植物类型Ⅲ聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型Ⅲ聚酮化合物合酶(PKS)在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型Ⅲ PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型Ⅲ PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型Ⅲ PKS超家族基因结构、功能及代谢产物研究进展。     

植物类型III聚酮合酶超家族基因结构、功能及代谢产物

植物聚酮类化合物主要包括酚类、芪类及类黄酮化合物等,在植物花色、防止紫外线伤害、预防病原菌、昆虫危害以及作为植物与环境互作信号分子方面行使着重要的生物学功能。该类化合物具有显著多样的生物学活性,对人体保健及疾病治疗有显著意义。植物类型III 聚酮化合物合酶 (PKS) 在该类化合物生物合成起始反应中行使着关键作用,决定该类化合物基本分子骨架建成和代谢途径碳硫走向,为合成途径关键酶和限速酶。以查尔酮合酶为原型酶的植物类型III PKS超家族是研究系统进化和蛋白结构与功能关系的模式分子家族,目前已经分离得到14种植物类型III PKS基因,这些同祖同源基因及其表达产物既有共性,也表现出许多独特个性,这些个性赋予此类次生代谢产物结构上的多样性。以下综述了植物类型III PKS超家族基因结构、功能及代谢产物研究进展。     

PKSI-AT结构域及在组合生物合成研究中的应用

I型聚酮合酶（PKSI）的模块型分子结构组织方式非常适合于组合生物合成研究.结构域和模块通过二级组织方式构成了PKSI的催化单元,其它结构多肽则作为“支架”.在“支架”上对结构域和模块两个水平进行突变、替换、插入、缺失等基因操作形成重组PKS,可以理性设计并获得复杂多样的新活性或高活性的聚酮化合物.利用PKSI进行组合生物合成以期获得新聚酮化合物的研究迄今已有约25年,但是目前仍不能够对PKS进行完美的理性设计,快速合成目标活性的新聚酮化合物.PKS中的酰基转移酶结构域的研究在PKS的组合生物合成研究中一直发挥着重要作用.本文结合本课题组的研究基础,对AT结构域的结构、功能及在组合生物合成研究中的最新研究成果作以分析总结.     

顺式和反式-AT型聚酮合酶研究进展及应用

聚酮类化合物因广泛应用于医药等方面而被大家所熟知,Ⅰ型聚酮合酶（Polyketide Synthase,PKS）在催化聚酮类化合物的生物合成中起着重要的作用。根据不同的酰基转移酶（Acyltransferase,AT）结构域,I型PKS可分为顺式-AT （cis-Acyltransferase,cis-AT）型PKS和反式-AT （trans-Acyltransferase,trans-AT）型PKS,目前cis-AT型PKS研究得比较透彻,trans-AT型PKS相关研究成为当今热点。本文总结了cis-AT型PKS和trans-AT型PKS的联系与区别、工程进展、相关应用以及目前存在的问题,以期为了解cis-AT型PKS和trans-AT型PKS在聚酮化合物合成中的作用提供参考。     

可培养海绵共附生微生物的PKS基因筛选

利用PCR技术对21株分离自我国南海澳大利亚厚皮海绵的放线菌及9株分离自贪婪倔海绵的芽孢杆菌进行了聚酮合酶(PKS)基因筛选。从芽孢杆菌C89中获得了一条669bp片段,BLAST比对结果表明该基因对应的氨基酸序列和枯草芽孢杆菌I型聚酮合成酶基因(PKS)KS域的相似性达96%。通过系统发育分析推测芽孢杆菌C89PKS基因属于trans-AT型。首次证明了贪婪倔海绵共附生微生物中存在PKS基因,这为海绵活性物质的微生物来源假说提供了证据;同时也为可以产生聚酮类化合物的微生物筛选以及聚酮类化合物的发酵制备奠定了基础。     

一个可介导链霉菌PKS基因 向植物转化的杂合质粒的构建

抗生素FR-008是由链霉菌FR-008所产生的一种七烯大环内酯类抗真菌抗生素。胡志浩等已克隆了长达约105kb的FR-008聚酮合酶(PKS)基因簇,对该基因簇中相邻于pabAB基因下游的3.8kbDNA进行序列分析,找到一个多功能聚酮合酶基因的起点,与数据库中蛋白质序列的比较分析揭示出一个尚未结束的大型开读框架的存在,它与抗细菌大环内酯类抗生素-红霉素生物合成所需的Ⅰ型聚酮合酶(PKS)基因中的乙酰转移酶(AT)和β-酮酰合酶(KS)的功能结构域显示出了高度的同源性,从分子水平上证实了FR-008抗生素由Ⅰ型PKS所合成。本实验将3.8kb中的编码聚酮合酶的部分开读框架通过基因工程的方法插入植物表达载体WRG2410上,从而成功构建了表达性质粒pHZ321。     

海洋链霉菌酮合成酶结构域基因的克隆及分析

海洋链霉菌通过聚酮合酶(PKS)合成许多结构和功能多样且具有药用价值的聚酮化合物(PKs),酮合成酶结构域(KS)作为PKS的核心结构域,可催化底物与伸长的聚酮之间的脱羧缩合,在聚酮化合物生物合成中起着重要作用。本文通过对从海洋链霉菌Streptomyces sp. X66基因组DNA克隆获得的ks基因的生物信息学分析表明,该ks基因序列长945 bp, BLAST序列比对显示其具有典型的酮合酶结构域的功能区域。理化分析显示其拟编码309个氨基酸,理论等电点为6.60,原子组成为C₁₄₀₁H₂₂₃₉N₄₂₅O₄₁₉S₈,不稳定指数为42.11,平均亲水系数为0.112,编码产物为酸性疏水不稳定蛋白,且不含信号肽和跨膜结构,二级结构以无规则卷曲和α-螺旋为主,SDS-PAGE显示其分子量约为55 kDa。通过对ks基因的研究,为进一步解析聚酮化合物合成代谢中的调控机制及组合生物学和体外酶系合成聚酮化合物提供参考。     

利玛原甲藻中聚酮合酶基因克隆与分析

为探讨聚酮合酶 (polyketide synthase, PKS)基因与藻毒素合成的关系,揭示PKS基因在赤潮毒素合成中的作用,采用兼并引物,通过PCR技术获得利玛原甲藻(Prorocentrum lima)可能存在的I型PKS基因;并对所获得PKS基因的同源性进行了分析,构建了基于PKS氨基酸序列的系统进化树;采用RT-PCR技术分析了PKS基因在利玛原甲藻中的表达状况;并通过多聚腺苷酸RNA的扩增、细菌的分离鉴定、限制性内切酶酶切、Southern blotting等技术对PKS基因进行了分析.结果表明,利玛原甲藻中PKS基因与海洋原甲藻聚为一支,在利玛原甲藻中有显著表达;以Oligo(T)引物进行RT-PCR扩增时,可出现18S rRNA和PKS基因相应条带;限制性内切酶酶切和Southern blotting结果显示,该基因中存在明显的甲基化;16S rRNA基因序列分析显示,从利玛原甲藻培养液中分离到的细菌与海洋放线菌假诺卡氏菌属(Pseudonocardia)基因序列同源性达到99%,该菌株中并不存在PKS基因.结果显示,所获得的PKS基因是利玛原甲藻聚酮合酶基因,基因序列已提交GenBank (EF521601);PKS可能在腹泻性贝毒合成中起着关键作用.     

长松萝中一个聚酮合酶基因簇的克隆和鉴定

【目的】探索药用地衣长松萝(Usnea longissima Ach)聚酮化合物的生物合成基因簇,克隆聚酮合酶(PKS)基因并分析其功能。【方法】以长松萝地衣型真菌为材料,通过巢氏PCR获得聚酮合酶基因片段和原位杂交筛选基因组文库获得聚酮合酶基因及相邻基因簇。并对获得聚酮合酶进行分子系统进化分析和基因表达分析。【结果】获得药用地衣长松萝中的编码聚酮合酶基因UlPKS5的全长序列以及相邻修饰基因β-内酰胺酶和脱水酶。聚酮合酶UlPKS5含有酮体合成酶(KS),酰基转移酶(AT),产物模板(PT)以及酰基载体蛋白(ACP)结构域。分子系统进化分析显示UlPKS5属于非还原型聚酮合酶中第五组,与蒽醌类化合物生物合成相关。通过半定量RT-PCR分析表明山梨醇(10%)和蔗糖(2%和10%)能够强烈诱导UlPKS5基因表达。【结论】聚酮合酶(UlPKS5)及相邻修饰基因β-内酰胺酶和脱水酶与长松萝中蒽醌类化合物生物合成相关。     

萘醌-氧吲哚生物碱coprisidins生物合成途径的研究

[目的] 新颖结构的天然萘醌-氧吲哚类生物碱coprisidins（A和B）分离自昆虫肠道相关链霉菌,具有预防癌症的活性。作为首例具有萘醌-氧吲哚骨架的生物碱,对其独特生物合成机理的研究可为II型聚酮类化合物生物合成途径提供新的认知。[方法] 本研究对coprisidins的产生菌Streptomyces sp.SNU607进行全基因组测序,并根据测序结果的生物信息学分析初步定位coprisidins的生物合成基因簇;通过基因敲除以及异源表达手段确定coprisidins的生物合成基因簇;基于体内遗传学实验与生物信息学分析初步推导coprisidins的生物合成途径。[结果] Streptomyces sp.SNU607中有23个基因簇可能参与次级代谢,其中4个基因簇与聚酮合酶（PKS）相关;通过基因敲除与异源表达实验,本研究证实1个II型PKS负责coprisidins的生物合成;基于生物信息学分析,我们推测copH/I/M/O/N构成了1个基因盒,并负责起始单元丁酰CoA的合成;KS_β（CopB）的序列比对表明coprisidins的II型PKS系统更倾向于合成C20的初始聚酮链。[结论] Coprisidins的萘醌-吲哚结构是由II型PKSs催化形成,我们推测丁酰CoA是coprisidins聚酮骨架的起始单元,在最小PKS、聚酮酶、环化酶的催化下先形成类似蒽环的四环系统,随后在后修饰酶与氧化重排的作用下生成萘醌-氧吲哚骨架。本研究为进一步探究萘醌-氧吲哚类生物碱的生物合成机制奠定了基础,同时增加了II型PKSs合成产物的结构多样性。     

A new reducing polyketide synthase gene from the lichen-forming fungus Cladonia metacorallifera

Lichens produce unique polyketide secondary metabolites including depsides, depsidones, dibenzofurans and depsones. The biosynthesis of these compounds is governed by polyketide synthase (PKS), but the mechanism via which they are produced has remained unclear until now. We reported the 6-methylsalicylic acid synthase (6-MSAS) type of PKS gene, which is a member of the fungal-reducing PKSs. A cultured mycobiont of Cladonia metacorallifera was employed in the isolation and characterization of a polyketide synthase gene (CmPKS1). The complete sequence information for CmPKS1 was acquired via the screening of a Fosmid genomic library with a 456 bp fragment corresponding to part of the acyl transferase (AT) domain as a probe. CmPKS1 contains β-ketoacyl synthase (KS), AT, dehydratase (DH), ketoreductase (KR) and phosphopantetheine attachment site (PP) domains.: The domain organization of CmPKS1 (KS-AT-DH-KR-PP) is a typical 6-MSAS-type PKS, and the results of phylogenetic analysis showed that CmPKS1 grouped with other fungal-reducing PKSs. Quantitative real time PCR analyses showed that CmPKS1 was expressed preferentially in the early growth stage of the axenically cultured mycobiont. Furthermore CmPKS1 expression was found to be dependent on the carbon sources and concentrations in the medium.     

Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei

The 200kb linear plasmid pSLA2-L was suggested to be involved in the production of two macrolide antibiotics, lankamycin (Lm) and lankacidin (Lc), in Streptomyces rochei 7434AN4. Hybridization experiments with the polyketide synthase (PKS) genes for erythromycin and actinorhodin identified two eryAI-homologous regions and an actI-homologous region on pSLA2-L. The nucleotide sequence of a 3.6kb SacI fragment carrying one of the eryAI-homologs revealed that it codes for part of a large protein with four domains for ketoreductase, acyl carrier protein, ketosynthase, and acyltransferase. Gene disruption confirmed that the two eryAI-homologs are parts of a large type-I PKS gene cluster for Lm. A 4.8kb DNA carrying the actI-homologous region contains four open reading frames (ORF1-ORF4) as well as an additional ORF, i.e. ORF5, which might code for a thioesterase. Deletion of the ORF2-ORF4 region showed that it is not involved in the synthesis of Lm or Lc. Thus, it was confirmed that pSLA2-L contains two PKS gene clusters for Lm and an unknown type-II polyketide.     

Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26

A genomic DNA region of over 80 kb that contains the complete biosynthetic gene cluster for the synthesis of the antifungal polyketide metabolite soraphen A was cloned from Sorangium cellulosum So ce26. The nucleotide sequence of the soraphen A gene region, including 67,523 bp was determined. Examination of this sequence led to the identification of two adjacent type I polyketide synthase (PKS) genes that encode the soraphen synthase. One of the soraphen A PKS genes includes three biosynthetic modules and the second contains five additional modules for a total of eight. The predicted substrate specificities of the acyltransferase (AT) domains, as well as the reductive loop domains identified within each module, are consistent with expectations from the structure of soraphen A. Genes were identified in the regions flanking the two soraphen synthase genes that are proposed to have roles in the biosynthesis of soraphen A. Downstream of the soraphen PKS genes is an O-methyltransferase (OMT) gene. Upstream of the soraphen PKS genes there is a gene encoding a reductase and a group of genes that are postulated to have roles in the synthesis of methoxymalonyl-acyl carrier protein (ACP). This unusual extender unit is proposed to be incorporated in two positions of the soraphen polyketide chain. One of the genes in this group contains distinct domains for an AT, an ACP, and an OMT.     

A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1-4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1-4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin-producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.     

Cloning and analysis of a type II polyketide synthase gene cluster from Streptomyces toxytricini NRRL 15,443

A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), beta-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.