1.Combinational expression of geranylgeranyl diphosphate synthase and taxadiene synthase in Coprinopsis cinerea.
Linfeng YOU ; Haixing YANG ; Junfang LIN ; Zhiwei YE ; Liqiong GUO ; Yanhua XIN
Chinese Journal of Biotechnology 2015;31(3):375-383
Taxa-4(5),11(12)-diene is the precursor for paclitaxel biosynthesis. The diterpenoid paclitaxel (marketed as Taxol), a plant secondary metabolite isolated from yew, is an effective drug widely used in the treatment of numerous cancers. However, further application of taxol has been restricted due to its low yield in plants and the difficulties in extraction. To increase the intact isoprene flux, we constructed the fusion gene plasmid pBgGGTS and individual cassette plasmid pBgGGgTS to enhance the expression levels of geranylgeranyl diphosphate synthase gene (ggpps) and a taxadiene synthase gene (ts) in Coprinopsis cinerea. These two plasmids were separately transformed into C. cinerea LT2 strain, resulting in several putative transformants. Putative transformants were determined by PCR technique, indicating that 5 out of 13 putative transformants transformed by pBgGGTS and 6 out of 13 putative transformants transformed by pBgGGgTS, respectively. Additionally, the Southern blotting analysis of these 10 transformants confirmed that both ggpps and ts gene were stably integrated into the genome of C. cinerea. Crude extracts from each of the transformants were analyzed. There is no difference in the mycelium extracts among the wild-type LT2 and two types of transformants. However, analysis of culture filtrates indicated that an additional GC peak was found at the retention time of 16.762 min which was absent in the wild type control. The mass fragmentation pattern of this peak had the same diagnostic ions with taxa-4(5),11(12)-diene. According to peak area, the amounts of taxa-4(5),11(12)-diene in each fermented broth were 44 ng/L (transformed with pBgGGgTS) and 30 ng/L (transformed with pBgGGTS), respectively. In conclusion, co-expression of the ggpps and ts gene could increase the taxadiene production in C. cinerea.
Agaricales
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metabolism
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Alkenes
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metabolism
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Diterpenes
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metabolism
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Farnesyltranstransferase
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genetics
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metabolism
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Genetic Engineering
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Isomerases
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genetics
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metabolism
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Paclitaxel
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Plasmids
2.Biotransformation of taxanes.
Li-ping ZHANG ; Ke-di CHENG ; Ping ZHU
Acta Pharmaceutica Sinica 2004;39(2):153-157
3.Research advances of diterpene synthase.
Yun-fei HE ; Wei GAO ; Ta-si LIU ; Wen-yuan LI ; Lu-qi HUANG
Acta Pharmaceutica Sinica 2011;46(9):1019-1025
Diterpenes, an important class of natural compounds, are widely distributed in nature. As the valuable diterpenoids continue to be found, diterpene synthase in the course of diterpene synthesis get as much attention as possible. The multiformity of end-product-diterpenoids were also due to the diversity of diterpene synthase. This paper focuses on the advances in recent biosynthesis pathway of diterpene and types, cloning, catalytic mechanism, synthetic biology application.
Alkyl and Aryl Transferases
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metabolism
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Biosynthetic Pathways
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Diterpenes
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metabolism
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Isomerases
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metabolism
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Phosphorus-Oxygen Lyases
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metabolism
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Plant Proteins
;
metabolism
4.High cell density culture of an engineered yeast strain for sclareol production.
Yehua SONG ; Hongwei SHEN ; Wei YANG ; Xiaobing YANG ; Zhiwei GONG ; Zongbao K ZHAO
Chinese Journal of Biotechnology 2015;31(1):147-151
Cell growth profiles were evaluated in shake-flask culture to improve sclareol production by the engineered yeast strain Saccharomyces cerevisiae S7. Product formation was tightly coupled with cell growth. High cell density cultures were performed with different carbon sources using a dissolved oxygen level feedback-control strategy in a 3 L bioreactor. The titers of sclareol were 253 mg/L, 386 mg/L and 408 mg/L, respectively, when glucose, ethanol and glucose/ethanol mixture were used as the carbons sources. The maximal titer was 27-fold higher than that obtained under shake-flask culture conditions. The results suggested that the presence of ethanol was beneficial to sclareol production. These results provided useful information for optimization of yeast cell factory and efficient production of terpenoids.
Bioreactors
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Culture Media
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Diterpenes
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metabolism
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Ethanol
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Glucose
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Industrial Microbiology
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methods
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Oxygen
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Saccharomyces cerevisiae
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metabolism
5.Correlation between expression level of functional genes and tanshinones' accumulation in Salvia miltiorrhiza from different areas.
Beining LI ; Xiaoli ZHOU ; Luqi HUANG ; Xueyong WANG ; Chunsheng LIU
China Journal of Chinese Materia Medica 2011;36(24):3406-3409
OBJECTIVETo study the relationship between functional gene expression in Salvia miltiorrhiza from different producing areas and active principles, which might provide scientific basis for the gene regulation of tanshinones.
METHODThe quantitative determination of cryptotanshinone and tanshinone II A was carried out by using HPLC method, expression level of 3 functional genes of SmAACT, SmCMK and SmIPPI were investigated by real-time PCR method.
RESULTThe content of active principles together with expression level of SmAACT and SmCMK were higher in S. miltiorrhiza from genuine producing areas including Henan and Shanxi, but lower in samples from Beijing which was non-genuine producing area.
CONCLUSIONExpression level of SmAACT and SmCMK had close relationships involving tanshinones' accumulation, but the SmIPPI gene had not.
Diterpenes, Abietane ; metabolism ; Gene Expression ; Real-Time Polymerase Chain Reaction ; Salvia miltiorrhiza ; genetics ; metabolism
6.Diterpenoid alkaloids from roots of Aconitum kongboense.
Chao-Yin XIE ; Shuai HUANG ; Lin CHEN ; Feng GAO ; Xian-Li ZHOU
China Journal of Chinese Materia Medica 2021;46(17):4424-4432
The chemical constituents from the roots of Aconitum kongboense were studied. Twenty-five diterpenoid alkaloids were isolated from the 95% methanol extract of the roots of A. kongboense by silica gel, reverse-phase silica gel and basic alumina column chromatography. They included a new aconitine-type diterpenoid alkaloid, named as kongboensenine(1), and twenty-four known ones(2-25), i.e., acotarine F(2), acotarine G(3), 14-acetyltalatisamine(4), talatisamine(5), indaconitine(6), yunaconitine(7), chasmanine(8), 6-epi-foresticine(9), homochasmanine(10), 8-deacetyl-yunaconitine(11), chasmaconitine(12), ajaconine(13), franchetine(14), ezochasmanine(15), crassicautine(16), 14-O-deacylcrassicausine(17), genicunine A(18), falconeridine(19), sachaconitine(20), liljestrandisine(21), 8-methyl-14-acetyltalatisamine(22), kongboendine(23), 14-benzoylchasmanine(24) and pseudaconine(25). Their structures were elucidated by common spectroscopic methods including high-resolution electrospray ionization mass spectrometry(HR-ESI-MS) and nuclear magnetic resonance(NMR) techniques. Compounds 2-4, 10, 13, 15-19 and 21-22 were isolated from this plant for the first time. Experimental results showed that all compounds did not have a significant inhibitory activity against acetylcholinesterase(AChE).
Acetylcholinesterase
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Aconitum/metabolism*
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Alkaloids
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Diterpenes
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Magnetic Resonance Spectroscopy
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Molecular Structure
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Plant Roots/metabolism*
7.Biosynthesis and regulation of diterpenoids in medicinal plants.
Junze REN ; Yu WU ; Zhanpin ZHU ; Ruibing CHEN ; Lei ZHANG
Chinese Journal of Natural Medicines (English Ed.) 2022;20(10):761-772
Plant diterpenoids are widely distributed and abundant natural products with diverse structures and functions in nature, which have been commonly used in pharmaceutical, agricultural and industrial production. In recent years, plant diterpenoids have attracted increasing attention, including their biosynthetic pathways, transcriptional regulatory networks, and biological functions. Herein, the biosynthetic pathways of diterpenoids are summarized in a modular fashion. Further, the regulatory network between diterpene biosynthesis and environmental factors is reviewed. Insights into diterpene metabolism may drive elucidation of complex active diterpene pathways and serve as a knowledge repository for metabolic engineering and cell factory construction.
Plants, Medicinal/metabolism*
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Diterpenes/chemistry*
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Metabolic Engineering
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Biosynthetic Pathways
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Biological Products/metabolism*
8.Metabolomics analysis of taxadiene producing yeasts.
Huifang YAN ; Mingzhu DING ; Yingjin YUAN
Chinese Journal of Biotechnology 2014;30(2):223-231
In order to study the inherent difference among terpenes producing yeasts from the point of metabolomics, we selected taxadiene producing yeasts as the model system. The changes of cellular metabolites during fermentation log phase of artificial functional yeasts were determined using metabolomics methods. The results represented that compared to W303-1A as a blank control, the metabolites in glycolysis, tricarboxylic acid cycle (TCA) cycle and several amino acids were influenced. And due to the changes of metabolites, the growth of cells was inhibited to a certain extent. Among the metabolites identified, citric acid content in taxadiene producing yeasts changed the most, the decreasing amplitude reached 90% or more. Therefore, citric acid can be a marker metabolite for the future study of artificial functional yeasts. The metabolomics analysis of taxadiene producing yeasts can provide more information in further studies on optimization of terpenes production in heterologous chassis.
Alkenes
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metabolism
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Amino Acids
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metabolism
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Citric Acid
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analysis
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Citric Acid Cycle
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Diterpenes
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metabolism
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Fermentation
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Glycolysis
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Metabolome
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Metabolomics
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Yeasts
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metabolism
9.Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi.
Yi-Qing WU ; Yue CAO ; Xin LIU ; Zhi-Hong CHENG
Chinese Journal of Natural Medicines (English Ed.) 2016;14(12):939-945
The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC values being 35.59 ± 5.37 μmol·L (Caco-2), 24.04 ± 4.70 μmol·L (MCF-7), and 22.24 ± 5.19 μmol·L (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.
Antineoplastic Agents
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chemistry
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metabolism
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Biotransformation
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Cell Line, Tumor
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Diterpenes
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chemistry
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metabolism
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Gibberella
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metabolism
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Humans
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Hydroxylation
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Molecular Structure
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Mortierella
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metabolism
;
Stereoisomerism
10.Mechanism of copper and zinc on tanshinones of cultivated Salvia miltiorrhiza.
Bin WANG ; De-hui LIU ; Xian-he TAN ; Kang-cai WANG ; Ya-qin GUO ; Jing-yu DAI
China Journal of Chinese Materia Medica 2008;33(17):2082-2087
OBJECTIVETo study the mechanism of microelement including Cu and Zn on the accumulation of three danshinones in Salviae miltiorrhizae root and build a theory basis for its good quality and high yield.
METHODSand culture experiments were conducted to study the effect of Cu and Zn on the accumulation of three danshinones and oxidase including peroxidase and polyphenol oxidase activity in the plant root. The correlation between available Cu and Zn contents in matrix and oxidase activity in the plant root and, the correlation between available Cu and Zn contents in matrix and contents of tanshinones in the root were discussed.
RESULTContents of danshinones in the root increased with the increasing of Cu and Zn concentration. Dynamic monitoring on contents of dan-shinones of the plant roots growing in the pots with different Cu and Zn concentration in the whole growing season showed that the contents of danshinones for 60 days were the lowest, for 120 days the highest and then dropped for 150 days. In addition, among available Cu and Zn contents of matrix, oxidase including peroxidase and polyphenol oxidase activity and contents of tanshinones in the root,the correlation between two factors were significant difference (P < 0.05 or P < 0.01).
CONCLUSIONThe mechanism of Cu and Zn on the accumulation of danshinones may be that Cu and Zn improve the activity of peroxidase and polyphenol oxidase, which promote transformation of phenolic compounds to terpenes and therefore to increase contents of danshinones.
Catechol Oxidase ; metabolism ; Copper ; metabolism ; Diterpenes, Abietane ; Drugs, Chinese Herbal ; analysis ; metabolism ; Peroxidase ; metabolism ; Phenanthrenes ; metabolism ; Plant Proteins ; metabolism ; Salvia miltiorrhiza ; enzymology ; metabolism ; Zinc ; metabolism