Research fields endophytic fungi taxol production
Pick to: paclitaxel endophytes from the yew requirements is higher, and expensive, in order to seek a more effective way to separate produce taxol, the research we conducted effective drug, this paper reviews the taxol production of endophytic fungi of taxus chinensis in recent years, the research progress of separation and screening, metabolism and fermentation.
Key words: paclitaxel; Taxus chinensis; Endophytic fungi
Paclitaxel (Taxol, commodity name Paclitaxel), molecular formula C47H51 NO14, molecular weight of 853, is a kind of plants of the genus from yew Taxus extracted natural anti-cancer drugs. Its anticancer mechanism is able to combine with micro protein, and promote its aggregation, inhibit cancer cell mitosis, effectively prevent the proliferation of cancer cells. At present, the main source of taxol are: tissue culture, cuttings and seedlings, seed liquid suspension culture, endogenous bacteria and taxol production of endogenous bacteria separation and cultivate the synthesis of taxol and genetic engineering and so on. Among them, the fungus in the production of taxol within method has broad development prospects, not only can obtain physiological activity consistent with plant sources of taxol, and conducive to industrial production, low cost accordingly. Nowadays, the main problem is that taxol yield is low, the price is expensive, if solve these problems, the industrialization of the endophytic fungi of taxol production can be achieved, the price will reduce accordingly, relatively to many cancer patients brought the Gospel, in recent years, the discovery of the endophytic fungi of taxus chinensis were introduced in this paper and the research progress of separation and taxol production.
1 Endophytic fungi of the separation of endophytic fungi of taxus chinensis (endophyticfungus) is a major categories are not fully aware of fungus, it refers to those who live in healthy plants do not cause any symptoms of disease all fungi. Endophytic fungi of producing taxol has found it, basically all is isolated from the plants of the genus taxus chinensis, also have individual isolated from the yew plant as an example
1.1 Species purification method now commonly used purification method is: put yew bark cut into small pieces in 75% alcohol disinfection, after washed with sterile water, placed in the potato solid plate culture medium (PDA), to be training for a period of time by explant grow around after hyphae, pick form different colony mycelia tip, inoculation on PDA solid medium culture, respectively, to grow new colonies after repeatedly purified again can get pure strains. If add a small amount of streptomycin in culture medium, can inhibit the growth of bacteria, fungi separation.
1.2 Is separation found that endophytic fungi are now separated endophytic fungi of taxus chinensis species found in different forms and also each are not identical, secondary metabolites with paclitaxel, barca pavilion O, taxol, aromatic amino acid decarboxylase, etc. The paclitaxel yield was 0.024 ~ 185.4 L g/L, the fermentation output level is very low, is obtained by biotechnology endophytic fungi of the fusion of taxol yield is only 448.52 L g/L, industrial production is still quite a distance away. In general, the fermentation output level to 1 mg/L only have industrial production value.
2 Reviewing secondary metabolites of endophytic fungi study. 69000 species of fungi have been found all over the world, and each year is increasing with more than 1000 kinds of new species, a conservative estimate is total up to more than 150, ten thousand. At the same time the secondary metabolites of fungi produce more than one thousand kinds, and are generally specific to species or strains. Scholars at home and abroad by TLC, MS, HPLC, UV, immune analysis and biological test, radioactive precursor method such as the mark of taxus endophytic fungi secondary metabolites identified, both found that some strains and become the research hotspot in recent years. Endosymbiont theory, secondary metabolism in the process of the continuous evolution of the biochemical pathways can lead to useful materials into symbionts, and once the secondary metabolism biochemical pathway has emerged, it can be other creatures use, interaction and co-evolution. The resulting assumption is: with the same secondary metabolites biosynthesis route is directly obtained related genes.
3 The influencing factors of fungal fermentation production of taxol production
3.1 Fermentation media Zhang Limin etc to separation from taxus yunnanensis endogenetic fungi M1 for experimental bacteria, liquid and solid fermentation culture medium for comparison, found that strains in solid culture medium, growth is better than liquid medium, estimates may be due to fungi exists in the bark, moisture content is low, so more suitable for solid medium. But the liquid culture medium used more widely. At present, generally using shake flask and small tank fermentation. Zhang Yani etc with T14 strains isolated from var. Chinensis is found during tests with ammonium chloride and ammonium sulfate for better N source, peptone makes taxol production has fallen sharply. This may be caused by different species differences. Because of taxol and its analogs in taxus chinensis synthesis in plants is a common diterpenoid precursor taureans pyrophosphate cyclization and generate a series of mainly oxidation modification effect, taxol biosynthesis and taxane skeleton is by glyceraldehyde 3 - phosphate/acetone acid pathway.
3.2 Medium additives radioactive isotope labeled precursor material feeding experimental results show: paclitaxel taxane tricyclic diterpene skeleton from hydroxymethyl valeric acid, acetyl acetate, and C - 13 ester base C6H5CH2CH2COHCOO - from phenylalanine in the side chain; Research also shows that phenylalanine is a precursor of synthesis of taxol. Add can promote taxol biosynthesis precursor. LiuXiaoLan are respectively in small spore disc, hairy spore and tree more than spore adding phenylalanine fermentation medium, the taxol production must be improved; At the same time to join a certain amount of sodium acetate can increase production. Cells in the synthesis of extensin, lignin, can use tyrosine and phenylalanine, partly resulting in phenylalanine to tyrosine conversion, this obviously bad for the synthesis of taxol. Some join tyrosine will inhibit the conversion reaction, so as to protect the effect of phenylalanine. Experiments have established that join tyrosine promoting effect on the synthesis of taxol, can improve the yield. In small spore disc, hairy spore culture medium after RNA enzyme inhibitor is added oxygen alum sulfate, found the taxol content in the fermented liquid to improve several times, but several other RNA enzyme inhibitors sulfur laurel ester and sulfur generation polyethylene resin effect is not obvious. RNA enzyme inhibitors vitriol sulphuric acid oxygen increase of taxol production specific mechanism is unclear.
3.3 Training time and ventilation 。LiuXiaoLan by measuring different incubation time of taxol content in the fermented liquid, the incubation time was 16 ~ 18 d, taxol content is higher, the estimates to be associated with fungal metabolic cycle. By the size of endophytic fungi for aerobic microorganisms, thus different concentrations of oxygen, carbon dioxide has a great influence on the cell growth and taxol production. Low concentration oxygen (10% volume) promote taxol, high concentrations of carbon dioxide (10% volume ratio) is to suppress it. In the tree more than spore in the fermentation process, oxygen is 150 ml / 500 ml effect is best, so adequate ventilation, keep suitable concentration of dissolved oxygen is helpful to improve yield.
4 Taxus chinensis endophytic fungi in the the application of fermentation way
4.1 Synthesis of taxol
Some fungus has a precursor to acetylation and sent as isomerization ability, its reason lies in the fungus in the fermentation process to produce the specificity of the enzyme. For the fungus in the synthesis of taxol opens a feasible way.
4.2 Induction of taxus chinensis cells increase the taxol production in recent ten years, both at home and abroad in the tissue cell culture using true bacteria inducer stimulate increased the production of secondary metabolites is a hot research. Preparation and endophytic fungi of taxus chinensis can be inducer taxus cell carries on the induction, the yield of taxol. Endophytic fungi not only can ferment produce taxol by themselves, can also through the preparation of paclitaxel inducer used in taxus chinensis cell culture.
5 Conclusion
Use of the advantages of endophytic fungi of taxus chinensis production of taxol is: easy to mass culture; Can grow in the simple culture medium; Specific growth rate of speed is high, can obtain a large number of products, etc. But now found the strains of the output failed to meet the requirements of industrial production (production in more than 1 mg/L). The key problem lies in the separation of the endophytic fungi screening and to increase the taxol production. In terms of technology, there are three ways to think is feasible:
One is the modified fermentation, optimization of fermentation conditions;
Secondly, improving bacterium;
A third is looking for a special fungus regulator. If it will be modern microbial mutation breeding and genetic engineering technology combining with the above mentioned methods, will cultivate high yield strains, screening for industrialized production, so as to solve the problem of shortage of yew alcohol, protect the ecological environment at the same time, positive role for human benefit.
