydroxy-beta-carotene (isocryptoxanthine). One of the products of oxidative degradation of beta-carotene, beta-apo-13-carotenone, underwent modification in the presence of enzyme preparations from Blakeslea trispora with the formation of trisporic acid precursors. It should be emphasized that beta-apo-13-carotenone transformation proceeded more rapidly than beta-carotene oxidation by carbon in the 4-position. Our findings suggest that under conditions of oxidative stress, oxidative degradation of beta-carotene into beta-apo-13-carotenone leads to the formation of considerable amounts of trisporic acids.     

添加氧载体及表面活性剂对番茄红素发酵的影响

通过添加氧载体（正十二烷、正己烷、过氧化氰）,有效改善发酵体系中的氧传递速率,从而促进了三孢布拉氏霉菌合成番茄红素的能力。实验结果表明,在第0d加入1．0％的正已烷、正十二烷时番茄红素的生成量分别提高了25．32％、72．84％,在第1d,添加50μL/100mL过氧化氢时番茄红素的生成量提高了40．35％,在加入正十二烷的同时,再加入表面活性剂Trilorl—x100,Tween20,Tween80,Span-20等,可使番茄红素的产量最多提高114．83％．     

Structure prediction and function characterization of WC-2 proteins in Blakeslea trispora

International Microbiology - Blakeslea trispora is known for its potential to produce an excess of carotenoids in mixed cultures of strains of opposite sex. The biosynthesis of β-carotene in...     

Transcriptome sequencing and global analysis of blue light-responsive genes provide clues for high carotenoid yields in Blakeslea trispora

International Microbiology - Blakeslea trispora has great potential uses in industrial production because of the excellent capability of producing a large quantity of carotenoids. However, the...     

Spectroscopy analysis for simultaneous determination of lycopene and β-carotene in fungal biomass of Blakeslea trispora

Blakeslea trispora is a good alternative source for producing such carotenoids as lycopene and β-carotene. The objective of this research was to elaborate a method for the simultaneous determination of lycopene and β-carotene in Blakeslea trispora products using a usual UV-vis spectrophotometer. The standard solutions of the mixture of different concentrations of β-carotene and lycopene were measured with the UV-vis method and correlation formula for the extinction coefficients of 1% standard solution of lycopene in the solvent (hexane) and the ratios of the optical densities at the character peaks of 470 and 502 nm was elaborated. This gives a possibility to calculate the concentrations of lycopene and β-carotene in the mixture. The prediction quality of the UV-vis method was sufficient and the obtained results were very close to the ones, being measured with the HPLC technique. The proposed method can be used for both routine industrial work and academic research, providing the rapid analysis for simultaneous measurements of lycopene and β-carotene.     

New insights into mechanisms of growth and beta-carotene production in Blakeslea trispora

Blakeslea trispora (Mucorales) has economic importance because of its ability to produce large amounts of beta-carotene. To shed light on the actual point of induction and to shorten the following production process, germination and growth of its two mating types, (-) and (+), were observed separately, and the mating point was investigated in lab scale experiments. The (-) mating type showed much faster germination than the (+) type on solid medium and in Erlenmeyer flasks. However, after a first period, the (-) mating type grew clearly slower than the (+) type. In addition, the (-) type branched more vividly than the (+) type. A ratio of 30:1 of (-) and (+) type at an age of 20 h was found to achieve highest beta-carotene yields. Our results provide a comprehensive overview of Blakeslea trispora growth and its product synthesis.     

代谢途径酶活促进剂对番茄红素发酵的影响

考察了几种代谢途径中关键酶活促进剂对番茄红素发酵的影响。通过添加代谢途径中HMg-CoA还原酶与MVA激酶酶活促进剂,提高类胡萝卜素前体物质的生物合成,从而促进三孢布拉霉菌合成番茄红素的能力。实验结果表明,发酵24 h分别加入质量分数0.05%的某醇及其代谢产物A、1 mg/L的青霉素和质量分数0.1%β-紫罗酮,番茄红素的产量分别提高了31%,32%及35%,最大产量达到1.54 g/L。