内生真菌紫杉醇生物合成的研究现状与展望

赵凯，宇璐，金昱言，马学玲，刘丹，王晓华，王歆

1 黑龙江大学 生命科学学院微生物重点实验室，黑龙江 哈尔滨 1500802 哈尔滨医科大学附属第四医院 神经内科，黑龙江 哈尔滨 150001

综 述

内生真菌紫杉醇生物合成的研究现状与展望

赵凯1，宇璐1，金昱言1，马学玲2，刘丹1，王晓华1，王歆1

1 黑龙江大学 生命科学学院微生物重点实验室，黑龙江 哈尔滨 150080
2 哈尔滨医科大学附属第四医院 神经内科，黑龙江 哈尔滨 150001

赵凯， 宇璐， 金昱言， 等. 内生真菌紫杉醇生物合成的研究现状与展望. 生物工程学报， 2016， 32（8）: 1038-1051.

Zhao K， Yu L， Jin YY， et al. Advances and prospects of taxol biosynthesis by endophytic fungi. Chin J Biotech， 2016， 32（8）: 1038-1051.

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。

内生真菌，紫杉醇，生物合成，相关基因

Taxol is a diterpenoid w ith anticancer activities，and was first isolated from the bark of Taxus brevifolia Nutt by Wani et al［1］. It is a natural anti-cancer drug w ith high efficiency， low toxicity，and broad-spectrum， and has been w idely used for the treatment of many malignant cancers， such as metastatic breast cancer， advanced ovarian cancer，nonsmall-cell lung cancer and Kaposi's sarcoma［2-3］. Up to now， taxol is extracted， isolated and purified mostly from the bark of Taxus species. Taxus grows very slow， sparse， and thus becomes the rare tree species； in addition， studies have showed that the taxol content in the Taxus species is fairly low. Therefore， it is very difficult to solve the taxol source if depending solely on extraction from yew. Chem ical synthesis of taxol has been also attempted，which has many disadvantages， such as complex synthesis route， uncontrollable reaction conditions， higher costs， and these lim ited the method to be used only in laboratory. In sem i-synthesis pathway， the precursors such as Baccatin Ⅲ and 10-Deacetylbaccatin Ⅲ have also to be extracted from Taxus. Plant cell culture or plant callus induction can only produce taxol at low output w ith high cost. The discovery of taxol producing endophytic fungi is a step stone towards the exploration of taxol sources. Taxol production by endophytic fungi fermentation has many advantages，including high grow th rate， short grow th period，simple medium composition， the controllable culture conditions and low costs， which therefore become the preference of the researchers. This method has now become a very effective method for exploring the taxol sources.

The use of endophytic fungi for biosynthesis of taxol by fermentation technique has yet on the lablevel， and there is still a gap towards industry-based level. The taxol yield from the taxol-producing stain is very low， and little has been known on the molecular biological basis of taxol synthesis， which contributed to its low output. Up to now， few reports on the taxol biosynthesis-related genes by endophytic fungi have been published. This promotes great difficulties and challenges for the use of modern biotechnology to modify the low-output strain genetically， therefore obtaining the high-output strain is necessary. In this view， the understanding of the molecular basis and genetically-regulating mechanisms of taxol biosynthesis by endophytic strain w ill make it possible to modulate the taxol biosynthesis by endophytic fungi at molecular level， elucidate the mechanisms of the taxol biosynthesis by endophytic fungi and construct high-output strain genetically. This may solve the sources of taxol and realize the production at industrial levels. This review stated the biosynthesis pathway of taxol by Taxus cells， the advantages of taxol production by endophytic fungi，the present advance of the isolation and biodiversities of taxol-producing endophytic fungi，and the taxol biosynthesis-related genes.

1　The biosynthetic pathway of taxol

The biosynthetic pathway of taxol from Taxus cells has been elucidated nowadays， while little is known about the taxol biosynthesis by endophytic fungi. The biosynthetic pathway of taxol from Taxus cells is generally divided into three stages， that is，the synthesis of isopentenylpyrophosphate （IPP）， a kind of terpene precursor， taxol carbocycle skeleton Baccatin III synthesis and taxol side chain synthesis. 1.1 Synthesis of isopentenyl pyrophosphate （IPP）

Rohmer indicated that the biosynthesis of terpenoid involved both the mevalonate （MVA） and non-MVA （MEP） pathways［4］. Eisenreich et al proved that the taxane was synthesized by MEP pathway［5］. Both the MVA and MEP pathways were related， although the former existed in cytoplasm，and the latter existed in plasm ids. IPP synthesized from these pathways is the precursor of the tricyclic diterpene in taxol biosynthesis.

1.2Biosynthesis of Baccatin III

IPP and its isomer dimethyl-propenepyrophosphoric acid （DMAPP） can form geranyl-pyrophosphoric acid （GPP） through condensation reaction. GPP may transform into FPP by adding one IPP. The FPP may condense w ith the third IPP to form geranylgeranyl diphosphate （GGPP）. GGPP can be catalyzed by taxol-dienecyclase for cyclization into taxa-4 （5） （12）-diene，which is the backbone of taxol-tricyclic-diterpene. This is the speed-lim iting step in taxol synthesis. Baccatin III， which is the last diterpene intermediate in taxol biosynthesis pathway and also the direct precursor of taxol biosynthesis was obtained after hydroxylation at C1， C2， C5， C7， C9，C10and C13， the formation of epoxypropane circle at C4and C5，acylation at C2， C5， C10， ketone at C9［6-9］.

1.3Synthesis of taxol side chain

The C13side chain of taxol is the key factor for ensuring the anticancer activities of taxol. The side chain structures have greater effects on the taxol synthesis speed than that of the backbone. Therefore，the study on the biosynthetic steps of side chains may be important for increasing taxol output. Phenylalanine is a key precursor for side chain synthesis. Under the catalysis of am inomutase，α-phenylalanine can transform into β-phenylalanine，which was then transformed into phenylisoserine after hydroxylation at C2site. The phenylisoserine is the precursor of taxol C13side chain. Phenylisoserine would then interact w ith taxol backbone to produce taxol［10］.

2　Isolation of taxol-p roducing strains

At present， great progress has been obtained in the synthesis of taxol and taxane-like compounds byfermentation using taxol and taxane-like compounds producing strain screened from yew endofungi. Stierle et al isolated Taxomyces andreanae， a taxol-producing endophytic fungus from T. brevifolia. From then on， the isolation and identification of taxol-producing endophytic fungi were carried out by many researchers. Up to now，more than 20 endophytic fungi genera have been found， which existed in many hosts， including yew，and non yew plants such as hazelnut， Wollemi and Torreya grandifolia indicating the biodiversity of taxol-producing fungi and their hosts （Table 1）.

Tab le 1 Taxol-p roducing endophytic fungi d iscovered

续表1

3　Advances on the taxol-biosynthesis related genes

The study on the taxol biosynthesis pathway using Taxus cells has achieved great advances in recent years， and some of the genes encoding key enzymes have been isolated， identified and cloned （Table 2）. However， there exist great differences in gene sequences encoding taxol biosynthesis between Taxus cells and endophytic fungi， which can be supported by the finding that candidate taxol biosynthetic genes from the taxol synthesizing in endophytic fungi were significantly different and had evolved independently from the host plants［53］. Up to now， few reports have been published on the isolation of taxol biosynthesis related genes from taxol-producing fungi.

3.1Clone of taxol-biosynthesis related genes from Taxus cells

3.1.1Taxane 14-β hyd roxylase gene

The expression inhibition of Taxane 14-β hydroxylase gene may block the taxane pathway ofthe intermediate product to C14oxygen substitution［69］. Jennewein et al［59］cloned genes expressing Taxane 14-β hydroxylase from Taxus， and pointed out that as no substitution at C14site exists in taxol， 14-β hydroxylase can not remain in the target drug pathway， and may be related to the transduction pathway of Taxus cells. Li et al［70］inhibited Taxane 14-β hydroxylase gene expression in Taxus media effectively by using the RNAi technique， which provides theoretical basis for improving the yield of taxol.

Tab le 2 Related enzymes of taxol biosynthesis from Taxus cells

3.1.2Deacetyl Baccatin III-10β-O-acetyltransferase gene

In the taxol biosynthesis pathway， Baccatin III was formed by catalyzing w ith deacetyl Baccatin III-10β-O-acetyl-transferase （DBAT）. This gene was first cloned by Walker et al［62］. Cheng et al also cloned the DBAT gene from Taxus chinensis var. mairei［71］.

3.1.3Geranylgeranyl diphosphate synthase gene

Geranylgeranyl diphosphate synthase （GGPPS）can catalyze to form Geranylgeranyl diphosphate （GGPP）， which is the common precursor of diterpenes. GGPPS is the key enzyme for taxol biosynthesis. Hefner et al discovered the gene from T. canadensis， which contains 393 am ino acid residues. GGPPS can provide necessary jasmonic methyl ester， inducing T. canadensis to produce the precursors for taxol synthesis［65］. Yu et al cloned 6 full-length cDNA encoding the important taxol genes including GGPPS gene［72］. Lan et al cloned GGPPS gene from T. wallichiana［73］. Wang et al cloned full-length sequence of GGPPS gene， and proved the high homogeneity of the protein w ith other plant-derived GGPPS［74］. Our group has obtained GGPPS gene fragments from T. cuspidate，which is 371 bp， and the gene has 99 % homogeneity w ith that of GGPPS gene recorded in GenBank［72］.

3.1.4Taxadiene synthase gene

Taxadiene synthase （TS） catalyze GGPP cyclization to form taxol-4（5），11（12）-diene， which is the backbone of taxol Tricyclic-diterpene. TS is the most important enzyme catalyzing taxol biosynthesis and the first oriental step of taxol biosynthesis，which aroused the investigator`s interest［76］. W ildung et al cloned the TS gene for the first timefrom T. brevifolia， which is 98 303 Da， containing 2 586 nucleotides-encoding ORF， and 862 am ino acid residues［77］. Liang et al cloned cDNA segments of TS gene from T. yunnanensis， w ith 98.42% identity to that reported by W ildung［78］. Xiao et al obtained the full length cDNA of TS gene from T. chinensis var. mairei［79］.

3.1.5Taxane 13α-hydroxylase gene

Taxane 13α-hydroxylase， w ith typical features of P450， is the key enzyme in the downstream of taxol biosynthesis by Taxus cells， which catalyzes the hydroxylation of C13side chain from taxol diene-5α-itol to form taxol diene-5α，13α-diol［80］. This gene is 1 458 bp in length and has high identity w ith Taxane 10β-hydroxylase gene. The gene was firstly cloned and sequenced from Taxus cells by Jennewein et al［76］. Teng et al cloned Taxane 13α hydroxylase gene from T. cuspidate and constructed plant expression vector and transformed into tobacco［81］. Li et al［82］and Huang et al［80］cloned the gene from T. cuspidate. These studies provided molecular basis for production of taxol and its precursor using metabolic engineering.

3.1.6Taxol diene-5α-itol-acety transferase gene

Taxol diene-5α-itol-acetyl transferase （TAT） is composed of 439 am ino acid residues， w ith molecular weight of 50 kDa. A t pH 9.0， TAT has good affinity to taxol dienetol and acetyl CoA. TAT is acetic taxol-4（20），11（12）-diene-5α-ester w ith a very low output in Taxus cells. Therefore， the taxol synthesis efficiency can be greatly affected by this enzyme. Walker et al firstly cloned the gene， and determ ined its important role in taxol biosynthesis，which can be used as the goal of improving taxol output［63］.

3.1.74C-13 phenylpropanoidoyl CoA transferease gene

4C-13 phenylpropanoidoyl CoA transferease （BAPT） can catalyze the formation of 3′-N-debenzoyl-taxol， which is the direct precursor of taxol biosynthesis from β-phenylalanoyl-CoA and Baccatin III. Further study should be performed to prove whether this is the lim iting step of taxol biosynthesis from yew. However， it is apparent that the enzyme used in this step is very important in taxol biosynthesis pathway. BAPT cDNA gene is 1 335 bp， encoding 445 am ino acid residues， which was first found and cloned from T. cuspidata by Walker et al［63］， who also pointed out that the gene could increase taxol output， activities and watersolubility when transferred into suitable host. Han et al cloned the BAPT full-length cDNA gene of 1 456 bp from three different Taxus， and there was 97.4 % identity among these sequences［83］.

3.1.8Taxadiene 5α-hydroxylase gene

Taxadiene 5α-hydroxylase is a multifunctional monooxygenase from m icrosome cytochrome P450. Jennewein et al clone the gene for the first time by screening cDNA library of Taxus［55］.

3.1.9Taxadiene 2α-hydroxylase gene

Taxadiene 2α-hydroxylase gene is 1 488 bp length， encoding 495 am ino acids. As Taxadiene 2α-hydroxylase and Taxadiene 7β-hydroxylase can catalyze products from the other side to form the same products， it can be speculated that the taxolbiosynthesis may involve complicated net. Chau et al discovered and cloned the gene from Taxus［54］.

3.2Genes related to taxol-biosynthesis by endophytic fungi

Although the study on taxol synthesis by endophytic fungi has acquired great progress， the work is still lim ited in laboratory. One reason is the low taxol output from the isolated taxol-producing endophytic fungi， making it difficult for industrial production. Therefore， the focus should be on how to increase the taxol output through endophytic fungi biosynthesis. Based on the documents reported，three ways may be used to increase the taxol output through endophytic fungi biosynthesis: firstly， the gene encoding rate-lim iting enzyme in taxol biosynthesis isolated from Taxus can be transferred into taxol-producing fungi to increase the expressionlevel of the key enzyme， and improve the taxol synthesis ability； secondly， optim ization of the fermentation culture using the metabolic engineering by filling several substances including carbon sources， nitrogen sources， precursors， inducer and the metabolic bypass inhibitors［84-85］. There are many reports using these measures on the taxol study from Taxus cells， while very few reports have been concerned w ith taxol biosynthesis by endophytic fungi fermentation. Our group has studied the factors that affect the biosynthesis of taxol from taxol-producing fungi， such as culture temperature， the initial pH of culture， the rotation speed， and dissolved oxygen， and then determ ined the optimal fermentation conditions. Our group has also studied the effects of adding different concentrations of carbon sources， nitrogen sources， precursors， inducers， the metabolic bypass inhibitors and their synergism on the metabolic regulation during the biosynthesis of taxol from taxol-producing fungi， and we have obtained the optimal medium composition［86］. Thirdly， the genes encoding key enzymes isolated from taxol-producing fungi may be induced into the m icrobes， and construct new taxol-high output engineered strain to produce taxol using other fungi， bacteria or even yeast. However， there is no report on the taxol biosynthesis-related genes from N. sylviforme. Our group has also constructed the differential expression cDNA subtracting library of taxol-high output engineered strain and starting strain w ith a low output of taxol， cDNA library of taxol synthesis period subsidizing non-synthesis period of taxol-producing strain， high ratio full-length cDNA library and its genetic transformed system w ith high efficiency， screened the mutants contributing taxol-output changes， isolated seven taxol synthesis related genes by N. sylviforme，which were genes encoding diterpene synthetase，diterpene-5α-hydroxylase， GGPP synthetase， taxane-10β-hydroxylase， diterpene-5α-itol-acetyl transferase，taxane-2α-hydroxylase， taxane-13α-hydroxylase，respectively. These works may provide new insight into constructing high-output taxol geneticengineering fungi by inducing the genes encoding the enzyme used for taxol biosynthesis by taxol-producing fungi through gene over-expression into m icrobes. And the study also provides a way for elucidating the taxol biosynthesis pathway by endophytic fungi and its biosynthesis mechanisms.

4　Prospective

It is possible to construct genetic-engineering strain w ith high yield of taxol， w ith the deep understanding of the screening of the taxol-synthesis related genes from endophytic fungi and the analysis of their functions. Simultaneously， using the constructed genetic-engineering strain w ith high yield of taxol as starting strain， the metabolic pathway and its mechanisms of biosynthesis of the genetic-engineered strain w ith high yield of taxol would be elucidated through classic methods for metabolic study such as inducer addition， resting cell，isotope tracer， blocked mutant， and mRNA differential display， transcription sequencing and protein expression differential analysis technique. It is believed that taxol production in large scale by taxol-producing endophytic fungi fermentation must be a leading direction， which would solve the taxol shortage and reduce the cost. This technique would become the important pathway for taxol resources.

On the other hand， w ith the developing of technology， many new research methods and ideas have been applied in the study of taxol production，such as high-throughput amplicon sequencing and ecological method［53， 87-89］. These results reveal that taxol biosynthetic pathway may differ between these m icrobes and Taxus， indicating that taxol biosynthesis in Taxus root endophytes may have evolved independently. These results also suggest that diversity of endophytes in Taxus is rich and the resident fungi w ithin a host plant interact w ith one another to stimulate taxol biosynthesis， eitherdirectly or through their metabolites and the endophyte secondary metabolism should be studied in the context of its native ecosystem.

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（本文责编 陈宏宇）

December 9， 2015； Accepted： January 19， 2016

Xin Wang. Tel: +86-451-86608586； Fax: +86-451-86609016； E-mail: tianronghaise@126.com

Advances and prospects of taxol biosynthesis by endophytic fungi

Kai Zhao1， Lu Yu1， Yuyan Jin1， Xueling M a2， Dan Liu1， Xiaohua Wang1， and Xin Wang1

1 Key Laboratory of Microbiology， School of Life Science， Heilongjiang University， Harbin 150080， Heilongjiang， China
2 Department of Neurosurgery， the Fourth Affiliated Hospital of Harbin Medical University， Harbin 150001， Heilongjiang， China

Taxol is one of the most important chemotherapeutic drugs against cancer. Taxol has been mainly extracted from the bark of yew s for a long time. However， methods for the extraction of taxol from the bark of Taxus species were inefficient and environmentally costly. As a result of the high ecological toll exacted on trees w ith the potential for Pacific yew extinction， investigators began to look for other methods of taxol production. Recently， increasing efforts have been made to develop alternative means of taxol production， such as using complete chem ical synthesis， sem i-synthesis， Taxus spp. plant cell culture and m icrobe fermentation. Using m icrobe fermentation in the production of taxol would be a very prospective method for obtaining a large amount of taxol. Therefore， it is necessary to understand the molecular basis and genetic regulation mechanisms of taxol biosynthesis by endophytic fungi， which may be helpful to construct the genetic engineering strain w ith high taxol output. In this paper， the taxol biosynthesis pathway from Taxus cells and the advantages of taxol biosynthesis by endophytic fungi were discussed. The study on the isolation and biodiversity of taxol-producing endophytic fungi and the taxol biosynthesis related genes are also discussed.

endophytic fungi， taxol， biosynthesis， related genes

Supported by： National Natural Science Foundation of China （No. 31270130）， Program for New Century Excellent Talents in University （No. NCET-12-0707）， Technological Innovation Talent of Special Funds for Outstanding Subject Leaders in Harbin （No. 2014RFXXJ081）.

国家自然科学基金 （No. 31270130），教育部新世纪优秀人才支持计划 （No. NCET-12-0707），哈尔滨市科技创新人才研究专项资金（优秀学科带头人） 项目 （No. 2014RFXXJ081） 资助。

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