Enhanced catalytic activities and modified substrate preferences for taxoid 10β-<Emphasis Type="Italic">O</Emphasis>-acetyl transferase mutants by engineering catalytic histidine residues

Objectives

Taxoid 10β-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis.

Results

Residues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activity of DBAT. Enzymatic activity assays revealed that the k_cat/K_M values of mutant H162A/R363H, D166H, R363H, D166H/R363H acting on 10-deacetylbaccatin III were about 3, 15, 26 and 60 times higher than that of the wild type of DBAT, respectively. Substrate preference assays indicated that these mutants (H162A/R363H, D166H, R363H, D166H/R363H) could transfer acetyl group from unnatural acetyl donor (e.g. vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-deacetylbaccatin III.

Conclusion

Taxoid 10β-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.

     

Expression of an acetyl-CoA synthase and a CoA-transferase in Escherichia coli to produce modified taxanes in vivo

Previous in vitro studies revealed that the 10-deacetylbaccatin III 10beta-O-acetyltransferase (DBAT) from Taxus can catalyze the transfer of acetyl, propionyl or n-butyryl from CoA to the C10-hydroxyl of 10-deacetylbaccatin III. Accordingly, Escherichia coli JM109 were transformed to recombinantly express dbat, and this enzyme function was coupled to that of acetyl-CoA synthase (acs, EC 6.2.1.1) expressed from and regulated by genes encoded on the bacterial chromosome. Incubation of the bacteria with 10-deacetylbaccatin III and increasing concentrations of acetic acid revealed an in vivo conversion ( approximately 10%) of substrate to natural product baccatin III (C10-acetylated), which was remarkably similar to the relative conversion without acid supplementation. Incubation of the modified E. coli with 5 mM propionic acid, revealed a fivefold increase in the conversion ( approximately 13%) of 10-deacetylbaccatin III to 10-deacetyl-10-propionylbaccatin III, compared to approximately 2% conversion in the absence of exogenous propionate. To produce the butyrylbaccatin III analog in vivo, bacteria were engineered to co-express the dbat and atoAD (EC 2.8.3.8) genes; the latter encodes an acetoacetate: acetyl-CoA CoA-transferase that activates butyrate to butyryl CoA. The bacteria were incubated with 10-deacetylbaccatin III and 25-100 mM butyrate, and a maximum of approximately 2.6% conversion to 10-butyrylbaccatin III was observed compared to approximately 0.6% conversion when no exogenous butyrate was supplied.     

Enzymatic acetylation of 10-deacetylbaccatin III to baccatin III by C-10 deacetylase from Nocardioides luteus SC 13913

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.     

南方红豆杉枝叶中药用抗癌活性物质含量

通过高效液相色谱（HPLC）,测定了人工栽培南方红豆杉枝叶中药用抗癌活性物质紫杉醇（taxol）、三尖杉宁碱（cephalomannine）和人工半合成紫杉醇的主要原料巴卡亭Ⅲ（baccatin Ⅲ）、10 去乙酰基巴卡亭Ⅲ（10-deacetylbaccatin Ⅲ,10-DAB）在一个生长季的含量变化.研究表明：从4月新枝叶萌发至11月枝叶基本停止生长,南方红豆杉枝叶中紫杉醇等药用活性物质含量季节性变化明显.其中紫杉醇和三尖杉宁碱含量的最高值都出现在5月;巴卡亭Ⅲ和10-去乙酰基巴卡亭Ⅲ含量的最高峰值分别出现在9月和4月.相关分析发现,南方红豆杉枝叶中4种药用活性物质之间有很好的相关性,其中紫杉醇与三尖杉宁碱含量呈显著正相关(P<0.05),三尖杉宁碱含量与10-去乙酰巴卡亭Ⅲ含量呈显著负相关(P<0.05).     

Seasonal changes in the concentrations of four taxoids in Taxus baccata L. during the autumn-spring period.

The concentrations of four common taxoids: baccatin III, paclitaxel, cephalomannine and 10-deacetylbaccatin III (10-DAB III) were measured in fresh needles and stems of Taxus baccata L. during the late autumn-spring period (November'96-April'97) which has not been investigated to date in this species. Baccatin III, paclitaxel and 10-DAB III were present on the surface of the twigs in concentrations of 8-26 micrograms/1000 g (fresh weight). Changes in the levels of baccatin III and paclitaxel inside the needles and stems showed similar trends over the investigated period. From November to March the total level of taxoids differed between the needles and stems, and were the same only in April. Total levels in fresh needles were stable from December to March. The highest concentrations of 10-DAB III in the whole analysed period in fresh stems were measured, as well as in the fresh needles except for samples collected in November and December when the levels of cephalomannine were higher. The concentrations of paclitaxel were usually the lowest. These results confirm that epigenetic factors--date of collection (and thus phyllogenesis) and kind of plant tissue--determine taxoid levels during the late autumn-spring period in T. baccata. The opposite patterns of changes for 10-DAB III and cephalomannine, especially in the fresh needles, suggest a possible role for 10-DAB III in the biosynthetic pathway to cephalomannine, a less polar taxoid with a side-chain at position C-13. As well, owing to the thermolability of taxoids, the influence of low temperatures in December and January could explain the highest observed concentrations of 10-DAB III in the fresh stems and needles, respectively.     

一株能水解巴卡亭ⅢC-10位乙酰基的成团泛菌的筛选和鉴定

10-脱乙酰巴卡亭Ⅲ是合成紫杉醇和多烯紫杉醇的前体。以巴卡亭Ⅲ为底物,结合TLC、HPLC、HPLC-MS分析方法,通过设计专门的筛选方法筛选产酶菌株,得到一株巴卡亭ⅢC-10位脱乙酰基酶产生菌株Z1-56。以形态特征、生理生化特征、16S rDNA序列分析作为菌株的鉴定手段,Z1-56被鉴定为成团泛菌(Pantoea agglomerans),首次发现成团泛菌产生巴卡亭ⅢC-10-脱乙酰酶。     

胸径、构件和季节对南方红豆杉中紫杉醇和10-DAB含量的影响

系统采集了12个不同胸径的南方红豆杉当年生针叶和树皮样品,用超高效液相色谱(UPLC)测定其紫杉醇(taxol)和10-去乙酰基巴卡亭Ⅲ(10-deacetylbaccatin Ⅲ,10-DAB)含量,结果显示针叶的两种被测试物质含量的平均值分别为0.0127mg·g-1和0.0805mg·g-1,树皮的这两种物质含量平均值分别为0.1164mg·g-1和0.4842mg·g-1。当年生针叶、树皮中紫杉醇含量与胸径相关性不显著。对植株不同构件(枝条、针叶、树皮和根)中紫杉醇和10-DAB含量的测定表明,根中紫杉醇和10-DAB含量最高,当年生针叶紫杉醇含量最低。天然和人工栽培南方红豆杉当年生针叶中紫杉醇和10-DAB含量呈明显的季节变化,各月份间含量差异显著,人工栽培和天然植株针叶在生长速度明显减缓的10月份,紫杉醇和10-DAB含量均出现一个明显的峰值。     

水杨酸在紫杉醇生物合成中诱导作用的研究

研究了水杨酸对红豆杉细胞培养中紫杉烷合成的影响。在适宜浓度的水杨酸诱导下,紫杉醇(Taxol)的产量提高了近3倍,同时10去乙酰基巴卡亭Ⅲ(10-DAB)与巴卡亭Ⅲ(Baccatin Ⅲ)相应上升。通过对紫杉醇合成代谢途径的动力学分析,初步推断水杨酸的加入提高了10-DAB合成速率。并通过水杨酸和硝酸银的配伍诱导,实现了诱导子之间的协同作用,获得了39 mg/L的紫杉醇含量,比两个诱导子单独作用时的最高含量之和还高出50%。     

南方红豆杉枝叶中6种紫杉烷类化合物含量季节变化

紫杉醇(taxol)是主要来源于红豆杉属植物的一种天然抗肿瘤药物,紫杉烷(taxane)是紫杉醇的代谢前体或支路代谢产物,同样具有开发成为抗肿瘤新药的潜质。本文采用高效液相色谱法研究了紫杉醇(Taxol)、10-去乙酰巴卡亭Ⅲ(10-DAB)、7-木-10-去乙酰紫杉醇(7-xyl-10-DAT)、10-去乙酰紫杉醇(10-DAT)、三尖杉宁碱(CE)和7-表-10-去乙酰紫杉醇(7-epi-10-DAT)6种紫杉烷类化合物在南方红豆杉枝叶中含量的季节变化,结果显示Taxol和10-DAT在8、9月份含量最低,10-DAB和7-xyl-10-DAT在8、9月份含量相对最高,Taxol含量季节变化和10-DAB呈负相关,与CE呈显著正相关。7-xyl-10-DAT含量季节变化和10-DAB、10-DAT分别呈正相关。本文为研究南方红豆杉中紫杉醇及相关紫杉烷的代谢、积累规律提供了依据,不但有助于阐明紫杉醇的生物合成的关键步骤及调控的生理机制,而且对红豆杉资源的深度开发具有指导意义。     

Partial purification and characterization of acetyl coenzyme A: taxa-4(20),11(12)-dien-5alpha-ol O-acetyl transferase that catalyzes the first acylation step of taxol biosynthesis

The acetylation of taxa-4(20),11(12)-dien-5alpha-ol is considered to be the third specific step of Taxol biosynthesis that precedes further hydroxylation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol-O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The reaction was dependent on both cosubstrates and active enzyme, and the product of this acetyl transferase was identified by radiochromatographic and GC-MS analysis. Following determination of the time course of acetyl transferase appearance in induced cell cultures, the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates, with Km values of 4.2 and 5.5 microM for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precursors, 10-deacetylbaccatin III or baccatin III. This acetyl transferase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resembles in general properties the few other O-acetyl transferases of higher plant origin that have been examined.     

Isolation and characterization of Stemphylium sedicola SBU-16 as a new endophytic taxol-producing fungus from Taxus baccata grown in Iran

In this study, a total of 25 endophytic fungi were successfully isolated from the inner bark of Taxus baccata grown in Iran by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (ts) gene, which encodes the enzyme catalyzing the first committed step of taxol biosynthesis. Four of 25 isolated endophytic fungi isolates showed PCR positive for the ts gene. Subsequently, taxol and 10-deacetylbaccatin III (10-DAB III) were extracted from culture filtrates and mycelia of the PCR positive isolates and analyzed by high-performance liquid chromatography and mass spectrometry. The analysis showed that one isolate (SBU-16) produced taxol (6.9?±?0.2?μg?L(-1) ) and its intermediate compound, 10-DAB III (2.2?±?0.1?μg?L(-1) ). The isolate SBU-16 was identified as Stemphylium sedicola SBU-16, according to its morphological characteristics as well as the internal transcribed spacer nuclear rDNA gene sequence analysis. Interestingly, this is the first report of the genus Stemphylium as a taxol-producing taxon.     

In vitro enzymatic synthesis of baccatin III with novel and cheap acetyl donors by the recombinant taxoid 10β-O-acetyl transferase

Despite the importance of baccatin III as a precursor to paclitaxel, a widely used chemotherapeutic agent, efficient enzymatic synthesis methods are lacking. Therefore, in this study, the recombinant taxoid 10β-O-acetyl transferase was prepared to produce baccatin III in vitro. The recombinant enzyme could use vinyl acetate, butyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate, and isoamyl acetate as novel and cheap alternative acetyl group donors to replace the expensive acetyl CoA for the enzymatic synthesis of baccatin III. A molecular docking study further confirmed that these acetyl donors could reasonably bind to the enzyme molecule. Using the aqueous two-phase bio-catalytic reaction system, hexane and ethyl acetate could increase the yield of product baccatin III by 2.8% and 1.1% respectively. This approach using novel and cheap acetyl donors is promising for the enzymatic synthesis of baccatin III for the future industrial production of paclitaxel.     

Transcript profiling of jasmonate‐elicited Taxus cells reveals a β‐phenylalanine‐CoA ligase

Plant cell cultures constitute eco‐friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate‐elicited Taxus baccata cell cultures by complementary DNA‐amplified fragment length polymorphism (cDNA‐AFLP) indicated a correlation between an extensive elicitor‐induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate‐induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl‐CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl‐CoA ligase that localizes to the cytoplasm and is able to convert β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐phenylalanyl‐CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.     

Molecular cloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from <Emphasis Type="Italic">Taxus x media</Emphasis>

A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family. Binhui Guo and Guoyin Kai contributed equally to this work.     

一株产紫杉醇内生真菌YN6的分离及鉴定

从云南红豆杉（Taxus yunnanensis）树皮内表皮中分离得到75株内生真菌,采用基于紫杉醇合成关键酶10-去乙酰巴卡亭Ⅲ-10-O-乙酰基转移酶（10-deacetylbaccatin Ⅲ-10-O-acetyl transferase,DBAT）和C-13苯丙氨基侧链CoA乙酰基转移酶（C-13-phenylpropanoid side chain-CoA acyltransferase,BAPT）基因为标志分子的快速筛选方法获得一株可产紫杉醇的内生真菌YN6,并通过高效液相色谱法和质谱法对其紫杉醇进行分析.同时,通过对内生真菌YN6的形态特征分析以及18S rDNA序列分析将其初步鉴定为拟盘多毛孢属（Pestalotiopsis sp.）真菌. 拟盘多毛孢YN6的紫杉醇产量约为120~140 μg/L,是目前已报道的紫杉醇产量较高的野生菌株之一. 拟盘多毛孢YN6的发现为微生物发酵生产紫杉醇提供了潜在的优良种质资源.     

Screening of the needles of different yew species and cultivars for paclitaxel and related taxoids

The needles of several yew species and cultivars were analysed by high-pressure liquid chromatography for paclitaxel, 10-deacetylpaclitaxel, cephalomannine, baccatin III, 10-deacetylbaccatin III and brevifoliol. About 750 samples were collected from five different locations in the Netherlands and the UK. The results of this screening show a large variation in taxane content between the different species and cultivars. The content of paclitaxel and 10-deacetylbaccatin III varied from 0 to 500 micrograms/g and 0 to 4800 micrograms/g dried needles, respectively. Brevifoliol was found in a very high concentration in Taxus brevifolia. 10-Deacetylpaclitaxel, cephalomannine and baccatin III were found in concentrations ranging from 0 to 500 micrograms/g dried needles.     

Microbial transformation of 10-deacetylbaccatin III (10-DAB) by Curvularia lunata and Trametes hirsuta

The microbial transformation of 10-deacetylbaccatin III (10-DAB) (1a) and 13-DeBAC (4b) was investigated. Trametes hirsuta induced 13-oxidation of 10-DAB to give (4a) in high yield, whereas incubation with Curvularia lunata resulted in the isolation of the 7-epi-10-DAB (2) and the 7-epi-10-oxo-10-DAB (3). 13-DeBAC (4b) was biotransformed into compounds (4a) and (4c) by Alternaria alternata.     

悬浮培养南方红豆杉细胞的紫杉醇生物合成路径中诱导子的作用位点

作者研究开发出一种基于分析中间响应模式确定悬浮培养诱导子作用位点的新方法.研究结果表明,一个诱导子的作用位点存在于浓度变化方向相反的相邻两个中间代谢物之间;该方法的有效性在悬浮培养南方红豆杉(Taxus chinensis(Pilg.)Rehd.var.mairei(Lemee et Levl.)Cheng et L. K.Fu)生物合成紫杉醇过程中得以证实;经确定,甲基茉莉酮酸、硝酸根和柠檬酸铵的作用位点在baccatinⅢ至10-去乙酰基紫杉醇之间;水杉酸、花生四烯酸的作用位点存在3种可能性,即增强10-去乙酰基紫杉醇的合成、防止紫杉醇和cephalomannine的降解.该方法对指导诱导子配伍优化紫杉醇生产具有指导意义.