Trisporoids and carotenogenesis in Blakeslea trispora

The recent data on the pathways of carotenoid biosynthesis, which resulted in revision of the earlier concepts, are analyzed. Trisporoid diversity and their role in the sexual process of mucoraceous fungi, resulting in formation of sexual cells (zygospores) are discussed. Special attention is paid to the role of trisporic acids in carotenogenesis of combined culture of the (+) and (?) strains, in which zygospores are not formed.     

The role of oxidative stress on carotene production by Blakeslea trispora in submerged fermentation

In aerobic metabolism, reactive oxygen species (ROS) are formed during the fermentation that can cause oxidative stress in microorganisms. Microbial cells possess both enzymatic and non-enzymatic defensive systems that may protect cells from oxidative damage. The antioxidant enzymes superoxide dismutase and catalase are the two key defensive enzymes to oxidative stress. The factors that induce oxidative stress in microorganisms include butylated hydroxytoluene (BHT), hydrogen peroxide, metal ions, dissolved oxygen tension, elevated temperature, menadione, junglone, paraquat, liquid paraffin, introduction to bioreactors of shake flask inocula and synthetic medium sterilized at initial pH 11.0. Carotenes are highly unsaturated isoprene derivatives. They are used as antioxidants and as coloring agents for food products. In fungi, carotenes are derived via the mevalonate biosynthesis pathway. The key genes in carotene biosynthesis are hmgR, ipi, isoA, carG, carRA and carB. Among microorganisms, Βlakeslea trispora is the main microorganism used for the production of carotenes on the industrial scale. Currently, the synthetic medium is considered the superior substrate for the production of carotenes in a pilot plant scale. The fermentation systems used for the production of carotenes include shake flasks, stirred tank fermentor, bubble column reactor and flat panel photobioreactor. This review summarizes the oxidative stresses in microorganisms and it is focused on the current status of carotene production by B. trispora including oxidative stress induced by BHT, enhanced dissolved oxygen levels, iron ions, liquid paraffin and synthetic medium sterilized at an initial pH 11.0. The oxidative stress induced by the above factors increases significantly the production of carotenes. However, to further reduce the cost of carotene production, new biotechnological methods with higher productivity still need to be explored.     

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。     

Mutants of carotene production in Blakeslea trispora

The filamentous fungus Blakeslea trispora, an industrial carotene source, contains -carotene and precursors of its synthesis — phytoene, phytofluene, lycopene, and -carotene. Strain improvement through mutagenesis is difficult because all life stages are multinucleate. Mutants have been obtained following exposure of wild-type spores to N-methyl-N-nitro-N-nitrosoguanidine. Changes in the colour of the mycelia reflect variations in the accumulation of various precursors and the final product. Quantitative analysis of the mutants leads to the conclusion that the biosynthetic pathway is similar to that of the related fungus Phycomyces blakesleeanus, but the regulation is completely different. In particular, interruption of the pathway does not lead to overacummulation of precursors.     

Hormonal Interactions in Mucor mucedo and Blakeslea trispora

Evidence is presented that progametangia in both the plus and the minus mating types of Mucor mucedo can be induced by one substance, namely (-)-trisporic acid B. A method is described for the determination of the concentration of the sex factors (trisporone, trisporic acid B, trisporic acid C) in mated cultures of Mucorales by polarography. It can be demonstrated that the amount of plus mycelium is limiting for the production of the sex factors in Blakeslea trispora. It is shown that the minus type of this organism is able to synthesize the sex factors when incubated in the filtered medium of a mated culture. Cycloheximide and 5-fluorouracil inhibit strongly the sex factor production in a mated culture of B. trispora at any time. This result suggests that sexual activity comprises the synthesis of proteins which are involved in the production of the sex factors.     

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％．     

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。     

Relationship Between Sexuality and Carotene Synthesis in Blakeslea trispora

When stimulated by equivalent amounts of progametangia-inducing hormones, cultures of the minus mating type of Blakeslea trispora produce about the same quantities of carotenoids as mated cultures of the fungus, which suggests that the stimulation of carotene synthesis during the sexual activity of mated cultures is the result of hormonal action. These hormones were isolated and purified. From spectroscopic analysis of purified samples, it appears that the hormones are identical with trisporic acids B and C. When both mating types of B. trispora were cultivated in one vessel but were kept apart by membrane filters, the formation of sex hormones was not inhibited. Physical contact between the mating types is obviously not required for the induction of sexual activity. The sex hormones also formed in combined cultures of B. trispora-plus and Zygorhynchus moelleri (a homothallic species), but not in combined cultures of B. trispora-minus and Z. moelleri. This is evidence for the hypothesis that the hormones are produced by B. trispora-plus only.     

Lipid composition of cells of heterothallic strains in the developmental cycle of Blakeslea trispora

Lipid compositions in mycelium and spores of Blakeslea trispora heterothallic strains were studied. Distinctions between the strains in the ability to synthesize linolenic acid and in optimal growth temperature were demonstrated. The (-) strain grew at a higher temperature and was unable to synthesize linolenic acid, whereas the (+) strain accumulated this acid up to 20% of total fatty acids. The distinctions between the strains remained at different developmental stages (mycelium and spores). A higher thermophilicity of the (-) strain correlated with a high sterol content, which is typical of thermophilic fungi. The lipid compositions of heterothallic strains studied differed in lipid content, their fractional composition, the degree of unsaturation, and carotenoid composition.     

三孢布拉霉发酵产番茄红素的研究进展

番茄红素是一种重要的类胡萝卜素,在生物体中具有抗氧化、抗衰老、提高免疫力等生理功能。虽然已经有部分企业实现了番茄红素的工业化生产,但产量仍然是制约三孢布拉霉发酵生产番茄红素的重要因素。在本实验室研究的基础上,本文结合近年来国内外学者的研究成果,从遗传育种、发酵工艺优化、化学调控等方面综述了提高三孢布拉霉中番茄红素产量的研究进展,并展望了未来的研究方向。     

Chemical regulators of carotenogenesis by Blakeslea trispora

The activation of the carotene biosynthetic pathway in Blakeslea trispora was found to occur by trisporic acid and many other compounds such as abscisic acid, β-ionone, α-ionone and vitamin A which share significant structural similarity with trisporic acid. The magnitude of stimulatory activities of these effectors was in the order trisporic acid > abscisic acid > β-ionone > α-ionone > vitamin A. Comparison of structures and stimulatory activities of all the effectors indicated that the short length of the side chain and the presence of a keto group in the ring structure of the trisporic acid molecule contributed significantly to the biological activity towards carotenogenesis.     

Effects of amines on the carotenogenesis in Blakeslea trispora

Six amines profoundly affected carotenogenesis in Blakeslea trispora. When cultures were treated with the amines, namely 4-[β-(diethylamino)-ethoxy]-benzaldehyde, 4-[β-(diethylamino)-ethoxy]-acetophenone hydrochloride, 4-β-(diethylamino)-ethoxy]-benzophenone hydrochloride, triethylamine hydrochloride, α-diethylaminopropiophenone hydrochloride and tributylamine hydrochloride, an increase in the lycopene accumulation was observed. The modes of action of these amines appear to be similar to that of 2-(4-chlorophenylthio)triethylamine hydrochloride (CPTA); however, they difrer in relative effectiveness.     

微生物发酵产生的番茄红素的分离纯化

研究了三孢布拉霉(Blakeslea trispora)发酵产生的番茄红素的分离纯化方法。与植物源的番茄红素相比,真菌发酵产物中含有更多的油脂成分,且提取物中含有的一些同分异构体与产物性质类似而不易分离。实验采用溶剂抽提、大孔吸附树脂吸附法纯化以及结晶的方法可获得番茄红素晶体,纯度达70.9%,收率为64.6%。产品经重结晶后可获得纯度90%以上的番茄红素,经红外、核磁、质谱等证实与植物来源的番茄红素相一致。