红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展

紫杉醇是一种四环二萜酰胺类化合物,是从红豆杉科红豆杉属植物中提取分离出来的次生代谢物,是世界公认广谱、活性强的天然抗癌新药。但直接从植物中提取紫杉醇的传统生产方式,不仅产量低,且会对野生红豆杉资源造成严重破坏,同时紫杉醇的化学全合成也由于其结构复杂而不具备商业价值。与之相反,细胞培养技术具有受外界影响少、生产成本低、次生代谢产物多、细胞生长周期短的优势,是目前最具前景的紫杉醇生产方式。近年来随着科研水平的不断提升,紫杉醇无论在生理代谢调控、关键基因挖掘,还是新药物制剂与剂型及其类似物的开发和运用等方面,都取得了进展,但要建立紫杉醇商业化高产体系,还必须和前人的研究经验相结合。该文对红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展进行了综述,主要包括前人对红豆杉属植物组织与细胞培养相关的外植体、培养基、激素、培养条件、褐化等问题的研究,以及从代谢调节、培养方式、基因工程等多方面提高紫杉醇含量的最新进展,最后总结了当前研究的不足,并对今后通过多种组合方式来提高紫杉醇含量的生产途径进行了展望。以期促进红豆杉组织培养技术的进步,为药用资源保护和利用提供一定的理论基础与生产指导。     

Taxol biosynthetic genes

The function and properties of heterologously expressed full-length cDNA clones, isolated from a Taxus cDNA library and specific to Taxol biosynthesis, are summarized. Recombinant enzymes are described that catalyze early steps of the pathway, including taxadiene synthase, taxadien-5alpha-ol-O-acetyltransferase and taxadien-5alpha-yl acetate 10beta-hydroxylase, and that catalyze late steps, including 10-deacetylbaccatin III-10beta-O-acetyltransferase and taxane 2alpha-O-benzoyltransferase. The properties of Taxus geranylgeranyl diphosphate synthase are also described; although this synthase does not mediate a committed step of Taxol biosynthesis, it does provide the universal plastidial diterpenoid precursor, geranylgeranyl diphosphate, for initiating Taxol biosynthesis.     

Recent advances in chlorophyll biosynthesis

The importance of chlorophyll (Chl) to the process of photosynthesis is obvious, and there is clear evidence that the regulation of Chl biosynthesis has a significant role in the regulation of assembly of the photosynthetic apparatus. The understanding of Chl biosynthesis has rapidly advanced in recent years. The identification of genetic loci associated with each of the biochemical steps has been accompanied by a greater appreciation of the role of Chl biosynthesis intermediates in intracellular signaling. The purpose of this review is to provide a source of information for all the steps in Chl and bacteriochlorophyll a biosynthesis, with an emphasis on steps that are believed to be key regulation points.     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

Recent advances in chlorophyll biosynthesis

The importance of chlorophyll (Chl) to the process of photosynthesis is obvious, and there is clear evidence that the regulation of Chl biosynthesis has a significant role in the regulation of assembly of the photosynthetic apparatus. The understanding of Chl biosynthesis has rapidly advanced in recent years. The identification of genetic loci associated with each of the biochemical steps has been accompanied by a greater appreciation of the role of Chl biosynthesis intermediates in intracellular signaling. The purpose of this review is to provide a source of information for all the steps in Chl and bacteriochlorophyll a biosynthesis, with an emphasis on steps that are believed to be key regulation points.     

Application of DNA methyltransferases in targeted DNA methylation

Taxol is a valuable plant-derived drug showing activity against various cancer types. Worldwide efforts had been made to overcome the supply problem, because the supply by isolation from the bark of the slow-growing yew trees is limited. Plant cell cultures as well as chemical and biotechnological semisynthesis are processes, which are intensively investigated for the production of taxanes paclitaxel (Taxol) and docetaxel (Taxotere) in the last few years. This article provides a comparison of the current research on taxane biosynthesis and production in yew cell cultures.     

内生真菌紫杉醇生物合成的研究现状与展望

紫杉醇是重要的抗癌药物之一,已经证明其对多种癌症具有显著疗效。目前,人们主要是从红豆杉的树皮中提取、分离和纯化紫杉醇,但由于红豆杉为生长缓慢、散生、濒危的珍稀植物,且随着紫杉醇临床用途的不断拓宽,市场需求的稳定增长,单纯依靠从红豆杉树皮中提取紫杉醇已经无法满足日益增长的市场需求。为了解决紫杉醇的药源不足,科学家已把目光从红豆杉树分离提取紫杉醇转向了其他替代方法,如化学全合成、半合成、组织培养与细胞培养、微生物发酵法生产紫杉醇等。因此,了解内生真菌紫杉醇生物合成的分子基础和遗传调控机制,对解析内生真菌紫杉醇生物合成机制、构建高产紫杉醇基因工程菌株和早日实现内生真菌紫杉醇工业化生产具有重要的科学意义和现实意义。结合本课题组多年来的科研工作,概述了红豆杉细胞紫杉醇生物合成途径、内生真菌发酵生产紫杉醇的优势、产紫杉醇内生菌的分离研究现状和生物多样性及紫杉醇生物合成相关基因的研究现状。内生真菌生物发酵合成紫杉醇是可以无限生产、大量获取紫杉醇、解决紫杉醇药源短缺问题的很有前景的方法之一。     

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone‐induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone‐triggered ABA generation and assayed the effect of fluridone on ozone‐induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone‐triggered ABA generation but also blocked the ozone‐induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose‐dependent. Interestingly, the suppression of fluridone on ozone‐induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone‐induced Taxol production of T. chinensis suspension cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

紫杉醇生物合成途径中相关酶的研究进展

抗癌新药紫杉醇是具有萜类环状结构的一种重要次生代谢产物 ,研究紫杉醇的生物合成对于通过基因工程手段提高紫杉醇的产量 ,解决目前资源紧缺造成的巨大供求矛盾具有重要意义 ,这就需要对紫杉醇生物合成途径中催化各步反应 (尤其是关键步骤 )的酶以及编码这些酶的基因有个全面的了解。对近年来紫杉醇生物合成途径中相关酶的研究进行了综述 ,大部分酶及相关基因已被分离、克隆 ,但还有一些酶及相关基因没有发现 ,有待继续深入研究。     

紫杉醇生物合成相关酶类的研究进展

紫杉醇是红豆杉属植物次生代谢产物之一,是近20年来抗癌药物研究领域的重要发现。弄清楚紫杉醇合成途径和相关酶的反应可以从根本上大大提高紫杉醇的产量。综述近几年来紫杉醇生物合成途径中相关酶的研究工作,包括已经得到相应cDNA克隆的紫杉二烯合成酶,细胞色素P450氧化酶和3个紫杉烷的乙酰转移酶,由于GGPP是紫杉醇合成的必需前体,HMGR和GGPP合成酶的相关情况也有简述。     

Intracellular accumulation of c-di-GMP and its regulation on self-flocculation of the bacterial cells of Zymomonas mobilis

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner–Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.     

Translocation of isopentenyl pyrophosphate for Taxol biosynthesis in suspension cultures of Taxus chinensis var. mairei

Two inhibitors of metabolite translocators, sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), respectively, were added into suspension cultures of Taxus chinensis var. mairei at the early and late growth phases to investigate the translocation of isopentenyl pyrophosphate (IPP) from cytoplasm to plastids for Taxol biosynthesis. NaPP and DLG hardly affected cell biomass and viability regardless of the phases of their introduction. The Taxol content varied less when NaPP or DLG was added at day 7 but decreased obviously when NaPP or DLG was introduced at day 14. It is thus inferred that NaPP and DLG inhibited Taxol biosynthesis by blocking IPP translocation at the late growth phase, suggesting that the translocation of IPP be involved only at the late growth phase of Taxus cells.     

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

The last few steps in the biosynthesis of the anticancer drug Taxol in yew (Taxus) species are thought to involve the attachment of β-phenylalanine to the C13-O-position of the advanced taxane diterpenoid intermediate baccatin III to yield N-debenzoyl-2′-deoxytaxol, followed by hydroxylation on the side chain at the C2′-position to afford N-debenzoyltaxol, and finally N-benzoylation to complete the pathway. A cDNA encoding the N-benzoyl transferase that catalyzes the terminal step of the reaction sequence was previously isolated from a family of transferase clones (derived from an induced Taxus cell cDNA library) by functional characterization of the corresponding recombinant enzyme using the available surrogate substrate N-debenzoyl-2′-deoxytaxol [K. Walker, R. Long, R. Croteau, Proc. Nat. Acad. Sci. USA 99 (2002) 9166–9171]. Semi-synthetic N-debenzoyltaxol was prepared by coupling of 7-triethylsilybaccatin III and (2R,3S)-β-phenylisoserine protected as the N-Boc N,O-isopropylidene derivative by means of carbodiimide activation and formic acid deprotections. The selectivity of the recombinant N-transferase for N-debenzoyltaxol was evaluated, and the enzyme was shown to prefer, by a catalytic efficiency factor of two, N-debenzoyltaxol over N-debenzoyl-2′-deoxytaxol as the taxoid co-substrate in the benzoyl transfer reaction, consistent with the assembly sequence involving 2′-hydroxylation prior to N-benzoylation. Selectivity for the acyl/aroyl-CoA co-substrate was also examined, and the enzyme was shown to prefer benzoyl-CoA. Transfer from tigloyl-CoA to N-debenzoyltaxol to afford cephalomannine (Taxol B) was not observed, nor was transfer observed from hexanoyl-CoA or butanoyl-CoA to yield Taxol C or Taxol D, respectively. These results support the proposed sequence of reactions for C13-O-side chain assembly in Taxol biosynthesis, and suggest that other N-transferases are responsible for the formation of related, late pathway, N-acylated taxoids.     

Indole-3-butyric acid in plant growth and development

Within the last ten years it has been established by GC-MS thatindole-3-butyric acid (IBA) is an endogenous compound in a variety ofplant species. When applied exogenously, IBA has a variety of differenteffects on plant growth and development, but the compound is stillmainly used for the induction of adventitious roots. Using moleculartechniques, several genes have been isolated that are induced duringadventitious root formation by IBA. The biosynthesis of IBA in maize(Zea mays L.) involves IAA as the direct precursor. Microsomalmembranes from maize are able to convert IAA to IBA using ATP andacetyl-CoA as cofactors. The enzyme catalyzing this reaction wascharacterized from maize seedlings and partially purified. The invitro biosynthesis of IBA seems to be regulated by several externaland internal factors: i) Microsomal membranes from light-grownmaize seedlings directly synthesize IBA, whereas microsomal membranesfrom dark-grown maize plants release an as yet unknown reaction product,which is converted to IBA in a second step. ii) Drought and osmoticstress increase the biosynthesis of IBA maybe via the increaseof endogenous ABA, because application of ABA also results in elevatedlevels of IBA. iii) IBA synthesis is specifically increased byherbicides of the sethoxydim group. iv) IBA and IBA synthesizingactivity are enhanced during the colonization of maize roots with themycorrhizal fungus Glomus intraradices. The role of IBA forcertain developmental processes in plants is discussed and somearguments presented that IBA is per se an auxin and does notact via the conversion to IAA.     

Taxol,an anticancer drug produced by an endophytic fungus <Emphasis Type="Italic">Bartalinia robillardoides</Emphasis> Tassi,isolated from a medicinal plant, <Emphasis Type="Italic">Aegle marmelos</Emphasis> Correa ex Roxb.

Taxol is an important anticancer drug widely used in the clinic. An endophytic fungus Bartalinia robillardoides (strain AMB-9) was isolated from Aegle marmelos, a medicinal plant and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed chromatographically and spectrometrically, for the presence of Taxol. The amount of taxol produced by this endophytic fungus was quantified by HPLC. It produced 187.6 μg/L of taxol which suggests that the fungus can serve as a potential material for genetic engineering to improve the production of Taxol. This fungal taxol isolated from the organic extract of this fungal culture, has strong cytotoxic activity towards BT 220, H116, Int 407, HL 251 and HLK 210 human cancer cells in vitro, tested by Apoptotic assay.     

Biomimetic Synthesis and Studies Toward Enantioselective Synthesis of Flindersial Alkaloids

A strategy allowing both stereocontrol and control over structural isomer formation has been defined for the antimalarial flindersial alkaloids. The recently reported flinderoles were demonstrated to be derived from the natural product borrerine. The structural isomers of flinderoles, the borreverines, were also produced in vitro along with the flinderoles through the dimerization of borrerine in acidic conditions. This result is thought to replicate the biosynthesis of these compounds. Flinderoles A, B, and C, desmethylflinderole C, isoborreverine, and dimethylisoborreverine can each be synthesized in three steps from tryptamine. Furthermore, progress toward a concise enantioselective synthesis of flinderoles A, B, and C is described. This work includes enantioselective conjugate addition to an unprotected indole‐appended enone. Chirality 27:14–17, 2015. © 2013 Wiley Periodicals, Inc.     

Taxol-induced apoptotic cell death in suspension cultures of Taxus cuspidata

Taxol caused apoptotic cell death of Taxus cuspidata in suspension cultures. Typical morphological and biochemical changes of apoptosis were observed by microscopy and total DNA agarose gel electrophoresis. Taxus cuspidata responded to the added Taxol by increasing the biosynthesis of Taxol. The percentage of apoptotic cells in total cells increased with the concentration of added Taxol. With Taxol added at 10 mg l^–1, the maximum concentration of Taxol produced was 23 mg l^–1, 3 times higher than that of the control culture.