Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.     

Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.     

Molecular cloning and characterization of a cytochrome P450 taxoid 2alpha-hydroxylase involved in Taxol biosynthesis

The Taxol biosynthetic pathway, arising from the primary isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in yew (Taxus), consists of approximately twenty steps, at least nine of which are thought to be cytochrome P450-mediated oxygenations. Several oxygenases involved in the early hydroxylation steps of the pathway have been identified and the corresponding genes have been cloned; however, defining the enzymes and their genes responsible for oxygenations in the central portion of the pathway is more difficult because neither the exact sequence of reactions nor the relevant intermediates are known. A surrogate substrate, (+)-taxusin (taxa-4(20),11(12)-dien-5alpha,9alpha,10beta,13alpha-tetraol tetraacetate), that was previously employed in the isolation of a taxoid 7beta-hydroxylase, was used here to functionally screen a family of cytochrome P450 oxygenases originating from a Taxus cell EST library. This in vivo screen in yeast led to the identification of a 1488bp cDNA clone (encoding a 495 residue protein) that was capable of producing 2alpha-hydroxytaxusin from taxusin with a K(m) value of 10.5 +/- 2.7 microM and k(cat) of about 0.05 s(-1) for the surrogate substrate. This structurally typical cytochrome P450 resembles most closely the previously isolated taxoid 7beta-hydroxylase, which also uses taxusin as a substrate, and both 2alpha- and 7beta-hydroxylases are capable of the reciprocal conversion of their respective pentaol tetraacetate products to the common hexaol tetraacetate. This C2-hydroxylase would appear to mediate the mid-pathway functionalization of the C2-position of the taxane core that ultimately bears a benzoyl group as an important Taxol pharmacophore. Overexpression of this cytochrome P450 taxoid 2alpha-hydroxylase in Taxus cells may improve Taxol yields and could prove useful in the production of other 2alpha-hydroxy taxoids as starting materials for subsequent acylation at this position.     

Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey

The literature concerning the tissue culture of Taxus sp. as an alternative source for taxoid production is reviewed. The aim of this review is to summarize and discuss the progress achieved with the approaches and methods used for the establishment of various Taxus culture systems, the methods used for the evaluation of taxoid production, the multiple factors which control taxoid production and the feasibility of the in vitro production of taxoids on a commercial scale.     

穗花杉中不存在紫杉醇的化学及分子生物学证据

穗花杉(Amentotaxus argotaenia(Hance)Pilger)是红豆杉科穗花杉属的一个种。由于穗花杉与红豆杉属植物具有较近的亲缘关系,有研究者认为穗花杉也可以合成紫杉醇,并利用HPLC法测到其含有紫杉醇。但这仅仅是依靠HPLC中出峰时间来判断,并没有质谱结果。该研究利用LC-MS对穗花杉茎叶的化学提取物分析结果显示,穗花杉的茎叶中均未发现紫杉醇。为了进一步证明穗花杉不能合成紫杉醇,该研究利用欧洲红豆杉(Taxus baccata L.)紫杉醇生物合成关键基因-紫杉二烯合成酶基因(Taxadiene synthase gene,TbTS)的序列TBLASTN比对穗花杉本地转录组,从中找出并克隆得到与TbTS同源性最高的AarTSL1基因;利用原核表达分析AarTSL1基因的编码蛋白,结果发现该基因不具有紫杉二烯合成酶编码基因的功能。该研究从化学和生物学两个方面均证明穗花杉无法合成紫杉醇,为以后通过植物学和化学分类学等研究穗花杉的分类学地位提供了新的有用信息。     

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。     

Characterization of Four Clustered and Coregulated Genes Associated with Fumonisin Biosynthesis in Fusarium verticillioides

Fumonisins are mycotoxins that cause several fatal animal diseases, including cancer in rats and mice. These toxins are produced by several Fusarium species, including the maize pathogen Fusarium verticillioides, and can accumulate in maize infected with the fungus. We have identified four F. verticillioides genes (FUM6, FUM7, FUM8, and FUM9) adjacent to FUM5, a previously identified polyketide synthase gene that is required for fumonisin biosynthesis. Gene disruption analysis revealed that FUM6 and FUM8 are required for fumonisin production and Northern blot analysis revealed that expression of all four recently identified genes is correlated with fumonisin production. Nucleotide sequence analysis indicated that the predicted FUM6 translation product is most similar to cytochrome P450 monooxygenase-P450 reductase fusion proteins and the predicted products of FUM7, FUM8, and FUM9 are most similar to type III alcohol dehydrogenases, class-II alpha-aminotransferases, and dioxygenases, respectively. Together, these data are consistent with FUM5 through FUM9 being part of a fumonisin biosynthetic gene cluster in F. verticillioides.     

Preliminary assessment of the C13-side chain 2'-hydroxylase involved in taxol biosynthesis

The biosynthesis of the anticancer drug Taxol in yew (Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13-O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts alpha-phenylalanine to beta-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield beta-phenylalanoyl baccatin III (i.e., N-debenzoyl-2'-deoxytaxol) that undergoes subsequent 2'-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both beta-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2'-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of beta-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of beta-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.     

薇甘菊颈盲蝽细胞色素P450 PmCYP4基因克隆及表达分析

【目的】薇甘菊颈盲蝽是入侵植物薇甘菊的天敌昆虫。CYP4家族基因在专食性昆虫与宿主植物的相互作用中发挥着极其重要的作用,探明其在不同部位的表达情况,可为薇甘菊生物控制提供科学依据。【方法】采用RACE技术克隆薇甘菊颈盲蝽CYP基因,实时荧光定量PCR检测其在不同部位的表达情况。【结果】PmCYP4C1基因全长1713 bp,其中ORF长1500 bp,共编码500个氨基酸,理论分子质量为57.44 ku,无信号肽;与其他昆虫CYP4家族基因的同源性大于40%,与温带臭虫CYP的亲缘关系最近。该基因在雌、雄虫各部位均有表达,且都是足部的表达量明显地高于其他部位;雌、雄虫的表达差异在于雄虫翅膀中的表达量明显地高于触角和残体,但在雌虫中这3个部位的表达量无显著差异,且雄虫翅膀中的表达量显著地高于雌虫,是其2.37倍。【结论】薇甘菊颈盲蝽PmCYP4基因除参与代谢有毒物质外,其主要功能可能是编码与薇甘菊颈盲蝽运动相关的酶。     

Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

Biodegradation of bisphenol A by cells and cell lysate from <Emphasis Type="Italic">Sphingomonas </Emphasis>sp. strain AO1

The capacity and pathway of bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] degradation in Sphingomonassp. strain AO1, which was isolated from the soil of a vegetable-growing field in Japan, were investigated. The bacterial strain was able to grow in a basal mineral salt medium containing BPA as the sole carbon source (BSMB medium), and was able to degrade 115 gml^–1 BPA in 6h in L medium. Several BPA metabolites were detected in the culture supernatant by HPLC and then identified by GC-MS and LC-MS-MS. These compounds were confirmed to be the same as those reported for other BPA-degrading bacteria. BPA degradation by cells in the basal mineral salt medium was induced by BPA, and activity was detected only in the intracellular soluble fraction in the presence of coenzymes, such as NADH, NAD⁺ , NADPH or NADP⁺. The addition of metyrapone, a cytochrome P450 inhibitor, to BSMB medium resulted in a decrease in BPA degradation and cell growth. The BPA-degradation activity of the intracellular soluble fraction was also inhibited by the cytochrome P450 inhibitor. Carbon monoxide difference spectra indicated that cytochrome P450 was present in the cells and that the amount of cytochrome P450 corresponded to the cellular BPA-degradation activity. Our results provide evidence that the cytochrome P450 system is involved in BPA metabolism in Sphingomonassp. strain AO1.     

Comparative study of topogenesis of cytochrome P450scc (CYP11A1) and its hybrids with adrenodoxin expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Hybrid proteins consisting of the mature form of cytochrome P450scc (mP) and adrenodoxin (Ad), attached to either the NH2- or COOH-terminus (Ad-mP and mP-Ad, respectively), were expressed in E. coli. Spectral and catalytic properties of P450scc were studied using the membrane fraction of E. coli cells. It has been shown that the Ad amino acid sequence attached to the termini of the P450scc-domain neither affects the insertion of a hybrid protein into the cytoplasmic membrane nor influences its heme binding ability. The results suggest that Ad attached to the NH2-terminus does not markedly affect the folding of the P450scc-domain, but cholesterol hydroxylase/lyase activity of the Ad-mP hybrid was found to be much lower than that of the native P450scc enzyme. The modification of the COOH-terminus does not alter the specific P450scc activity, but results in a dramatic increase in the amount of hybrid protein with incorrectly folded P450scc domain.     

Toxin-binding properties of cytochrome P450 in Saccharomyces cerevisiae and Kluyveromyces marxianus

Microsomes from Kluyveromyces marxianus GK1005 examined by carbon monoxide difference spectroscopy showed no evidence of cytochrome P450, in contrast to microsomes isolated from a control strain of Saccharomyces cerevisiae. Benzo[a]pyrene produced a typical Type I-binding spectrum with microsomes of both yeasts, with K _s values of 82 M (S. cerevisiae) and 70 M (K. marxianus). While aflatoxin B₁ generated a typical Type I-binding spectrum with microsomes from S. cerevisiae (K _s of 178 M), the toxin did not produce a recognisable binding spectrum with microsomes from K. marxianus.     

Expression of cytochrome P450scc in Escherichia coli cells: Intracellular location and interaction with bacterial redox proteins

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.     

Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea

Polysaccharides (5, 10, 25, 50 and 100 microg ml(-1)) from soybeans and soybeans fermented with Phellinus igniarius or Agrocybe cylindracea inhibited cytochrome P450 1A1, cytochrome P450 1A2 and cytochrome P450 2B1 activities in rat liver microsomes. The polysaccharides (5, 10 and 25 microg ml(-1)) also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity. The most potent inhibitors of cytochrome P450 isozymes and ornithine decarboxylase activities were the polysaccharides from soybeans fermented with Agrocybe cylindracea.     

Pest and disease resistance enhanced by heterologous suppression of a <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> cytochrome P450 gene

The functional role of theNicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.     

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.     

Gibberellin Biosynthetic Pathway inGibberella fujikuroi:Evidence for a Gene Cluster

Differential screening of aGibberella fujikuroicDNA library was used to successfully clone and identify genes involved in the pathway of gibberellin biosynthesis. Several cDNA clones that hybridized preferentially to a cDNA probe prepared from mycelium induced for gibberellin production were isolated and characterized. The deduced amino acid sequences of two (identical) clones contained the conserved heme-binding motif of cytochrome P450 monooxygenases (FXXGXXXCXG). One of these cDNA fragments was used as a homologous probe for the screening of a genomic library. A hybridizing 6.7-kb genomicSalI fragment was cloned into pUC19. The sequencing of this clone revealed that a second cytochrome P450 monooxygenase gene was closely linked to the first one. Since at least four cytochrome P450 monooxygenase-catalyzed steps are involved in the synthesis of gibberellins, chromosome walking was performed to find a further gene of this family or other genes involved in gibberellin pathway. Next to the two P450 monooxygenase genes, a putative geranylgeranyl diphosphate synthase gene, the copalyl diphosphate synthase gene, which is the first specific gene of the gibberellin pathway, and a third P450 monooxygenase gene were identified. These results suggest that at least some of the genes involved in the biosynthesis of gibberellins are closely linked in a gene cluster inG. fujikuroi,as has been recently found for other “dispensable” pathways in fungi.     

Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4

The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4. Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences. One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases. Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions     

Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria

During an incompatible interaction between tobacco and the bacterial phytopathogen Pseudomonas solanacearum, 2 classes of genes, the so-called hsr (hypersensitivity-related) genes, activated preferentially during the hypersensitive reaction, and the str (sensitivity-related) genes, expressed strongly during compatible and incompatible interactions, have been identified. In this report, two hsr cDNA clones, hsr515 and hsr201, as well as their expression patterns are presented. Hsr515 was found to encode a P450 monooxygenase and is most similar to the ripening-related avocado gene CYP71A1 (40.6% amino acid identity). Hsr201 presents 58.6% amino acid identity with pTom36, a tomato gene expressed during fruit maturation. The putative functions of the hsr gene products appear to be quite diverse and their characteristics of activation were found to be very conserved: accumulation of the corresponding mRNAs primarily in leaf areas in contact with the avirulent P. solanacearum strain or with a Pseudomonas fluorescens strain containing the hrpZ gene encoding a necrotizing polypeptide, harpin and absence of expression during normal plant development. Our results also suggest that, in a tobacco line expressing NahG, a lower level of salicylic acid, a compound associated with systemic acquired resistance, and also possibly involved in the development of necrotic lesions characteristic of the HR, does not affect the hsr gene expression.