中国东海微生物来源新聚酮合酶基因簇/基因片段的筛选及其新酰基转移酶结构域的功能研究
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摘要
聚酮合酶(polyketide synthase,PKS)催化产生多种类型的聚酮化合物及其复杂的衍生物,其中部分化合物具有重要的药理学活性。分离、表达这些合成生物活性化合物的新PKS成为生物技术药物研究领域的重点。另外,PKS由于其结构上的特点更成为研究开发组合生物合成药物的理想材料。但是目前已知的PKS数量限制了这些研究深入进行,研究者需要更多的新PKS基因簇序列来充实PKS数据库,以期从中分析获得更多的信息。
本研究提取了中国东海海洋沉积物样本的宏基因组DNA,从中通过简并PCR分离得到了26条新PKS基因片段,并进行了进化分析,推测了它们的来源菌株;通过构建海泥宏基因组DNA文库,用探针筛选得到了1个长度为7981 bp的PKS部分基因簇,并通过数据库和生物信息学软件对其基因结构进行了解析;在体外通过底物结合实验推测出其中1个新酰基转移酶(acyltransferase,GenBank登录号EF080951)的底物特异性;avermectin合成酶系基因簇中PKS基因簇的aveAl基因中模块2编码的AT2(3)结构域用AT-EF080951结构域通过DNA同源重组替换掉,检测突变株产物的变化,在体内进一步分析了AT-EF080951结构域的底物特异性。
通过以上研究我们初步建立了新PKS基因簇分离、结构解析、功能研究的技术平台,通过底物结合实验和结构域替换实验鉴定了1个新酰基转移酶的功能,为进一步的聚酮化合物组合生物合成研究打下基础。
一、海洋沉积物样本宏基因组DNA的提取
海洋微生物资源丰富,大部分微生物属于人类未知的品种。本研究即选择海洋沉积物为材料,2005年9月于中国东海洋山港东南1km处,约10m深度海底不同地点采集沉积物样本M1、2、3、4、5各约10ml。
通过微生物学分离和培养操作分析各样本中的微生物种群丰富度,选取丰富度最高的样本M1提取DNA。由于海洋沉积物成分复杂,传统的方法(如苯酚/氯仿法)提取其中DNA比较困难,本研究中采用了Mo Bio公司的土壤DNA抽提试剂盒提取海洋沉积物中总DNA,并在实验中加以改进。
抽提得到的总DNA通过脉冲场电泳鉴定长度;通过紫外分光光度法测定浓度度:设计了细菌16S rDNA引物,以总DNA为模板进行PCR扩增,检验是否抑制PCR反应。
二、简并引物设计和筛选酮缩酶基因片段
酮缩酶(ketosynthase,KS)结构域在PKS酶系中功能和序列保守,适合设计简并引物。引物设计方法参考Rose等提出的CODEHOP PCR(consensus-degeneratehybrid oligonucleotide primers PCR)。通过NCBI的Conserved Domains Database找出保守并适合设计CODEHOP引物的蛋白模序DPQQR和HGTGT,两个模序之间对应的核苷酸在600～900 bp之间,针对它们各设计若干条引物并两两配对,共计112对引物。
PCR筛选实验以第一部分中提取的宏基因组DNA为模板。由于目的DNA片段的GC含量较高(多为80%以上),PCR反应缓冲液选用Takara公司的GC buffer。符合理论长度的阳性PCR扩增片段回收、连接入测序载体pMD18-T送公司测序,结果与GenBank数据库比对,确认是否目的DNA片段。PCR扩增共得到15条阳性片段,其中3条为新KS基因片段,至此本课题组共从海洋沉积物样本中获得26条新KS基因片段。
通过比对这些KS基因片段的蛋白序列,构建出进化树系图将它们分为两大PKS基因簇类群:Ⅰ型KS基因片段和来自杂合PKS/NRPS基因簇的KS基因片段;并且分析出这些片段的可能来源菌属种类为放线菌门、δ变形菌纲、蓝细菌门和β变形菌纲。
三、海洋沉积物样本宏基因组文库的构建及PKS基因簇分离
简并PCR虽然可以有效地分离新PKS基因片段,但是无法获得全基因簇。而构建宏基因组DNA文库则可以解决这个问题。根据统计现在已经发现的PKS基因簇大小大部分低于50 kb,本研究选用Epicentre公司的fosmid文库构建宏基因组文库,其容量20～50 kb,符合PKS基因簇筛选的条件。构建文库的模版即为第一部分中提取的海洋沉积物样本M1的宏基因组DNA。文库构建中也对其方法进行了改进。
通过实验和公式计算出文库的滴度,按照预计密度进行铺板;文库菌落转移到硝酸纤维素膜上;用第二部分中分离得到的KS基因片段DQ924531作为探针,以DIG标记后对硝酸纤维素膜上的菌落进行Southern杂交,化学显色;阳性克隆提取质粒测序,得到1个7981 bp的PKS部分基因簇(GenBank登录号EF568935),但是由于其自身频繁的高级结构和测序条件的限制,未能继续测通;根据已知序列进行基因组步移也未能得到更多序列。
通过GenBank数据库比对以及运用生物信息学软件对EF568935进行了结构解析发现具有典型的Ⅰ型PKS结构,如AT、KS、ACP等结构域,确定出各结构域及启动子的位置。
四、通过底物结合实验对新酰基转移酶AT-EF080951功能进行体外分析
AT结构域在PKS酶系中起选择加载起始和延伸单位的作用,其底物特异性(substrate specificity)是组合生物合成研究中的关注点。PKS部分基因簇EF568935中编码的酰基转移酶AT-EF080951通过生物信息学软件分析发现具有结合malonyl-CoA的特征。为确定其底物,本研究选择CoA、acetyl-CoA、malonyl-CoA和methylmalonyl-CoA共4种底物对AT-EF080951进行体外底物结合实验。
通过pMAL-c2x蛋白表达和纯化系统表达出MBP-AT-EF080951融合蛋白,并通过Xa因子酶切、分离出单独的AT-EF080951蛋白。用MBP、BSA蛋白以及空白作为对照,通过ELISA法做底物结合实验,然后通过公式计算4种蛋白对4种底物的结合常数,分析AT-EF080951的底物特异性。通过对比对照蛋白与底物的结合常数,可知AT-EF080951与4种底物的亲和力依次为Ka_((acetyl-CoA))>Ka_((malonyl-CoA))≈Ka_((Methylmalonyl coenzyme A))≈Ka_((CoA)),对后3种底物为非特异性吸附。底物结合实验结果表明MBP-AT-EF080951和AT-EF080951都特异性结合acetyl-CoA,Ka_((acetyl-CoA))>Ka_((malonyl-CoA)),这与软件分析结果不符。
通过异源表达,产生可溶的、具有底物选择结合功能的MBP融合酰基转移酶MBP-AT-EF080951和单体酰基转移酶AT-EF080951,说明酰基转移酶在没有N端KS结构域、C端ACP结构域及细胞内其它因子存在的情况下仍然对底物具有选择结合的能力。
五、通过结构域替换实验对AT-EF080951功能进行体内分析
菌株S.avermitilis ATCC31271中的avermectin合成酶系的全基因簇的序列已测通,各结构域功能均已有研究。为了进一步确定AT-EF080951体外结合实验的结果,本研究用AT-EF080951替换掉avermectin合成酶系基因簇的aveA1基因中模块2编码的AT2(3)结构域(结合malonyl-CoA),通过HPLC检测其产物变化。
本部分制备了S.avermitilis原生质体,构建同源打靶载体,电转化入原生质体中;通过双交换重组,用AT-EF080951替换掉aveA1基因中模块2的AT2(3)结构域基因;通过PCR筛选克隆并通过DNA测序鉴定重组正确;正确的突变株用种子培养基培养,培养液经甲醇抽提后用HPLC分析;结果显示在突变株中仍然存在avermectin,但含量减少。
实验结果说明AT-EF080951在体内可以利用malonyl-CoA延伸聚酮链,结果支持软件分析结论。结合类似的研究结果,本研究结果支持起始AT结构域的底物特异性在体内可能受其它因子作用,通过体外底物结合实验鉴定其特异性底物可能产生不准确的结论。
Polyketide synthase(PKS) catalyze certain substrates to produce structurally different polyketides and their vast analogues.Some of them have important pharmaceutical activities.In biotechnological drug research,it is important to isolate and express new PKSs which produce bioactive compound.Because of its structure characteristics,PKS has become an ideal material for research and development of combinatorial biosynthetic drugs.The already known PKSs are still not enough to further support these researches. Scientists need more new sequences of PKS genes to enlarge the PKS gene clusters data base to get more valuable information.
In this study we isolated 26 new PKS gene fragments from metagenomic DNA of a marine sediment sample in the East China Sea.Phylogenetic relations of the protein sequences of all the new KS fragments were analyzed and the originating strains were predicted;a 7981 bp partial PKS gene cluster was isolated through construction of metagenomic DNA library.We analyzed the sequence of the partial PKS gene cluster and also predicted the substrate specificity of a new acyltransferase(AT,accession number EF080951) in it through substrate binding test in vitro;an AT domain AT2(3) in module 2 of the PKS in avermectin biosynthesis enzyme system was replaced with AT-EF080951 to testify the substrate specificity of AT-EF080951 in vivo.
Through this study we preliminarily established a technical platform for gene isolation, structural and functional analysis of new PKS;through substrate binding test and domain replacement we validated the function of a new acyltransferase,thus made a ground for further study in polyketide combinatorial biosynthesis.
1.Metagenomic DNA extraction from a marine sediment sample
Marine environment has the most enormous phylogenetic and metabolic diversity in the Earth but remain undersampled and essentially uncharted.In this study we choose the marine sediment as target and got marine sediment samples M1～5 approximately 10 ml of each from different sites about 10 m below the surface in the ECS in September 2005.
Through microbiological isolation and culture we compared the population variety of each sample and choose M1(the one with the richest varieties of microbes among them) to extract its metagenomic.Because of the abundant components in marine sediment, traditional method(e.g.phenol/chloroform) is not suitable for the sample's DNA extraction. In this study we used the soil DNA isolation kit of Mo Bio Company to extract the metagenomic DNA from marine sediment sample M1 with modifications.
The length of metagenomic DNA extracted was determined though pulsed-field gel electrophoresis;the concentration was determined through ultra-violet spectrometry;we used the metagenomic DNA as the template and designed 16S rDNA primers to run PCR to verify whether the DNA makes inhibition.
2.Degenerative primers design and ketosynthase gene fragments isolation
The function and sequence of ketosynthase(KS) domain is conserved in PKS,so KS domain is appropriate for degenerative primer design.The method of degenerative primer design was referred to CODEHOP PCR invented by Rose and colleagues.For primer design,conserved and appropriate motifs DPQQR and HGTGT were selected through searching the Conserved Domain Database of NCBI.The nucleotide sequence distance between two motifs ranges from 600 to 900 bp.Several primers were designed targeting the two motifs and paired resulting totally 112 pairs of primers.
The metagenomic DNA extracted in Part one was used as PCR template.Because of the high GC content of the target genes(mostly higher than 80%),GC buffer of Takara company was selected in PCR.The amplicons coincided with theoretical prediction were purified and ligated into vector pMD18-T for sequencing.The sequences were analyzed through GenBank database and software of NCBI.By degenerative PCR we isolated total 15 amplicons of correct molecular size,and 3 of them were new KS gene fragments.So far, our research team has isolated 26 new KS gene fragments from marine sediment samples.
By construction of phylogenetic tree,all the new KS domains were classified into 2 PKS groups:typeⅠPKS and those from hybrid or mixed PKS/NRPS enzyme complexes primarily originating from marine actinobacteria,delta-proteobacteria,cyanobateria,and beta-proteobacteria.
3.Construction of marine sediment sample metagenomic DNA library and isolation of PKS gene clusters.
Although new PKS gene fragments can be isolated effectively by degenerative PCR, we can not acquire the whole sequences of the gene clusters,while by construction of metagenomic DNA library we can solve this problem.Since the molecular sizes of all the already known PKS genes are not above 50 kb,fosmid vector(range of inserts,20～50 kb) is appropriate for cloning of PKS gene clusters.Therefore we choose the fosmid library kit of Epicentre Company for metagenomic DNA library construction.The template for library construction was the metagenomic DNA extracted in Part 1.The protocol was also modified in our study.
By tests and formula we calculated the titer of the library and plated library clones according to desired density;the library clones were transferred to nitrocellulose(NC) membrane;DNA fragment DQ924531 isolated from Part 2 was used as a probe and labeled with DIG;positive clones were screened out by Southern blotting and chemical staining;a 7981 bp gene cluster was obtained by sequencing the fosmid plasmids extracted from positive clones,but the whole sequence of the cluster could not obtained because of the high frequent super structure and the limitation of sequencing techniques;by genome walking basing on the known sequence we did not get longer sequence of the cluster either.
We analyzed the structure of gene EF568935 by using GenBank database and bioinformatics software;typical domains like AT,KS and ACP were identified;locations of the domains and a promotor in the EF568935 sequence were also analyzed.
4.The functional analysis of new acyltransferase AT-EF080951 in vitro through substrate binding test
The function of AT domain in a PKS is to select and incorporate extender unit in polyketide biosynthetic pathway.The substrate specificity has become a hot research point. The acyltransferase AT-EF080951 in the partial PKS gene cluster EF568935 was found to have the ability to incorporate malonyl-CoA by bioinformatic softwares.To analyze its substrate specificity,substrates CoA,acetyl-CoA,malonyl-CoA and methylmalonyl-CoA were used to carry the substrate binding test of AT-EF080951.
MBP-AT-EF080951 fusion protein were expressed and purified,and single AT-EF080951 was separated through factor Xa cleavage reaction.Using MBP,BSA protein as controls we tested the binding ability of the 4 proteins to 4 substrates through ELISA method and calculated the binding constants of them.Through comparison to the binding constants of control proteins,we found the binding ability of AT-EF080951 to the substrates is Kα_((acetyl-CoA))> Kα_((malonyl-CoA))≈Kα_((Methylmalonyl coenzyme A))≈Kα_((CoA)),(the later three belong to non-specific absorption).The result of binding test indicate that both MBP-AT-EF080951 and AT-EF080951 bind acetyl-CoA specifically and Kα_((acetyl-CoA))> Kα _((malonyl-CoA)),which conflicts with the result by bioinformatics software.
Soluble and specific substrate-binding active fusion protein MBP-AT-EF080951 and single AT-EF080951 acyltransferase were obtained through heterologous expression in pMAL-c2x system,which indicated the function of an acyltransferase selectively binding to a specific substrate still remains when the N-terminal KS domain,the C-terminal ACP domain and some intracellular factors are not present.The joining regions at both sides of AT-EF080951 remained no more than 14 amino acids,which also indicated that its function does not depend on the space structure of the joining regions flanking the AT domain.
5.The functional analysis of new acyltransferase AT-EF080951 in vivo through domain replacement
The sequence and domains' functions of the avermectin biosynthetic gene cluster have already known.To further testify the result by substrate binding test in vitro,the acyltransferase AT2(3) gene(malonyl-CoA specific,within module 2) in aveA1 of avermectin PKS gene of S.avermitilis ATCC31271 was replaced with AT-EF080951.The products of avermectin PKS in S.avermitilis ATCC31271 were analyzed by HPLC.If AT-EF080951 only binds malonyl-CoA the component and content of avermectin will remain the same;if AT-EF080951 only binds acetyl-CoA the polyketide chain will terminate,and avermectin will not be detected;if AT-EF080951 binds to both of them the component of avermectin will remain the same while the content of it will decrease.
We prepared protoplast of ATCC31271 and constructed targeting vector which was electrotransformated into the protoplast;through double crossover the module 2 AT2(3) domain gene in aveA1 was replaced by AT-EF080951;we used PCR to verify the mutants; the correct mutant was cultivated with fermentation medium and the broth was extracted with methanol;we applied the supernatant to liquid chromatography mass spectrometry; the components of avermectin were the same while the content decreased.
The results showed that AT-EF080951 can incorporate malonyl-CoA to extend the polyketide chain.Based on results in this study and others',we concluded that the specificity of starter AT domain in vivo might be interfered or regulated by other factors and the substrate binding test of starter AT domain in vitro may lead to inaccurate
conclusions.
本研究提取了中国东海海洋沉积物样本的宏基因组DNA,从中通过简并PCR分离得到了26条新PKS基因片段,并进行了进化分析,推测了它们的来源菌株;通过构建海泥宏基因组DNA文库,用探针筛选得到了1个长度为7981 bp的PKS部分基因簇,并通过数据库和生物信息学软件对其基因结构进行了解析;在体外通过底物结合实验推测出其中1个新酰基转移酶(acyltransferase,GenBank登录号EF080951)的底物特异性;avermectin合成酶系基因簇中PKS基因簇的aveAl基因中模块2编码的AT2(3)结构域用AT-EF080951结构域通过DNA同源重组替换掉,检测突变株产物的变化,在体内进一步分析了AT-EF080951结构域的底物特异性。
通过以上研究我们初步建立了新PKS基因簇分离、结构解析、功能研究的技术平台,通过底物结合实验和结构域替换实验鉴定了1个新酰基转移酶的功能,为进一步的聚酮化合物组合生物合成研究打下基础。
一、海洋沉积物样本宏基因组DNA的提取
海洋微生物资源丰富,大部分微生物属于人类未知的品种。本研究即选择海洋沉积物为材料,2005年9月于中国东海洋山港东南1km处,约10m深度海底不同地点采集沉积物样本M1、2、3、4、5各约10ml。
通过微生物学分离和培养操作分析各样本中的微生物种群丰富度,选取丰富度最高的样本M1提取DNA。由于海洋沉积物成分复杂,传统的方法(如苯酚/氯仿法)提取其中DNA比较困难,本研究中采用了Mo Bio公司的土壤DNA抽提试剂盒提取海洋沉积物中总DNA,并在实验中加以改进。
抽提得到的总DNA通过脉冲场电泳鉴定长度;通过紫外分光光度法测定浓度度:设计了细菌16S rDNA引物,以总DNA为模板进行PCR扩增,检验是否抑制PCR反应。
二、简并引物设计和筛选酮缩酶基因片段
酮缩酶(ketosynthase,KS)结构域在PKS酶系中功能和序列保守,适合设计简并引物。引物设计方法参考Rose等提出的CODEHOP PCR(consensus-degeneratehybrid oligonucleotide primers PCR)。通过NCBI的Conserved Domains Database找出保守并适合设计CODEHOP引物的蛋白模序DPQQR和HGTGT,两个模序之间对应的核苷酸在600～900 bp之间,针对它们各设计若干条引物并两两配对,共计112对引物。
PCR筛选实验以第一部分中提取的宏基因组DNA为模板。由于目的DNA片段的GC含量较高(多为80%以上),PCR反应缓冲液选用Takara公司的GC buffer。符合理论长度的阳性PCR扩增片段回收、连接入测序载体pMD18-T送公司测序,结果与GenBank数据库比对,确认是否目的DNA片段。PCR扩增共得到15条阳性片段,其中3条为新KS基因片段,至此本课题组共从海洋沉积物样本中获得26条新KS基因片段。
通过比对这些KS基因片段的蛋白序列,构建出进化树系图将它们分为两大PKS基因簇类群:Ⅰ型KS基因片段和来自杂合PKS/NRPS基因簇的KS基因片段;并且分析出这些片段的可能来源菌属种类为放线菌门、δ变形菌纲、蓝细菌门和β变形菌纲。
三、海洋沉积物样本宏基因组文库的构建及PKS基因簇分离
简并PCR虽然可以有效地分离新PKS基因片段,但是无法获得全基因簇。而构建宏基因组DNA文库则可以解决这个问题。根据统计现在已经发现的PKS基因簇大小大部分低于50 kb,本研究选用Epicentre公司的fosmid文库构建宏基因组文库,其容量20～50 kb,符合PKS基因簇筛选的条件。构建文库的模版即为第一部分中提取的海洋沉积物样本M1的宏基因组DNA。文库构建中也对其方法进行了改进。
通过实验和公式计算出文库的滴度,按照预计密度进行铺板;文库菌落转移到硝酸纤维素膜上;用第二部分中分离得到的KS基因片段DQ924531作为探针,以DIG标记后对硝酸纤维素膜上的菌落进行Southern杂交,化学显色;阳性克隆提取质粒测序,得到1个7981 bp的PKS部分基因簇(GenBank登录号EF568935),但是由于其自身频繁的高级结构和测序条件的限制,未能继续测通;根据已知序列进行基因组步移也未能得到更多序列。
通过GenBank数据库比对以及运用生物信息学软件对EF568935进行了结构解析发现具有典型的Ⅰ型PKS结构,如AT、KS、ACP等结构域,确定出各结构域及启动子的位置。
四、通过底物结合实验对新酰基转移酶AT-EF080951功能进行体外分析
AT结构域在PKS酶系中起选择加载起始和延伸单位的作用,其底物特异性(substrate specificity)是组合生物合成研究中的关注点。PKS部分基因簇EF568935中编码的酰基转移酶AT-EF080951通过生物信息学软件分析发现具有结合malonyl-CoA的特征。为确定其底物,本研究选择CoA、acetyl-CoA、malonyl-CoA和methylmalonyl-CoA共4种底物对AT-EF080951进行体外底物结合实验。
通过pMAL-c2x蛋白表达和纯化系统表达出MBP-AT-EF080951融合蛋白,并通过Xa因子酶切、分离出单独的AT-EF080951蛋白。用MBP、BSA蛋白以及空白作为对照,通过ELISA法做底物结合实验,然后通过公式计算4种蛋白对4种底物的结合常数,分析AT-EF080951的底物特异性。通过对比对照蛋白与底物的结合常数,可知AT-EF080951与4种底物的亲和力依次为Ka_((acetyl-CoA))>Ka_((malonyl-CoA))≈Ka_((Methylmalonyl coenzyme A))≈Ka_((CoA)),对后3种底物为非特异性吸附。底物结合实验结果表明MBP-AT-EF080951和AT-EF080951都特异性结合acetyl-CoA,Ka_((acetyl-CoA))>Ka_((malonyl-CoA)),这与软件分析结果不符。
通过异源表达,产生可溶的、具有底物选择结合功能的MBP融合酰基转移酶MBP-AT-EF080951和单体酰基转移酶AT-EF080951,说明酰基转移酶在没有N端KS结构域、C端ACP结构域及细胞内其它因子存在的情况下仍然对底物具有选择结合的能力。
五、通过结构域替换实验对AT-EF080951功能进行体内分析
菌株S.avermitilis ATCC31271中的avermectin合成酶系的全基因簇的序列已测通,各结构域功能均已有研究。为了进一步确定AT-EF080951体外结合实验的结果,本研究用AT-EF080951替换掉avermectin合成酶系基因簇的aveA1基因中模块2编码的AT2(3)结构域(结合malonyl-CoA),通过HPLC检测其产物变化。
本部分制备了S.avermitilis原生质体,构建同源打靶载体,电转化入原生质体中;通过双交换重组,用AT-EF080951替换掉aveA1基因中模块2的AT2(3)结构域基因;通过PCR筛选克隆并通过DNA测序鉴定重组正确;正确的突变株用种子培养基培养,培养液经甲醇抽提后用HPLC分析;结果显示在突变株中仍然存在avermectin,但含量减少。
实验结果说明AT-EF080951在体内可以利用malonyl-CoA延伸聚酮链,结果支持软件分析结论。结合类似的研究结果,本研究结果支持起始AT结构域的底物特异性在体内可能受其它因子作用,通过体外底物结合实验鉴定其特异性底物可能产生不准确的结论。
Polyketide synthase(PKS) catalyze certain substrates to produce structurally different polyketides and their vast analogues.Some of them have important pharmaceutical activities.In biotechnological drug research,it is important to isolate and express new PKSs which produce bioactive compound.Because of its structure characteristics,PKS has become an ideal material for research and development of combinatorial biosynthetic drugs.The already known PKSs are still not enough to further support these researches. Scientists need more new sequences of PKS genes to enlarge the PKS gene clusters data base to get more valuable information.
In this study we isolated 26 new PKS gene fragments from metagenomic DNA of a marine sediment sample in the East China Sea.Phylogenetic relations of the protein sequences of all the new KS fragments were analyzed and the originating strains were predicted;a 7981 bp partial PKS gene cluster was isolated through construction of metagenomic DNA library.We analyzed the sequence of the partial PKS gene cluster and also predicted the substrate specificity of a new acyltransferase(AT,accession number EF080951) in it through substrate binding test in vitro;an AT domain AT2(3) in module 2 of the PKS in avermectin biosynthesis enzyme system was replaced with AT-EF080951 to testify the substrate specificity of AT-EF080951 in vivo.
Through this study we preliminarily established a technical platform for gene isolation, structural and functional analysis of new PKS;through substrate binding test and domain replacement we validated the function of a new acyltransferase,thus made a ground for further study in polyketide combinatorial biosynthesis.
1.Metagenomic DNA extraction from a marine sediment sample
Marine environment has the most enormous phylogenetic and metabolic diversity in the Earth but remain undersampled and essentially uncharted.In this study we choose the marine sediment as target and got marine sediment samples M1～5 approximately 10 ml of each from different sites about 10 m below the surface in the ECS in September 2005.
Through microbiological isolation and culture we compared the population variety of each sample and choose M1(the one with the richest varieties of microbes among them) to extract its metagenomic.Because of the abundant components in marine sediment, traditional method(e.g.phenol/chloroform) is not suitable for the sample's DNA extraction. In this study we used the soil DNA isolation kit of Mo Bio Company to extract the metagenomic DNA from marine sediment sample M1 with modifications.
The length of metagenomic DNA extracted was determined though pulsed-field gel electrophoresis;the concentration was determined through ultra-violet spectrometry;we used the metagenomic DNA as the template and designed 16S rDNA primers to run PCR to verify whether the DNA makes inhibition.
2.Degenerative primers design and ketosynthase gene fragments isolation
The function and sequence of ketosynthase(KS) domain is conserved in PKS,so KS domain is appropriate for degenerative primer design.The method of degenerative primer design was referred to CODEHOP PCR invented by Rose and colleagues.For primer design,conserved and appropriate motifs DPQQR and HGTGT were selected through searching the Conserved Domain Database of NCBI.The nucleotide sequence distance between two motifs ranges from 600 to 900 bp.Several primers were designed targeting the two motifs and paired resulting totally 112 pairs of primers.
The metagenomic DNA extracted in Part one was used as PCR template.Because of the high GC content of the target genes(mostly higher than 80%),GC buffer of Takara company was selected in PCR.The amplicons coincided with theoretical prediction were purified and ligated into vector pMD18-T for sequencing.The sequences were analyzed through GenBank database and software of NCBI.By degenerative PCR we isolated total 15 amplicons of correct molecular size,and 3 of them were new KS gene fragments.So far, our research team has isolated 26 new KS gene fragments from marine sediment samples.
By construction of phylogenetic tree,all the new KS domains were classified into 2 PKS groups:typeⅠPKS and those from hybrid or mixed PKS/NRPS enzyme complexes primarily originating from marine actinobacteria,delta-proteobacteria,cyanobateria,and beta-proteobacteria.
3.Construction of marine sediment sample metagenomic DNA library and isolation of PKS gene clusters.
Although new PKS gene fragments can be isolated effectively by degenerative PCR, we can not acquire the whole sequences of the gene clusters,while by construction of metagenomic DNA library we can solve this problem.Since the molecular sizes of all the already known PKS genes are not above 50 kb,fosmid vector(range of inserts,20～50 kb) is appropriate for cloning of PKS gene clusters.Therefore we choose the fosmid library kit of Epicentre Company for metagenomic DNA library construction.The template for library construction was the metagenomic DNA extracted in Part 1.The protocol was also modified in our study.
By tests and formula we calculated the titer of the library and plated library clones according to desired density;the library clones were transferred to nitrocellulose(NC) membrane;DNA fragment DQ924531 isolated from Part 2 was used as a probe and labeled with DIG;positive clones were screened out by Southern blotting and chemical staining;a 7981 bp gene cluster was obtained by sequencing the fosmid plasmids extracted from positive clones,but the whole sequence of the cluster could not obtained because of the high frequent super structure and the limitation of sequencing techniques;by genome walking basing on the known sequence we did not get longer sequence of the cluster either.
We analyzed the structure of gene EF568935 by using GenBank database and bioinformatics software;typical domains like AT,KS and ACP were identified;locations of the domains and a promotor in the EF568935 sequence were also analyzed.
4.The functional analysis of new acyltransferase AT-EF080951 in vitro through substrate binding test
The function of AT domain in a PKS is to select and incorporate extender unit in polyketide biosynthetic pathway.The substrate specificity has become a hot research point. The acyltransferase AT-EF080951 in the partial PKS gene cluster EF568935 was found to have the ability to incorporate malonyl-CoA by bioinformatic softwares.To analyze its substrate specificity,substrates CoA,acetyl-CoA,malonyl-CoA and methylmalonyl-CoA were used to carry the substrate binding test of AT-EF080951.
MBP-AT-EF080951 fusion protein were expressed and purified,and single AT-EF080951 was separated through factor Xa cleavage reaction.Using MBP,BSA protein as controls we tested the binding ability of the 4 proteins to 4 substrates through ELISA method and calculated the binding constants of them.Through comparison to the binding constants of control proteins,we found the binding ability of AT-EF080951 to the substrates is Kα_((acetyl-CoA))> Kα_((malonyl-CoA))≈Kα_((Methylmalonyl coenzyme A))≈Kα_((CoA)),(the later three belong to non-specific absorption).The result of binding test indicate that both MBP-AT-EF080951 and AT-EF080951 bind acetyl-CoA specifically and Kα_((acetyl-CoA))> Kα _((malonyl-CoA)),which conflicts with the result by bioinformatics software.
Soluble and specific substrate-binding active fusion protein MBP-AT-EF080951 and single AT-EF080951 acyltransferase were obtained through heterologous expression in pMAL-c2x system,which indicated the function of an acyltransferase selectively binding to a specific substrate still remains when the N-terminal KS domain,the C-terminal ACP domain and some intracellular factors are not present.The joining regions at both sides of AT-EF080951 remained no more than 14 amino acids,which also indicated that its function does not depend on the space structure of the joining regions flanking the AT domain.
5.The functional analysis of new acyltransferase AT-EF080951 in vivo through domain replacement
The sequence and domains' functions of the avermectin biosynthetic gene cluster have already known.To further testify the result by substrate binding test in vitro,the acyltransferase AT2(3) gene(malonyl-CoA specific,within module 2) in aveA1 of avermectin PKS gene of S.avermitilis ATCC31271 was replaced with AT-EF080951.The products of avermectin PKS in S.avermitilis ATCC31271 were analyzed by HPLC.If AT-EF080951 only binds malonyl-CoA the component and content of avermectin will remain the same;if AT-EF080951 only binds acetyl-CoA the polyketide chain will terminate,and avermectin will not be detected;if AT-EF080951 binds to both of them the component of avermectin will remain the same while the content of it will decrease.
We prepared protoplast of ATCC31271 and constructed targeting vector which was electrotransformated into the protoplast;through double crossover the module 2 AT2(3) domain gene in aveA1 was replaced by AT-EF080951;we used PCR to verify the mutants; the correct mutant was cultivated with fermentation medium and the broth was extracted with methanol;we applied the supernatant to liquid chromatography mass spectrometry; the components of avermectin were the same while the content decreased.
The results showed that AT-EF080951 can incorporate malonyl-CoA to extend the polyketide chain.Based on results in this study and others',we concluded that the specificity of starter AT domain in vivo might be interfered or regulated by other factors and the substrate binding test of starter AT domain in vitro may lead to inaccurate
conclusions.
引文
[1]Sogin ML,Morrison HG,Huber JA.Microbial diversity in the deep sea and the underexplored "rare biosphere".Proc Natl Acad Sci.2006,103(32):12115-12120.
[2]伯杰细菌鉴定手册(第八版).科学出版社.1984.
[3]中国科学院微生物研究所放线菌组.链霉菌鉴定手册.北京:科学出版社.1975.
[4]Blackwood CB,Waldrop MP,Zak DR,et al.Molecular Analysis of Fungal Communities and Laccase Genes in Decomposing Litter Reveals Differences Among Forest Types But No Impact of Nitrogen Deposition.Environ Microbiol.2007,9:1306-1316.
[5]Herrera A,Cockell CS.Exploring microbial diversity in volcanic environments:a review of methods in DNA extraction.J Microbiol Methods.2007,70(1):1-12.
[6]Hortal S,Pera J,Galipenso L,et al.Molecular identification of the edible ectomycorrhizal fungus Lacterius in the symbiotic and extraradical mycelium stages.J Biotechnol.2006,126(2):123-134.
[7]Marchetti E,Accinelli C,Talame V,et aL Persistence of Cry Toxins and Cry Gene From Genetically Modified Plants in Two Agricultural Soils.Agron Sustain Dev.2007,27:231-236.
[8]Mayer KM,Ford J,Macpherson GR,et al.Exploring the diversity of marine-derived fungal polyketide synthases.Can J Microbiol.2007,53(2):291-302.
[9]Mummey DL,Rillig MC,et al.Endogeic Earthworms Differentially Influence Bacterial Communities Associated with Different Soil Aggregate Size Fractions.Soil Biol Biochem.2006,35:1608-1614.
[10]Mummey DL,Rillig MC.Evaluation of LSU rRNA-gene PCR Primers for the Study of Arbuscular Mycorrhizal Fungal Communities via Terminal Restrictions Fragment Length Polymorphism Analysis.J Microbiol Methods.2007,70(1):200-204.
[11]Mummey DL,Rillig MC.The Invasive Plant Species Centaurea maculosa Alters Arbuscular Mycorrhizal Fungal Communities in the Field.Plant Soil.2006,288:81-90.
[12]Robe P,Nalin R,Capellano C,et al.Extraction of DNA from soil.Eur J Soil Biol.2003,(39)4:183-190
[13]Webster G,Watt LC,Rinna J,et al.A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries.Environ Microbiol.2006,8(9):1575-1589.
[14]Weiss A,Jerome V,Freitag R.Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.J Chromatogr B Analyt Technol Biomed Life Sci.2007,853(1-2):190-197.
[15]李会荣,俞勇,曾胤新,等.北极太平洋扇区海洋沉积物细菌多样性的系统发育分析.微生物学报.2006,46(2):177-183.
[16]吴少杰,朱丽华,杨志娟.不同培养基对海洋放线菌抗微生物活性的影响.华北煤炭医学院学报.2006,(8)5:600-602.
[1]Barrios-Llerena ME,Burja AM,et al.Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites.J Ind Microbiol Biotechnol.2007,34(6):443-456.
[2]Castonguay R,He W,Chen AY,et al.Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase.J Am Chem Soc.2007,129(44):13758-13769.
[3]Atoui A,Dao HP,Mathieu F,et al.Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase genes in Aspergillus ochraceus and Aspergillus carbonarius producers ofochratoxin A.Mol Nutr Food Res.2006,50(6):488-493.
[4]Savic M,Vasiljevic B.Targeting polyketide synthase gene pool within actinomycetes:new degenerate primers.J Ind Microbiol Biotechnol.2006,33(6):423-430.
[5]Khosla C,Gokhale RS,Jacobsen JR,et al.Tolerance and specificity of polyketide synthases.Annu Rev Biochern,1999.68:219-25.
[6]Jenke-Kodama H,Sandmann A,Muller R,et al.Evolutionary implications of bacterial polyketide synthases.Mol Biol Evol.2005,22(10):2027-2039.
[7]Rose TM,Henikoff JG,Henikoff S.CODEHOP(COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.Nucleic Acids Res.2003,31(13):3763-3766.
[8]Rose TM.CODEHOP-mediated PCR-a powerful technique for the identification and characterization of viral genomes.Virol J.2005,2:20.
[9]Gokhale RS,Sankaranarayanan R,Mohanty D.Versatility of polyketide synthases in generating metabolic diversity.Curr Opin Struct Biol.2007,17(6):736-743.
[10]Kim TK,Fuerst JA.Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata:culture-dependent and culture-independent approaches.Environ Microbiol.2006,8(8):1460-1470.
[11]Piel J,Hui D,Wen G,et al.Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci USA. 2004, 101(46): 16222-16227.
[12]Sudek S, Lopanik NB, Waggoner LE, et al. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. J Nat Prod. 2007, 70(1): 67-74.
[13]Ginolhac A, Jarrin C, Gillet B, et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol. 2004, 70(9): 5522-5527.
[14]Baines JE, McGovern RM, Persing D, et al. Consensus-degenerate hybrid oligonucleotide primers (CODEHOP) for the detection of novel papillomaviruses and their application to esophageal and tonsillar carcinomas. J Virol Methods. 2005, 123(1): 81-87.
[15]Beck BJ, Aldrich CC, Fecik RA, et al. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J Am Chem Soc. 2003, 125(41): 12551-12557.
[16]Kim BC, Lee JM, Ahn JS, et al. Cloning, sequencing, and characterization of the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2. J Microbiol Biotechnol. 2007, 17(5): 830-839.
[17]Kurosaki F, Mitsuma S, Arisawa M. Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme, by free coenzyme A. Phytochemistry. 2002, 61(6): 597-604.
[18]Morant M, Hehn A, Werck-Reichhart D. Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants. BMC Plant Biol. 2002, 2: 7.
[19]O'Hare HM, Baerga-Ortiz A, Popovic B, et al. High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase. Chem Biol. 2006, 13(3): 287-296.
[20] Wu J, Kinoshita K, Khosla C, et al. Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase. Biochemistry. 2004, 43(51): 16301-16310.
[21]Zhu X, Yu F, Li XC, et al. Production of dihydroisocoumarins in Fusarium verticillioides by swapping ketosynthase domain of the fungal iterative polyketide synthase Fumlp with that of lovastatin diketide synthase. J Am Chem Soc. 2007, 129(1): 36-37.
[1]Lopez-Garcia P,Brochier C,Moreira D,et al.Comparative analysis of a genome fragment of an uncultivated mesopelagic.Environ Microbiol.2004,6:19-34.
[2]Babcock DA,Wawrik B,Paul JH,et al.Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP.J Microbiol Methods.2007,71(2):156-161.
[3]Schirmer A,Gadkari R,Reeves CD,et al.Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta.Appl Environ Microbiol.2005,71(8):4840-4849.
[4]Hardeman F, Sjoling S. Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol. 2007, 59(2): 524-534.
[5]Sata N, Abinsay H, Yoshida WY, et al. Lehualides A-D, metabolites from a Hawaiian sponge of the genus Plakortis. J Nat Prod. 2005, 68(9): 1400-1403.
[6]Kubota T, Endo T, Takahashi Y, et al. Amphidinin B, a new polyketide metabolite from marine dinoflagellate Amphidinium sp. J Antibiot (Tokyo). 2006, 59(8): 512-516.
[7]Lu CK, Chou HN, Lee CK, et al. Prorocentin, a new polyketide from the marine dinoflagellate Prorocentrum lima. Org Lett. 2005, 7(18): 3893-3896.
[8]Kwon HC, Kauffman CA, Jensen PR, et al. MarinomycinsA-D, antitumor - antibiotics of a new structure class from a marine actinomycete of the recently discovered genus "marinispora". J Am Chem Soc. 2006, 128(5): 1622-1632.
[9]Campbell BJ, Stein JL, Cary SC, et al. Evidence of chemolithoautotrophy in the bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete. Appl Environ Microbiol. 2003, 69: 5070-5078.
[10]Cottrell MT, Yu L, Kirchman DL. Sequence and expression analyses of Cytophaga-like hydrolases in a Western arctic metagenomic library and the Sargasso Sea. Appl Environ Microbiol. 2005, 71(12): 8506-8513.
[11]Hallam SJ, Girguis PR, Preston CM, et al. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing Archaea. Appl Environ Microbiol. 2003, 69:5483-5491.
[12]Hughes DS, Felbeck H, Stein JL, et al. A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila. Appl Environ Microbiol. 1997, 63: 3494-3498.
[13]Martin-Cuadrado AB, Lopez-Garcia P, et al. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat. PLoS ONE. 2007, 2(9): e914.
[14]Sabehi G, Massana R, Bielawski JP, et al. Novel Proteorhodopsin variants from the Mediterranean and Red Seas. Environ Microbiol. 2003, 5(10): 842-849.
[15]Schleper C, DeLong EF, Preston CM, et al. Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum. J Bacteriol. 1998, 180: 5003-5009.
[16]Stein JL, Marsh TL, Wu KY, et al. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J
1 Bacteriol.1996,178:591-599,
[17]Xu M,Wang F,Meng J,et al.Construction and preliminary analysis of a metagenomic library from a deep-sea sediment of east Pacific Nodule Province.FEMS Microbiol Ecol.2007,62(3):233-241.
[18]吴杰,李志勇,张戌升.海绵宏基因组文库构建及抗菌肽功能基因的初步筛选.生物技术通报.2006,103(3):95-98.
[19]杨官品,茅云翔.环境细菌宏基因组研究及海洋细菌生物活性物质BAC文库筛选.青岛海洋大学学报:自然科学版.2001,(31)5:718-722.
[20]周通,牛荣丽,林秀坤.宏基因组学在海洋药物研究中的应用.中国海洋药物.2006,25(2):1-6.
[21]王延华,冯忠堂.分子杂交理论与技术.北京:科学出版社,2005.
[1]Kim TK, Fuerst JA. Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ Microbiol. 2006, 8(8): 1460-1470.
[2]Sudek S, Lopanik NB, Waggoner LE, et al. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. J Nat Prod. 2007, 70(1): 67-74.
[3]Ginolhac A, Jarrin C, Gillet B, et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol. 2004, 70(9):5522-5527.
[4]Aparicio JF, Colina AJ, Ceballos, et al. The Biosynthetic Gene Cluster for the 26-Membered ring Polyene Macrolide Pimaricin. J Biol Chem. 1999, 274: 10133-10139.
[5]Aparicio JF, Fouces R, Mendes MV, et al. A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis. Chem Biol. 2000, 7: 895-905.
[6]August PR, Tang L, Yoon YJ, et al. Biosynthesis of the ansamycin antibiotic rifamycin, deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem Biol. 1998, 5: 69-79.
[7]Bevitt DJ, Cortes J, Haydock SF, et al. 6-Deoxyerythronolide-B synthase 2 from Saccharpolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme. Eur J Biochem. 1992, 204: 39-49.
[8]Brauteset T, Sekurova ON, Stella H, et al. Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol. 2000, 7: 395-403.
[9]Caffrey P, Lynch S, Flood E, et al. Amphotericin biosynthesis in Sterptomyces nodosus: deductions from analysis of polyketide synthase and late genes. Chem Biol. 2001, 8: 713-723.
[10]Cortes J, Haydock SF, Bevitt DJ. An unusually large multifuctional polypeptide in the erythromycin-producing PKS of Saccharopolyspora erythraea. Nature. 1997, 348: 176-178.
[11]Floss HG, Yu TW. Lessons from the rifamycin biosynthetic gene cluster. Curr Opin Chem Biol. 1999, 3: 592-597.
[12]Gaitatzis N, Silakowski B, Kunze B, et al. The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase. J Biol Chem. 2002, 277: 13082-13090.
[13]Ikeda H, Nonomiya T, Usami M, et al. Organisation of the biosynthetic gene cluster for the polyketide antihelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci USA. 1999, 96: 9509-9514.
[14]Kakavas SJ, Katz L, Stassi D. Identification and Characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. J Bact. 1997, 179: 7515-7522.
[15]Molnar I, Schupp T, Ono M, et al. The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90. Chem Biol. 2000, 7: 97-109.
[16]Schupp T, Toupet C, Cluzel B, et al. A Sorangium cellulosum (myxobacterium) gene cluster for biosynthesis of macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes. J Bact. 1995, 177(13): 3673-3679.
[17]Schwecke T, Aparicio JF, Molnar I, et al. The biosynthetic gene cluster for the polyketide immunosuppresant rapamycin. Proc Natl Acad Sci USA. 1995, 92: 7839-7843.
[18]Silakowski B, Nordsiek G, Kunze B, et al. Novel features in a combined Polyketide synthase/ non-ribosomal peptide synthetase: The myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sgal5. Chem Biol.2001, 8(1): 59-69.
[19]Silakowski B, Schairer HU, Ehret H, et al. New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1. J Biol Chem. 1999, 274(52): 37391-37399.
[20]Tang L, Shah S, Chung L, et al.Cloning and heterologous expression of the epothilone gene cluster. Science. 2000, 287(5453): 640-642.
[21] Tang Li, Yoon YJ, Choi CY, et al. Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterrannei. Gene. 1998, 216(2): 255-265
[22]Thompson BN, Chaney N, Wing JS, et al. Characterization of the Pyoluteorin Biosynthetic Gene Cluster of Pseudomonas flourescens Pf-5. J Bact. 1999, 181(7): 2166-2174.
[23]Thompson BN, Gould SJ, Loper JE. Identification and sequence analysis of the genes encoding a polyketide synthase required for pyoluteorin biosynthesis in Pseudomonas flourescens Pf-5. Gene. 1997, 204(1-2): 17-24.
[24]Volchegursky Y, Hu Z, Katz L, et al. Biosynthesis of the anti-parasitic agent megalomycin: transformation of erythromycin to megalomycin in Saccharopolyspora erythraea. Mol Microbiol. 2000, 37: 752-762.
[25]Waldron C, Matsushima P, Rosteck PR, et al. Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem Biol. 2001, 8: 487-499.
[26]Wilkinson CJ, Frost EJ, Staunton J, et al. Chain initiation on the soraphen producing modular polyketide synthase from Sorangium cellulosum. Chem Biol. 2001, 8: 1192-1208.
[27]Wu K, Chung L, Revill WP, et al. The Fk520 gene cluster of Strptomycetes hygroscopiocus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene. 2000, 251: 81-90.
[28]Yadav G, Gokhale RS, Mohanty D. SEARCHPKS: A program for detection and analysis of polyketide synthase domains. Nucleic Acids Res. 2003, 31(13): 3654-3658.
[1]Hopwood DA.Cracking the polyketide code. PLoS Biol. 2004, 2(2): E35.
[2]Lau J, Cane DE, Khosla C. Substrate specificity of the loading didomain of the erythromycin polyketide synthase. Biochemistry. 2000, 39(34): 10514-10520.
[3]Abe I, Watanabe T, Lou W, et al. Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase. FEBS J. 2006, 273(1): 208-18.
[4]Crawford JM, Dancy BC, Hill EA, et at. Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase. Proc Natl Acad Sci USA. 2006, 103(45): 16728-16733.
[5]Alekseyev VY, Liu CW, Cane DE, et al. Solution structure and proposed domain domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase. Protein Sci. 2007, 16(10): 2093-2107.
[6]Beck BJ, Aldrich CC, Fecik RA, et al. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J Am Chem Soc. 2003, 125(41): 12551-12557.
[7]Beltran-Alvarez P, Cox RJ, Crosby J, et al. Dissecting the component reactions catalyzed by the actinorhodin minimal polyketide synthase. Biochemistry. 2007, 46(50): 14672-14681.
[8]Chan YA, Boyne MT 2nd, Podevels AM, et al. Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units. Proc Natl Acad Sci USA. 2006, 103(39): 14349-14354.
[9]Funa N, Ohnishi Y, Ebizuka Y, et al. Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochem J. 2002, 367(Pt 3): 781-789.
[10]Gu L, Geders TW, Wang B, et al. GNAT-like strategy for polyketide chain initiation. Science. 2007, 318(5852):970-974.
[11]Lowden PA, Wilkinson B, Bohm GA, et al. Origin and True Nature of the Starter Unit for the Rapamycin Polyketide Synthase. Angew Chem Int Ed Engl. 2001,40(4): 777-779.
[12]Moldenhauer J, Chen XH, Borriss R, et al. Biosynthesis of the antibiotic bacillaene, the product of a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl. 2007,46(43): 8195-8197.
[13]Reeves CD, Chung LM, Liu YQ, et al. A New Substrate Specificity for Acyl Transferase Domains of the Ascomycin Polyketide Synthase in Streptomyces hygroscopicus. J Biol Chem. 2002, (11)277: 9155-9159
[14]Tae H,Kong EB,Park K,et al.ASMPKS:an analysis system for modular polyketide synthases.BMC Bioinformatics.2007,8:327.
[15]Thattai M,Burak Y,Shraiman BI.The origins of specificity in polyketide synthase protein interactions.PLoS Comput Biol.2007,3(9):1827-1835.
[16]Wu J,Kinoshita K,Khosla C,et al.Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase.Biochemistry.2004,43(51):16301-16310.
[17]Yadav G,Gokhale RS,Mohanty D.Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases.J Mol Biol.2003,328(2):335-363.
[18]Yadav G,Gokhale RS,Mohanty D.SEARCHPKS:A program for detection and analysis of polyketide synthase domains.Nucleic Acids Res.2003,31(13):3654-3658.
[19]Yin Y,Lu H,Khosla C,et al.Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase.J Am Chem Soc.2003,125(19):5671-5676.
[20]朱峰,乔建军.聚酮合酶底物专一性的研究进展.中国抗生素杂志.2006,31(11):641-645.
[1]Ikeda H,Nonomiya T,Usami M,et al.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis.Proc Natl Acad Sci USA.1999,96(17):9509-9514,
[2]Kieser T,Bibb MJ,Buttner MJ,et al.Practical Streptomyces Genetics.The John Innes Foundation,2000.
[3]Bisang C,Long PF,Cortes J,et al.A chain initiation factor common to both modular and aromatic polyketide synthases.Nature.1999,401:502-505.
[4]Rajgarhia VB,Priestley ND,Strohl WR.The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp.strain C5.Metab Eng.2001,3(1):49-63.
[5]Chen AY,Cane DE,Khosla C.Structure-based dissociation of a type Ⅰ polyketide synthase module.Chem Biol.2007,14(7):784-792.
[6]Gu L,Geders TW,Wang B,et al.GNAT-like strategy for polyketide chain initiation.Science.2007,318(5852):970-4.
[7]Hans M,Hornung A,Dziarnowski A,et al.Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules.J Am Chem Soc.2003,125(18):5366-5374.
[8]Hopwood DA,Bibb M J,Chater KF,et al.Genetic Manipulation of Streptomyces.A Laboratory Manual.Norwich:The John Innes Foundation,1985.
[9]Hopwood DA.Cracking the polyketide code.PLoS Biol.2004,2(2):E35.
[10]Lau J,Cane DE,Khosla C.Substrate specificity of the loading didomain of the erythromycin polyketide synthase.Biochemistry.2000,39(34):10514-10520.
[11]Long PF, Wilkinson CJ, Bisang CP, et al. Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase. Mol Microbiol. 2002,43(5): 1215-1225.
[12]Lowden PA, Wilkinson B, Bohm GA, et al. Origin and True Nature of the Starter Unit for the Rapamycin Polyketide Synthase. Angew Chem Int Ed Engl. 2001,40(4): 777-779.
[13]Moldenhauer J, Chen XH, Borriss R, et al. Biosynthesis of the antibiotic bacillaene, the product of a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl. 2007,46(43): 8195-8197.
[14]Moore BS, Hertweck C. Biosynthesis and attachment of novel bacterial polyketide synthase starter units. Nat Prod Rep. 2002, 19(1): 70-99.
[15]Reeves CD, Murli S, Ashley GW, et al. Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry. 2001,40(51): 15464-15470.
[16]Renier A, Vivien E, Cociancich S. Substrate specificity-conferring regions of the nonribosomal peptide synthetase adenylation domains involved in albicidin pathotoxin biosynthesis are highly conserved within the species Xanthomonas albilineans. Appl Environ Microbiol. 2007, 73(17): 5523-5530.
[17]Thattai M, Burak Y, Shraiman BI. The origins of specificity in polyketide synthase protein interactions. PLoS Comput Biol. 2007, 3(9): 1827-1835.
[18]Watanabe K, Wang CC, Boddy CN, et al. Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases. J Biol Chem. 2003, 278(43): 42020-42026.
[19]Wu N, Tsuji SY, Cane DE, et al. Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J Am Chem Soc. 2001, 123(27): 6465-6474.
[20]Zhang X, Chen Z, Li M, et al. Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis. Appl Microbiol Biotechnol. 2006, 72(5): 986-994.
[1]Bentley R,Bennett JW.Constructing polyketides:from Collie to combinatorial biosynthesis.Annu Rev Microbiol.1999,53:411-446.
[2]Hill AM.The biosynthesis,molecular genetics and enzymology of the polyketide-derived metabolites.Nat Prod Rep.2006,23:256-320
[3]Hopwood DA.Cracking the polyketide code.PLoS Biol.2004,2(2):E35.
[4]Hranueli D,Peric N,Borovicka B,et al.Molecular Biology of Polyketide Biosynthesis.Food technol biotechnol.2001,39(3):203-213
[5]Fullbeck M,Michalsky E,Dunkel M,et al.Natural products:sources and databases.Nat Prod Rep.2006,23(3):347-356.
[6]Weissman KJ,Leadlay PF.Combinatorial biosynthesis of reduced polyketides.Nat Rev Microbiol.2005,3(12):925-936.
[7]Shen B.Polyketide biosynthesis beyond the type Ⅰ,Ⅱ and Ⅲ polyketide synthase paradigms.Curr Opin Chem Biol.2003,7(2):285-295.
[8]Chan YA,Boyne MT 2nd,Podevels AM,et al.Hydroxymalonyl-acyl carrier protein(ACP)and aminomalonyl-ACP are two additional type Ⅰ polyketide synthase extender units.Proc Natl Acad Sci USA.2006,103(39):14349-14354.
[9]Crawford JM,Dancy BC,Hill EA,et al.Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type Ⅰ polyketide synthase.Proc Natl Acad Sci USA,2006,103(45):16728-16733.
[10]Floss HG.Combinatorial biosynthesis-potential and problems.J Biotechnol.2006,124(1):242-257.
[11]Menzella HG, Reid R, Carney JR, et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol. 2005, 23(9): 1171-1176.
[12]Khosla C, Tang Y, Chen AY, et al. Structure and mechanism of the 6-deoxyerythronolide B synthase. Annu Rev Biochem. 2007, 76: 195-221.
[13]Yadav G, Gokhale RS, Mohanty D. SEARCHPKS: A program for detection and analysis of polyketide synthase domains. Nucleic Acids Res. 2003, 31(13): 3654-3658.
[14]Helen L, Steele, Streit WR. Metagenomics: Advances in ecology and biotechnology. FEMS Microbiol Lett. 2005, 247(2): 105-111.
[15]Ginolhac A, Jarrin C, Gillet B, et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol. 2004, 70(9): 5522-5527.
[16]Sogin ML, Morrison HG, Huber JA. Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci. 2006, 103(32): 12115-12120.
[17]Kim TK, Hewavitharana AK, Shaw PN, et al. Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction. Appl Environ Microbiol. 2006, 72(3): 2118-2125.
[18]Sata N, Abinsay H, Yoshida WY, et al. Lehualides A-D, metabolites from a Hawaiian sponge of the genus Plakortis. J Nat Prod. 2005, 68(9): 1400-1403.
[19]Schirmer A, Gadkari R, Reeves CD, et al. Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta. Appl Environ Microbiol. 2005, 71(8): 4840-4849.
[20]Sujatha P, Bapi RKV, Ramana T. Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus. Microbiol Res. 2005, 160(2): 119-126.
[21]Sudek S, Lopanik NB, Waggoner LE, et al. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. J Nat Prod. 2007, 70(1): 67-74.
[22]Kubota T, Endo T, Takahashi Y, et al. Amphidinin B, a new polyketide metabolite from marine dinoflagellate Amphidinium sp. J Antibiot (Tokyo). 2006, 59(8): 512-516.
[23]Lu CK, Chou HN, Lee CK, et al. Prorocentin, a new polyketide from the marine dinoflagellate Prorocentrum lima. Org Lett. 2005, 7(18): 3893-3896.
[24]Kwon HC, Kauffman CA, Jensen PR, et al. MarinomycinsA-D, antitumor - antibiotics of a new structure class from a marine actinomycete of the recently discovered genus "marinispora". J Am Chem Soc. 2006, 128(5): 1622-1632.
[25]Jenke-Kodama H, Sandmann A, Muller R, et al. Evolutionary implications of bacterial polyketide synthases. Mol Biol Evol. 2005, 22(10): 2027-2039.
[26]Khosla C, Gokhale RS, Jacobsen JR, et al. Tolerance and specificity of polyketide synthases. Annu Rev Biochem. 1999. 68: 219-25.
[27]Savic M, Vasiljevic B. Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers. J Ind Microbiol Biotechnol. 2006, 33(6): 423-430.
[28]Ayuso-Sacido A, Genilloud O. New PCR Primers for the Screening of NRPS and PKS-I Systems in Actinomycetes: Detection and Distribution of These Biosynthetic Gene Sequences in Major Taxonomic Groups. Microbial Ecology. 2005, 49(1): 10-24.
[29]Rose TM, Henikoff JG, Henikoff S. CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 2003, 31(13): 3763-3766.
[30]Rose TM. CODEHOP-mediated PCR - a powerful technique for the identification and characterization of viral genomes. Virol J. 2005, 2: 20.
[31]Beja O, Aravind L, Koonin EV, et al. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science. 2000, 289: 1902-1906.
[32]Preston CM, Suzuki MT, Beja O, et al. Quantitative phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay. Microb Ecol. 2004, 48: 473-488.
[33]de la Torre RJ, Christianson L, Beja O, et al. Proteorhodopsin genes are widely distributed among divergent bacterial taxa. Proc Natl Acad Sci USA. 2003, 100: 12830-12835.
[34]Babcock DA, Wawrik B, Paul JH, et al. Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP. J Microbiol Methods. 2007, 71(2): 156-161.
[35]Suzuki MT, Preston CM, Beja O, et al. Phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay. Microb Ecol. 2004, 48(4): 473-488.
[36]Cottrell MT, Yu L, Kirchman DL. Sequence and expression analyses of Cytophaga-like hydrolases in a Western arctic metagenomic library and the Sargasso Sea. Appl Environ Microbiol. 2005, 71(12): 8506-8513.
[37]Stein JL, Marsh TL, Wu KY, et al. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J Bacteriol. 1996, 178: 591-599.
[38]Martin-Cuadrado AB, Lopez-Garcia P, et al. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat. PLoS ONE. 2007, 2(9): e914.
[39]Beja O, Suzuki MT, Heidelberg JF, et al. Unsuspected diversity among marine aerobic anoxygenic phototrophs. Nature. 2002,415: 630-633.
[40]Hardeman F, Sjoling S. Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol. 2007, 59(2): 524-534.
[41]Kim TK, Fuerst JA. Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ Microbiol. 2006, 8(8): 1460-1470.
[42]Schleper C, DeLong EF, Preston CM, et al Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum. J Bacteriol. 1998, 180: 5003-5009.
[43]Sabehi G, Massana R, Bielawski JP, et al. Novel Proteorhodopsin variants from the Mediterranean and Red Seas. Environ Microbiol. 2003, 5(10): 842-849.
[44]Hallam SJ, Girguis PR, Preston CM, et al. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing Archaea. Appl Environ Microbiol. 2003, 69:5483-5491.
[45]Kruger M, Meyerdierks A, Glockner FO, et al. A conspicuous nickel protein in microbial mats that oxidize methane anaerobically. Nature. 2003,426: 878-881.
[46]Grzymski JJ, Carter BJ, DeLong EF, et al. Comparative genomics of DNA fragments from six Antarctic marine planktonic bacteria. Appl Environ Microbiol. 2006, 72(2): 1532-1541.
[47]Hughes DS, Felbeck H, Stein JL, et al. A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila. Appl Environ Microbiol. 1997, 63: 3494-3498.
[48]Campbell BJ, Stein JL, Cary SC, et al. Evidence of chemolithoautotrophy in the bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete. Appl Environ Microbiol. 2003, 69: 5070-5078.
[49]Lopez-Garcia P, Brochier C, Moreira D, et al. Comparative analysis of a genome fragment of an uncultivated mesopelagic. Environ Microbiol. 2004, 6: 19-34.
[50]Xu M, Wang F, Meng J, et al. Construction and preliminary analysis of a metagenomic library from a deep-sea sediment of east Pacific Nodule Province. FEMS Microbiol Ecol. 2007, 62(3): 233-241.
[51]Zhang Y, Muyerers JPP, Testa G, et al. DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol. 2000, 18: 1314-1317.
[52]Reeves CD, Chung LM, Liu YQ, et al. A New Substrate Specificity for Acyl Transferase Domains of the Ascomycin Polyketide Synthase in Streptomyces hygroscopicus. J Biol Chem, 2002, (11)277: 9155-9159.
[53]Lau J, Cane DE, Khosla C. Substrate specificity of the loading didomain of the erythromycin polyketide synthase. Biochemistry. 2000, 39(34): 10514-10520.
[54]Kim BS, Sherman DH, Reynolds KA. An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases. Protein Eng Des Sel. 2004, (17)3: 277-284.
[55]Kittendorf JD, Sherman DH. Developing tools for engineering hybrid polyketide synthetic pathways. Curr Opin Biotechnol. 2006, 17(6): 597-605.
[56]Thomas I, Martin CJ, Wilkinson CJ, et al. Skipping in a hybrid polyketide synthase-Evidence for ACP-to-ACP chain transfer. Chem Biol. 2002, 9(7): 781-787.
[57]Gokhale RS, Sankaranarayanan R, Mohanty D. Versatility of polyketide synthases in generating metabolic diversity. Curr Opin Struct Biol. 2007, 17(6): 736-743.
[58]Weissman KJ. Single amino acid substitutions alter the efficiency of docking in modular polyketide biosynthesis. Chem Bio Chem. 2006, 7: 1334-1342.
[59]Arthur CJ, Szafranska AE, Long J, et al. The malonyl transferase activity of type II polyketide synthase acyt carrier proteins.Chem Biol. 2006, 13(6): 587-596.
[60]Pfeifer BA, Admiraal SJ, Gramajo H, et al. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science. 2001, 291:1790-1792.
[61]McDaniel R, Ebert-Khosla S, Hopwood DA, et al. Engineered biosynthesis of novel polyketides. Science. 1993,262(5139): 1546-1550.
[62]Ziermann R, Betlach MC. Recombinant polyketide synthesis in Streptomyces: engineering of improved host strains. Biotechniques. 1999,26(1): 106-110.
[63]Kealey JT, Liu L, Santi DV, et al. Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA. 1998, 95(2): 505-509.
[64]Kao CM, Katz L, Khosla C. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science. 1994, 265(5171): 509.
[65]Wang Y, Boghigian BA, Pfeifer BA. Improving heterologous polyketide production in Escherichia coli by overexpression of an S-adenosylmethionine synthetase gene. Appl Microbiol Biotechnol. 2007, 77: 367-373.
[66]Kennedy J, Auclair K, Kendrew SG, et al. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science. 1999, 284(5418): 1368-72.
[67]Reynolds KA. Combinatorial biosynthesis: Lesson learned from nature. Proc. Natl. Acad. Sci. USA. 1998,95: 12744-12746.
[68]Hutchinson CR. Polyketide and non-ribosomal peptide synthases: falling together by coming apart. Proc Natl Acad Sci USA. 2003, 100(6): 3010-3012.
[69]Zotchev SB, Stepanchikova AV, Sergeyko AP, et al. Rational design of macrolides by virtual screening of combinatorial libraries generated through in silico manipulation of polyketide synthases. J Med Chem. 2006, 49(6): 2077-2087.
[70]Pfeifer BA, Khosla C. Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev. 2001,65(1): 106-118.
[71] Watanabe K, Wang CC, Boddy CN, et al. Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases. J Biol Chem. 2003, 278(43): 42020-42026.
[72]McDaniel R, Welch M, Hutchinson CR. Genetic Approaches to Polyketide Antibiotics. Chem Rev. 2005, 105: 543-558.
[73]Katz L, McDaniel R. Novel macrolides through genetic engineering. Med Res Rev. 1999, 19(6): 543-558.
[74]Ruan XR, Pereda A, Stassi D L, et al. Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel "unnatural" natural products. Proc Natl Acad Sci USA. 1999, 96(5): 1846-1851.
[75]Liu L, Thamchaipenet A, Fu H, et al. Biosynthesis of 2-nor-6-deoxyerythronolide B by rationally designed domain substitution. J Am Chem Soc. 1997, 119(43): 10553-10554.
[76]Petkovic H, Lill RE, Sheridan RM, et al. A novel erythromycin, 6-desmethylerythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase. J Antibiot (Tokyo). 2003, 56(6): 543-551.
[77]Oliynyk M, Brown MJ, Cortes J, et al. A hybrid modular polyketide synthase obtained by domain swapping. Chem Biol. 1996, 3(10): 833-839.
[78]Reeves CD, Murli S, Ashley GW, et al. Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry. 2001,40(51): 15464-15470.
[79]Del Vecchio F, Petkovic H, Kendrew SG, et al. Active-site residue, domain and module swaps in modular polyketide synthases. J Ind Microbiol Biotechnol. 2003, 30(8): 489-494.
[80]Kumar P, Koppisch AT, Cane DE, et al. Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA: ACP transacylase. J Am Chem Soc. 2003, 125(47): 14307-14312.
[81]Corte's J, Haydock SF, Roberts GA, et al. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature. 1990, 348(6297): 176-178.
[82]Rajgarhia VB, Priestley ND, Strohl WR. The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5. Metab Eng. 2001, 3(1): 49-63.
[83]Lopez JV. Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus. Mol Genet Genomics. 2003, 270(5): 420-431.
[84]Rodriguez E, McDaniel R. Combinatorial biosynthesis of antimicrobials and other natural products. Curr Opin Microbiol. 2001, 4(5): 526-534.
[85]Dorrestein PC, Kelleher NL. Dissecting non-ribosomal and polyketide biosynthetic machineries using electrospray ionization Fourier-Transform mass spectrometry. Nat Prod Rep. 2006, 23(6): 893-918.
[86]Abe I, Watanabe T, Lou W, et al. Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase. FEBS J. 2006, 273(1): 208-218.
[87]Barrios-Llerena ME, Burja AM, Wright PC. Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites. J Ind Microbiol Biotechnol. 2007, 34(6): 443-456.
[88]Beck BJ, Aldrich CC, Fecik RA, et al. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J Am Chem Soc. 2003, 125(41): 12551-12557.
[89]Beja O, Koonin EV, Aravind L, et al. Comparative genomic analysis of archaeal genotypic variants in a single population, and in two different oceanic provinces. Appl Environ Microbiol. 2002, 68: 335-345.
[90]Beja O, Suzuki MT, Koonin EV, et al. Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environ Microbiol. 2000, 2: 516-529.
[91]Beja O, Spudich EN, Spudich JL, et al. Proteorhodopsin phototrophy in the ocean. Nature. 2001,411:786-789.
[92]Beja O. To BAC or not to BAC: marine ecogenomics. Curr Opin Biotechnol. 2004, 15(3): 187-190.
[93]Bisang C, Long PF, Cortes J, et al. A chain initiation factor common to both modular and aromatic polyketide synthases. Nature. 1999, 401: 502-505.
[94]Castonguay R, He W, Chen AY, et al. Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase. J Am Chem Soc. 2007, 129(44): 13758-13769.
[95]Cheng YQ, Tang GL, Shen B. Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA, 2003, (100) 6: 3149-3154
[96]Chin YW, Balunas MJ, Chai HB, et al. Drug Discovery From Natural Sources. J AAPS. 2006, 8(2): 28
[97]Donadio S, Monciardini P, Sosio M. Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics. Nat Prod Rep. 2007, 24(5): 1073-1109.
[98]Fritzler JM, Zhu G. Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase. Int J Parasitol. 2007, 37: 307-316
[99]Funa N, Ohnishi Y, Ebizuka Y, et al. Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus. J Biol Chem. 2002, 277(7): 4628-4635.
[100]Hans M, Hornung A, Dziarnowski A, et al. Mechanistic analysis of acyltransferase domain exchange in polyketide synthase modules. J Am Chem Soc. 2003, 125(18): 5366-5374.
[101]Homburg S, Oswald E, Hacker J, et al. Expression analysis of the colibactin gene cluster coding for a novel polyketide in Escherichia coli. FEMS Microbiol Lett. 2007, 275(2): 255-262.
[102]Ikeda H, Nonomiya T, Usami M, et al. Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci USA. 1999, 96(17): 9509-9514.
[103]John U, Beszteri B, Derelle E, et al. Novel Insights into Evolution of Protistan Polyketide Synthases through Phylogenomic Analysis. Protist. 2008, 159(1): 21-30.
[104]Khatun S, Waugh AC, Redpath MB, et al. Distribution of polyketide synthase genes in bacterial populations. J Antibiot (Tokyo). 2002, 55(1): 107-108.
[105]Kroken S, Glass NL, Taylor JW, et al. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc Natl Acad Sci U S A. 2003, 100(26): 15670-15675.
[106]Kurosaki F, Mitsuma S, Arisawa M. Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme, by free coenzyme A. Phytochemistry. 2002, 61(6): 597-604.
[107]Kwon HJ, Smith WC, Xiang L, et al. Cloning and heterologous expression of the macrotetrolide biosynthetic gene cluster revealed a novel polyketide synthase that lacks an acyl carrier protein. J Am Chem Soc. 2001, 123(14): 3385-3386.
[108]Ma Y, Smith LH, Cox RJ, et al. Catalytic relationships between type I and type II iterative polyketide synthases: The Aspergillus parasiticus norsolorinic acid synthase. Chembiochem. 2006,7(12): 1951-1958.
[109]Mayer KM, Ford J, Macpherson GR, et al. Exploring the diversity of marine-derived fungal polyketide synthases. Can J Microbiol. 2007, 53(2): 291-302.
[110]McDaniel R, Ebert-Khosla S, Fu H, et al. Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature. 1995, 375: 549-554.
[111]Mikkelsen J, Hojrup P, Rasmussen MM, et al. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase. Biochem J. 1985, 227(1): 21-27.
[112]Moldenhauer J, Chen XH, Borriss R, et al. Biosynthesis of the antibiotic bacillaene, the product of a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl. 2007, 46(43): 8195-8197.
[113]Nicholson TP, Winfield C, Westcott J, et al. First in vitro directed biosynthesis of new compounds by a minimal type II polyketide synthase: evidence for the mechanism of chain length determination. Chem Commun (Camb). 2003, (6): 686-687.
[114]O'Hare HM, Baerga-Ortiz A, Popovic B, et al. High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase. Chem Biol. 2006, 13(3): 287-296.
[115]Piel J, Hui D, Wen G, et al. Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci USA. 2004, 101(46): 16222-16227.
[116]Sherman DH, Smith JL. Clearing the skies over modular polyketide synthases. Chem Biol. 2006, 1(8): 505-9.
[117]Smith S, Tsai SC. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep. 2007, 24(5): 1041-1072.
[118]Stacevic A, Jaspars M, Cullum J, et al. Predicting the Nature and Timing of Epimerisation on a Modular Polyketide Synthase. ChemBioChem. 2007, 8(1): 28-31.
[119]Strohl WR. The role of natural products in a modern drug discovery program. Drug Discov Today. 2000, 5(2): 39-41.
[120]Strohl, W. R. Industrial antibiotics: today and the future. In W. R. Strohl (ed.), Biotechnology of antibiotics, 2nd ed, 1997. Marcel Dekker, NewYork, N.Y.
[121]Tae H, Kong EB, Park K, et al. ASMPKS: an analysis system for modular polyketide synthases. BMC Bioinformatics. 2007, 8: 327.
[122]Tsai SC, Miercke LJ, Krucinski J, et al. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc Natl Acad Sci USA. 2001, 98(26): 14808-14813.
[123]Vecchio FD, Petkovic H, Kendrew SG, et al. Active-site residue, domain and module swaps in modular polyketide synthases. J Ind Microbiol Biotechnol. 2003, 30(8): 489-494.
[124]Watanabe CM, Townsend CA. Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B(1). Chem Biol. 2002, 9(9): 981-988.
[125]Wawrik B, Kerkhof L, Zylstra GJ, et al. Identification of Unique Type II Polyketide Synthase Genes in Soil. App Environ Microbiol, 2005, (71)5: 2232-2238
[126]Weissman KJ. The structural basis for docking in modular polyketide biosynthesis. Chembiochem. 2006, 7(3): 485-494.
[127]Wu J, Kinoshita K, Khosla C, et al. Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase. Biochemistry. 2004,43(51): 16301-16310.
[128]Wu N, Tsuji SY, Cane DE, et al. Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J Am Chem Soc. 2001, 123(27): 6465-6474.
[129]Yadav G, Gokhale RS, Mohanty D. Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. J Mol Biol. 2003, 328(2): 335-363.
[130]Yin Y, Lu H, Khosla C, et al. Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. J Am Chem Soc. 2003, 125(19): 5671-5676.
[131]Zha W, Rubin-Pitel SB, Zhao H. Characterization of the substrate specificity of PhlD, a type III poiyketide synthase from Pseudomonas fluorescens. J Biol Chem. 2006, 281(42): 32036-47.
[132]Zhang H, White-Phillip JA, Melancon CE 3rd, et al. Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars. J Am Chem Soc. 2007, 129(47): 14670-14683.
[133]Zhang WJ, Ames BD, Tsai SC, et al. Engineered Biosynthesis of a Novel Amidated Poiyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Poiyketide Synthase. App Environ Microbiol. 2006, (72) 4: 2573-2580
[134]Zhang X, Chen Z, Li M, et al. Construction of ivermectin producer by domain swaps of avermectin poiyketide synthase in Streptomyces avermitilis. Appl Microbiol Biotechnol. 2006, 72(5): 986-994.
[135]Zhou P, Florova G, Reynolds KA. Poiyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis. Chem Biol. 1999, 6: 577-584.
[136]Zhu X, Yu F, Li XC, et al. Production of dihydroisocoumarins in Fusarium verticillioides by swapping ketosynthase domain of the fungal iterative poiyketide synthase Fumlp with that of lovastatin diketide synthase. J Am Chem Soc. 2007, 129(1): 36-37.
[2]伯杰细菌鉴定手册(第八版).科学出版社.1984.
[3]中国科学院微生物研究所放线菌组.链霉菌鉴定手册.北京:科学出版社.1975.
[4]Blackwood CB,Waldrop MP,Zak DR,et al.Molecular Analysis of Fungal Communities and Laccase Genes in Decomposing Litter Reveals Differences Among Forest Types But No Impact of Nitrogen Deposition.Environ Microbiol.2007,9:1306-1316.
[5]Herrera A,Cockell CS.Exploring microbial diversity in volcanic environments:a review of methods in DNA extraction.J Microbiol Methods.2007,70(1):1-12.
[6]Hortal S,Pera J,Galipenso L,et al.Molecular identification of the edible ectomycorrhizal fungus Lacterius in the symbiotic and extraradical mycelium stages.J Biotechnol.2006,126(2):123-134.
[7]Marchetti E,Accinelli C,Talame V,et aL Persistence of Cry Toxins and Cry Gene From Genetically Modified Plants in Two Agricultural Soils.Agron Sustain Dev.2007,27:231-236.
[8]Mayer KM,Ford J,Macpherson GR,et al.Exploring the diversity of marine-derived fungal polyketide synthases.Can J Microbiol.2007,53(2):291-302.
[9]Mummey DL,Rillig MC,et al.Endogeic Earthworms Differentially Influence Bacterial Communities Associated with Different Soil Aggregate Size Fractions.Soil Biol Biochem.2006,35:1608-1614.
[10]Mummey DL,Rillig MC.Evaluation of LSU rRNA-gene PCR Primers for the Study of Arbuscular Mycorrhizal Fungal Communities via Terminal Restrictions Fragment Length Polymorphism Analysis.J Microbiol Methods.2007,70(1):200-204.
[11]Mummey DL,Rillig MC.The Invasive Plant Species Centaurea maculosa Alters Arbuscular Mycorrhizal Fungal Communities in the Field.Plant Soil.2006,288:81-90.
[12]Robe P,Nalin R,Capellano C,et al.Extraction of DNA from soil.Eur J Soil Biol.2003,(39)4:183-190
[13]Webster G,Watt LC,Rinna J,et al.A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries.Environ Microbiol.2006,8(9):1575-1589.
[14]Weiss A,Jerome V,Freitag R.Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.J Chromatogr B Analyt Technol Biomed Life Sci.2007,853(1-2):190-197.
[15]李会荣,俞勇,曾胤新,等.北极太平洋扇区海洋沉积物细菌多样性的系统发育分析.微生物学报.2006,46(2):177-183.
[16]吴少杰,朱丽华,杨志娟.不同培养基对海洋放线菌抗微生物活性的影响.华北煤炭医学院学报.2006,(8)5:600-602.
[1]Barrios-Llerena ME,Burja AM,et al.Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites.J Ind Microbiol Biotechnol.2007,34(6):443-456.
[2]Castonguay R,He W,Chen AY,et al.Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase.J Am Chem Soc.2007,129(44):13758-13769.
[3]Atoui A,Dao HP,Mathieu F,et al.Amplification and diversity analysis of ketosynthase domains of putative polyketide synthase genes in Aspergillus ochraceus and Aspergillus carbonarius producers ofochratoxin A.Mol Nutr Food Res.2006,50(6):488-493.
[4]Savic M,Vasiljevic B.Targeting polyketide synthase gene pool within actinomycetes:new degenerate primers.J Ind Microbiol Biotechnol.2006,33(6):423-430.
[5]Khosla C,Gokhale RS,Jacobsen JR,et al.Tolerance and specificity of polyketide synthases.Annu Rev Biochern,1999.68:219-25.
[6]Jenke-Kodama H,Sandmann A,Muller R,et al.Evolutionary implications of bacterial polyketide synthases.Mol Biol Evol.2005,22(10):2027-2039.
[7]Rose TM,Henikoff JG,Henikoff S.CODEHOP(COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.Nucleic Acids Res.2003,31(13):3763-3766.
[8]Rose TM.CODEHOP-mediated PCR-a powerful technique for the identification and characterization of viral genomes.Virol J.2005,2:20.
[9]Gokhale RS,Sankaranarayanan R,Mohanty D.Versatility of polyketide synthases in generating metabolic diversity.Curr Opin Struct Biol.2007,17(6):736-743.
[10]Kim TK,Fuerst JA.Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata:culture-dependent and culture-independent approaches.Environ Microbiol.2006,8(8):1460-1470.
[11]Piel J,Hui D,Wen G,et al.Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci USA. 2004, 101(46): 16222-16227.
[12]Sudek S, Lopanik NB, Waggoner LE, et al. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. J Nat Prod. 2007, 70(1): 67-74.
[13]Ginolhac A, Jarrin C, Gillet B, et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol. 2004, 70(9): 5522-5527.
[14]Baines JE, McGovern RM, Persing D, et al. Consensus-degenerate hybrid oligonucleotide primers (CODEHOP) for the detection of novel papillomaviruses and their application to esophageal and tonsillar carcinomas. J Virol Methods. 2005, 123(1): 81-87.
[15]Beck BJ, Aldrich CC, Fecik RA, et al. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J Am Chem Soc. 2003, 125(41): 12551-12557.
[16]Kim BC, Lee JM, Ahn JS, et al. Cloning, sequencing, and characterization of the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2. J Microbiol Biotechnol. 2007, 17(5): 830-839.
[17]Kurosaki F, Mitsuma S, Arisawa M. Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme, by free coenzyme A. Phytochemistry. 2002, 61(6): 597-604.
[18]Morant M, Hehn A, Werck-Reichhart D. Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants. BMC Plant Biol. 2002, 2: 7.
[19]O'Hare HM, Baerga-Ortiz A, Popovic B, et al. High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase. Chem Biol. 2006, 13(3): 287-296.
[20] Wu J, Kinoshita K, Khosla C, et al. Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase. Biochemistry. 2004, 43(51): 16301-16310.
[21]Zhu X, Yu F, Li XC, et al. Production of dihydroisocoumarins in Fusarium verticillioides by swapping ketosynthase domain of the fungal iterative polyketide synthase Fumlp with that of lovastatin diketide synthase. J Am Chem Soc. 2007, 129(1): 36-37.
[1]Lopez-Garcia P,Brochier C,Moreira D,et al.Comparative analysis of a genome fragment of an uncultivated mesopelagic.Environ Microbiol.2004,6:19-34.
[2]Babcock DA,Wawrik B,Paul JH,et al.Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP.J Microbiol Methods.2007,71(2):156-161.
[3]Schirmer A,Gadkari R,Reeves CD,et al.Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta.Appl Environ Microbiol.2005,71(8):4840-4849.
[4]Hardeman F, Sjoling S. Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol. 2007, 59(2): 524-534.
[5]Sata N, Abinsay H, Yoshida WY, et al. Lehualides A-D, metabolites from a Hawaiian sponge of the genus Plakortis. J Nat Prod. 2005, 68(9): 1400-1403.
[6]Kubota T, Endo T, Takahashi Y, et al. Amphidinin B, a new polyketide metabolite from marine dinoflagellate Amphidinium sp. J Antibiot (Tokyo). 2006, 59(8): 512-516.
[7]Lu CK, Chou HN, Lee CK, et al. Prorocentin, a new polyketide from the marine dinoflagellate Prorocentrum lima. Org Lett. 2005, 7(18): 3893-3896.
[8]Kwon HC, Kauffman CA, Jensen PR, et al. MarinomycinsA-D, antitumor - antibiotics of a new structure class from a marine actinomycete of the recently discovered genus "marinispora". J Am Chem Soc. 2006, 128(5): 1622-1632.
[9]Campbell BJ, Stein JL, Cary SC, et al. Evidence of chemolithoautotrophy in the bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete. Appl Environ Microbiol. 2003, 69: 5070-5078.
[10]Cottrell MT, Yu L, Kirchman DL. Sequence and expression analyses of Cytophaga-like hydrolases in a Western arctic metagenomic library and the Sargasso Sea. Appl Environ Microbiol. 2005, 71(12): 8506-8513.
[11]Hallam SJ, Girguis PR, Preston CM, et al. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing Archaea. Appl Environ Microbiol. 2003, 69:5483-5491.
[12]Hughes DS, Felbeck H, Stein JL, et al. A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila. Appl Environ Microbiol. 1997, 63: 3494-3498.
[13]Martin-Cuadrado AB, Lopez-Garcia P, et al. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat. PLoS ONE. 2007, 2(9): e914.
[14]Sabehi G, Massana R, Bielawski JP, et al. Novel Proteorhodopsin variants from the Mediterranean and Red Seas. Environ Microbiol. 2003, 5(10): 842-849.
[15]Schleper C, DeLong EF, Preston CM, et al. Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum. J Bacteriol. 1998, 180: 5003-5009.
[16]Stein JL, Marsh TL, Wu KY, et al. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J
1 Bacteriol.1996,178:591-599,
[17]Xu M,Wang F,Meng J,et al.Construction and preliminary analysis of a metagenomic library from a deep-sea sediment of east Pacific Nodule Province.FEMS Microbiol Ecol.2007,62(3):233-241.
[18]吴杰,李志勇,张戌升.海绵宏基因组文库构建及抗菌肽功能基因的初步筛选.生物技术通报.2006,103(3):95-98.
[19]杨官品,茅云翔.环境细菌宏基因组研究及海洋细菌生物活性物质BAC文库筛选.青岛海洋大学学报:自然科学版.2001,(31)5:718-722.
[20]周通,牛荣丽,林秀坤.宏基因组学在海洋药物研究中的应用.中国海洋药物.2006,25(2):1-6.
[21]王延华,冯忠堂.分子杂交理论与技术.北京:科学出版社,2005.
[1]Kim TK, Fuerst JA. Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ Microbiol. 2006, 8(8): 1460-1470.
[2]Sudek S, Lopanik NB, Waggoner LE, et al. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. J Nat Prod. 2007, 70(1): 67-74.
[3]Ginolhac A, Jarrin C, Gillet B, et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol. 2004, 70(9):5522-5527.
[4]Aparicio JF, Colina AJ, Ceballos, et al. The Biosynthetic Gene Cluster for the 26-Membered ring Polyene Macrolide Pimaricin. J Biol Chem. 1999, 274: 10133-10139.
[5]Aparicio JF, Fouces R, Mendes MV, et al. A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis. Chem Biol. 2000, 7: 895-905.
[6]August PR, Tang L, Yoon YJ, et al. Biosynthesis of the ansamycin antibiotic rifamycin, deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem Biol. 1998, 5: 69-79.
[7]Bevitt DJ, Cortes J, Haydock SF, et al. 6-Deoxyerythronolide-B synthase 2 from Saccharpolyspora erythraea. Cloning of the structural gene, sequence analysis and inferred domain structure of the multifunctional enzyme. Eur J Biochem. 1992, 204: 39-49.
[8]Brauteset T, Sekurova ON, Stella H, et al. Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol. 2000, 7: 395-403.
[9]Caffrey P, Lynch S, Flood E, et al. Amphotericin biosynthesis in Sterptomyces nodosus: deductions from analysis of polyketide synthase and late genes. Chem Biol. 2001, 8: 713-723.
[10]Cortes J, Haydock SF, Bevitt DJ. An unusually large multifuctional polypeptide in the erythromycin-producing PKS of Saccharopolyspora erythraea. Nature. 1997, 348: 176-178.
[11]Floss HG, Yu TW. Lessons from the rifamycin biosynthetic gene cluster. Curr Opin Chem Biol. 1999, 3: 592-597.
[12]Gaitatzis N, Silakowski B, Kunze B, et al. The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase. J Biol Chem. 2002, 277: 13082-13090.
[13]Ikeda H, Nonomiya T, Usami M, et al. Organisation of the biosynthetic gene cluster for the polyketide antihelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci USA. 1999, 96: 9509-9514.
[14]Kakavas SJ, Katz L, Stassi D. Identification and Characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. J Bact. 1997, 179: 7515-7522.
[15]Molnar I, Schupp T, Ono M, et al. The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90. Chem Biol. 2000, 7: 97-109.
[16]Schupp T, Toupet C, Cluzel B, et al. A Sorangium cellulosum (myxobacterium) gene cluster for biosynthesis of macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes. J Bact. 1995, 177(13): 3673-3679.
[17]Schwecke T, Aparicio JF, Molnar I, et al. The biosynthetic gene cluster for the polyketide immunosuppresant rapamycin. Proc Natl Acad Sci USA. 1995, 92: 7839-7843.
[18]Silakowski B, Nordsiek G, Kunze B, et al. Novel features in a combined Polyketide synthase/ non-ribosomal peptide synthetase: The myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella aurantiaca Sgal5. Chem Biol.2001, 8(1): 59-69.
[19]Silakowski B, Schairer HU, Ehret H, et al. New lessons for combinatorial biosynthesis from myxobacteria. The myxothiazol biosynthetic gene cluster of Stigmatella aurantiaca DW4/3-1. J Biol Chem. 1999, 274(52): 37391-37399.
[20]Tang L, Shah S, Chung L, et al.Cloning and heterologous expression of the epothilone gene cluster. Science. 2000, 287(5453): 640-642.
[21] Tang Li, Yoon YJ, Choi CY, et al. Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterrannei. Gene. 1998, 216(2): 255-265
[22]Thompson BN, Chaney N, Wing JS, et al. Characterization of the Pyoluteorin Biosynthetic Gene Cluster of Pseudomonas flourescens Pf-5. J Bact. 1999, 181(7): 2166-2174.
[23]Thompson BN, Gould SJ, Loper JE. Identification and sequence analysis of the genes encoding a polyketide synthase required for pyoluteorin biosynthesis in Pseudomonas flourescens Pf-5. Gene. 1997, 204(1-2): 17-24.
[24]Volchegursky Y, Hu Z, Katz L, et al. Biosynthesis of the anti-parasitic agent megalomycin: transformation of erythromycin to megalomycin in Saccharopolyspora erythraea. Mol Microbiol. 2000, 37: 752-762.
[25]Waldron C, Matsushima P, Rosteck PR, et al. Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem Biol. 2001, 8: 487-499.
[26]Wilkinson CJ, Frost EJ, Staunton J, et al. Chain initiation on the soraphen producing modular polyketide synthase from Sorangium cellulosum. Chem Biol. 2001, 8: 1192-1208.
[27]Wu K, Chung L, Revill WP, et al. The Fk520 gene cluster of Strptomycetes hygroscopiocus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene. 2000, 251: 81-90.
[28]Yadav G, Gokhale RS, Mohanty D. SEARCHPKS: A program for detection and analysis of polyketide synthase domains. Nucleic Acids Res. 2003, 31(13): 3654-3658.
[1]Hopwood DA.Cracking the polyketide code. PLoS Biol. 2004, 2(2): E35.
[2]Lau J, Cane DE, Khosla C. Substrate specificity of the loading didomain of the erythromycin polyketide synthase. Biochemistry. 2000, 39(34): 10514-10520.
[3]Abe I, Watanabe T, Lou W, et al. Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase. FEBS J. 2006, 273(1): 208-18.
[4]Crawford JM, Dancy BC, Hill EA, et at. Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase. Proc Natl Acad Sci USA. 2006, 103(45): 16728-16733.
[5]Alekseyev VY, Liu CW, Cane DE, et al. Solution structure and proposed domain domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase. Protein Sci. 2007, 16(10): 2093-2107.
[6]Beck BJ, Aldrich CC, Fecik RA, et al. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J Am Chem Soc. 2003, 125(41): 12551-12557.
[7]Beltran-Alvarez P, Cox RJ, Crosby J, et al. Dissecting the component reactions catalyzed by the actinorhodin minimal polyketide synthase. Biochemistry. 2007, 46(50): 14672-14681.
[8]Chan YA, Boyne MT 2nd, Podevels AM, et al. Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units. Proc Natl Acad Sci USA. 2006, 103(39): 14349-14354.
[9]Funa N, Ohnishi Y, Ebizuka Y, et al. Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis. Biochem J. 2002, 367(Pt 3): 781-789.
[10]Gu L, Geders TW, Wang B, et al. GNAT-like strategy for polyketide chain initiation. Science. 2007, 318(5852):970-974.
[11]Lowden PA, Wilkinson B, Bohm GA, et al. Origin and True Nature of the Starter Unit for the Rapamycin Polyketide Synthase. Angew Chem Int Ed Engl. 2001,40(4): 777-779.
[12]Moldenhauer J, Chen XH, Borriss R, et al. Biosynthesis of the antibiotic bacillaene, the product of a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl. 2007,46(43): 8195-8197.
[13]Reeves CD, Chung LM, Liu YQ, et al. A New Substrate Specificity for Acyl Transferase Domains of the Ascomycin Polyketide Synthase in Streptomyces hygroscopicus. J Biol Chem. 2002, (11)277: 9155-9159
[14]Tae H,Kong EB,Park K,et al.ASMPKS:an analysis system for modular polyketide synthases.BMC Bioinformatics.2007,8:327.
[15]Thattai M,Burak Y,Shraiman BI.The origins of specificity in polyketide synthase protein interactions.PLoS Comput Biol.2007,3(9):1827-1835.
[16]Wu J,Kinoshita K,Khosla C,et al.Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase.Biochemistry.2004,43(51):16301-16310.
[17]Yadav G,Gokhale RS,Mohanty D.Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases.J Mol Biol.2003,328(2):335-363.
[18]Yadav G,Gokhale RS,Mohanty D.SEARCHPKS:A program for detection and analysis of polyketide synthase domains.Nucleic Acids Res.2003,31(13):3654-3658.
[19]Yin Y,Lu H,Khosla C,et al.Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase.J Am Chem Soc.2003,125(19):5671-5676.
[20]朱峰,乔建军.聚酮合酶底物专一性的研究进展.中国抗生素杂志.2006,31(11):641-645.
[1]Ikeda H,Nonomiya T,Usami M,et al.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis.Proc Natl Acad Sci USA.1999,96(17):9509-9514,
[2]Kieser T,Bibb MJ,Buttner MJ,et al.Practical Streptomyces Genetics.The John Innes Foundation,2000.
[3]Bisang C,Long PF,Cortes J,et al.A chain initiation factor common to both modular and aromatic polyketide synthases.Nature.1999,401:502-505.
[4]Rajgarhia VB,Priestley ND,Strohl WR.The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp.strain C5.Metab Eng.2001,3(1):49-63.
[5]Chen AY,Cane DE,Khosla C.Structure-based dissociation of a type Ⅰ polyketide synthase module.Chem Biol.2007,14(7):784-792.
[6]Gu L,Geders TW,Wang B,et al.GNAT-like strategy for polyketide chain initiation.Science.2007,318(5852):970-4.
[7]Hans M,Hornung A,Dziarnowski A,et al.Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules.J Am Chem Soc.2003,125(18):5366-5374.
[8]Hopwood DA,Bibb M J,Chater KF,et al.Genetic Manipulation of Streptomyces.A Laboratory Manual.Norwich:The John Innes Foundation,1985.
[9]Hopwood DA.Cracking the polyketide code.PLoS Biol.2004,2(2):E35.
[10]Lau J,Cane DE,Khosla C.Substrate specificity of the loading didomain of the erythromycin polyketide synthase.Biochemistry.2000,39(34):10514-10520.
[11]Long PF, Wilkinson CJ, Bisang CP, et al. Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase. Mol Microbiol. 2002,43(5): 1215-1225.
[12]Lowden PA, Wilkinson B, Bohm GA, et al. Origin and True Nature of the Starter Unit for the Rapamycin Polyketide Synthase. Angew Chem Int Ed Engl. 2001,40(4): 777-779.
[13]Moldenhauer J, Chen XH, Borriss R, et al. Biosynthesis of the antibiotic bacillaene, the product of a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl. 2007,46(43): 8195-8197.
[14]Moore BS, Hertweck C. Biosynthesis and attachment of novel bacterial polyketide synthase starter units. Nat Prod Rep. 2002, 19(1): 70-99.
[15]Reeves CD, Murli S, Ashley GW, et al. Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry. 2001,40(51): 15464-15470.
[16]Renier A, Vivien E, Cociancich S. Substrate specificity-conferring regions of the nonribosomal peptide synthetase adenylation domains involved in albicidin pathotoxin biosynthesis are highly conserved within the species Xanthomonas albilineans. Appl Environ Microbiol. 2007, 73(17): 5523-5530.
[17]Thattai M, Burak Y, Shraiman BI. The origins of specificity in polyketide synthase protein interactions. PLoS Comput Biol. 2007, 3(9): 1827-1835.
[18]Watanabe K, Wang CC, Boddy CN, et al. Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases. J Biol Chem. 2003, 278(43): 42020-42026.
[19]Wu N, Tsuji SY, Cane DE, et al. Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J Am Chem Soc. 2001, 123(27): 6465-6474.
[20]Zhang X, Chen Z, Li M, et al. Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis. Appl Microbiol Biotechnol. 2006, 72(5): 986-994.
[1]Bentley R,Bennett JW.Constructing polyketides:from Collie to combinatorial biosynthesis.Annu Rev Microbiol.1999,53:411-446.
[2]Hill AM.The biosynthesis,molecular genetics and enzymology of the polyketide-derived metabolites.Nat Prod Rep.2006,23:256-320
[3]Hopwood DA.Cracking the polyketide code.PLoS Biol.2004,2(2):E35.
[4]Hranueli D,Peric N,Borovicka B,et al.Molecular Biology of Polyketide Biosynthesis.Food technol biotechnol.2001,39(3):203-213
[5]Fullbeck M,Michalsky E,Dunkel M,et al.Natural products:sources and databases.Nat Prod Rep.2006,23(3):347-356.
[6]Weissman KJ,Leadlay PF.Combinatorial biosynthesis of reduced polyketides.Nat Rev Microbiol.2005,3(12):925-936.
[7]Shen B.Polyketide biosynthesis beyond the type Ⅰ,Ⅱ and Ⅲ polyketide synthase paradigms.Curr Opin Chem Biol.2003,7(2):285-295.
[8]Chan YA,Boyne MT 2nd,Podevels AM,et al.Hydroxymalonyl-acyl carrier protein(ACP)and aminomalonyl-ACP are two additional type Ⅰ polyketide synthase extender units.Proc Natl Acad Sci USA.2006,103(39):14349-14354.
[9]Crawford JM,Dancy BC,Hill EA,et al.Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type Ⅰ polyketide synthase.Proc Natl Acad Sci USA,2006,103(45):16728-16733.
[10]Floss HG.Combinatorial biosynthesis-potential and problems.J Biotechnol.2006,124(1):242-257.
[11]Menzella HG, Reid R, Carney JR, et al. Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol. 2005, 23(9): 1171-1176.
[12]Khosla C, Tang Y, Chen AY, et al. Structure and mechanism of the 6-deoxyerythronolide B synthase. Annu Rev Biochem. 2007, 76: 195-221.
[13]Yadav G, Gokhale RS, Mohanty D. SEARCHPKS: A program for detection and analysis of polyketide synthase domains. Nucleic Acids Res. 2003, 31(13): 3654-3658.
[14]Helen L, Steele, Streit WR. Metagenomics: Advances in ecology and biotechnology. FEMS Microbiol Lett. 2005, 247(2): 105-111.
[15]Ginolhac A, Jarrin C, Gillet B, et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol. 2004, 70(9): 5522-5527.
[16]Sogin ML, Morrison HG, Huber JA. Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci. 2006, 103(32): 12115-12120.
[17]Kim TK, Hewavitharana AK, Shaw PN, et al. Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction. Appl Environ Microbiol. 2006, 72(3): 2118-2125.
[18]Sata N, Abinsay H, Yoshida WY, et al. Lehualides A-D, metabolites from a Hawaiian sponge of the genus Plakortis. J Nat Prod. 2005, 68(9): 1400-1403.
[19]Schirmer A, Gadkari R, Reeves CD, et al. Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta. Appl Environ Microbiol. 2005, 71(8): 4840-4849.
[20]Sujatha P, Bapi RKV, Ramana T. Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus. Microbiol Res. 2005, 160(2): 119-126.
[21]Sudek S, Lopanik NB, Waggoner LE, et al. Identification of the Putative Bryostatin Polyketide Synthase Gene Cluster from "Candidatus Endobugula sertula", the Uncultivated Microbial Symbiont of the Marine Bryozoan Bugula neritina. J Nat Prod. 2007, 70(1): 67-74.
[22]Kubota T, Endo T, Takahashi Y, et al. Amphidinin B, a new polyketide metabolite from marine dinoflagellate Amphidinium sp. J Antibiot (Tokyo). 2006, 59(8): 512-516.
[23]Lu CK, Chou HN, Lee CK, et al. Prorocentin, a new polyketide from the marine dinoflagellate Prorocentrum lima. Org Lett. 2005, 7(18): 3893-3896.
[24]Kwon HC, Kauffman CA, Jensen PR, et al. MarinomycinsA-D, antitumor - antibiotics of a new structure class from a marine actinomycete of the recently discovered genus "marinispora". J Am Chem Soc. 2006, 128(5): 1622-1632.
[25]Jenke-Kodama H, Sandmann A, Muller R, et al. Evolutionary implications of bacterial polyketide synthases. Mol Biol Evol. 2005, 22(10): 2027-2039.
[26]Khosla C, Gokhale RS, Jacobsen JR, et al. Tolerance and specificity of polyketide synthases. Annu Rev Biochem. 1999. 68: 219-25.
[27]Savic M, Vasiljevic B. Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers. J Ind Microbiol Biotechnol. 2006, 33(6): 423-430.
[28]Ayuso-Sacido A, Genilloud O. New PCR Primers for the Screening of NRPS and PKS-I Systems in Actinomycetes: Detection and Distribution of These Biosynthetic Gene Sequences in Major Taxonomic Groups. Microbial Ecology. 2005, 49(1): 10-24.
[29]Rose TM, Henikoff JG, Henikoff S. CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 2003, 31(13): 3763-3766.
[30]Rose TM. CODEHOP-mediated PCR - a powerful technique for the identification and characterization of viral genomes. Virol J. 2005, 2: 20.
[31]Beja O, Aravind L, Koonin EV, et al. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science. 2000, 289: 1902-1906.
[32]Preston CM, Suzuki MT, Beja O, et al. Quantitative phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay. Microb Ecol. 2004, 48: 473-488.
[33]de la Torre RJ, Christianson L, Beja O, et al. Proteorhodopsin genes are widely distributed among divergent bacterial taxa. Proc Natl Acad Sci USA. 2003, 100: 12830-12835.
[34]Babcock DA, Wawrik B, Paul JH, et al. Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP. J Microbiol Methods. 2007, 71(2): 156-161.
[35]Suzuki MT, Preston CM, Beja O, et al. Phylogenetic screening of ribosomal RNA gene-containing clones in Bacterial Artificial Chromosome (BAC) libraries from different depths in Monterey Bay. Microb Ecol. 2004, 48(4): 473-488.
[36]Cottrell MT, Yu L, Kirchman DL. Sequence and expression analyses of Cytophaga-like hydrolases in a Western arctic metagenomic library and the Sargasso Sea. Appl Environ Microbiol. 2005, 71(12): 8506-8513.
[37]Stein JL, Marsh TL, Wu KY, et al. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J Bacteriol. 1996, 178: 591-599.
[38]Martin-Cuadrado AB, Lopez-Garcia P, et al. Metagenomics of the deep Mediterranean, a warm bathypelagic habitat. PLoS ONE. 2007, 2(9): e914.
[39]Beja O, Suzuki MT, Heidelberg JF, et al. Unsuspected diversity among marine aerobic anoxygenic phototrophs. Nature. 2002,415: 630-633.
[40]Hardeman F, Sjoling S. Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol. 2007, 59(2): 524-534.
[41]Kim TK, Fuerst JA. Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ Microbiol. 2006, 8(8): 1460-1470.
[42]Schleper C, DeLong EF, Preston CM, et al Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum. J Bacteriol. 1998, 180: 5003-5009.
[43]Sabehi G, Massana R, Bielawski JP, et al. Novel Proteorhodopsin variants from the Mediterranean and Red Seas. Environ Microbiol. 2003, 5(10): 842-849.
[44]Hallam SJ, Girguis PR, Preston CM, et al. Identification of methyl coenzyme M reductase A (mcrA) genes associated with methane-oxidizing Archaea. Appl Environ Microbiol. 2003, 69:5483-5491.
[45]Kruger M, Meyerdierks A, Glockner FO, et al. A conspicuous nickel protein in microbial mats that oxidize methane anaerobically. Nature. 2003,426: 878-881.
[46]Grzymski JJ, Carter BJ, DeLong EF, et al. Comparative genomics of DNA fragments from six Antarctic marine planktonic bacteria. Appl Environ Microbiol. 2006, 72(2): 1532-1541.
[47]Hughes DS, Felbeck H, Stein JL, et al. A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila. Appl Environ Microbiol. 1997, 63: 3494-3498.
[48]Campbell BJ, Stein JL, Cary SC, et al. Evidence of chemolithoautotrophy in the bacterial community associated with Alvinella pompejana, a hydrothermal vent polychaete. Appl Environ Microbiol. 2003, 69: 5070-5078.
[49]Lopez-Garcia P, Brochier C, Moreira D, et al. Comparative analysis of a genome fragment of an uncultivated mesopelagic. Environ Microbiol. 2004, 6: 19-34.
[50]Xu M, Wang F, Meng J, et al. Construction and preliminary analysis of a metagenomic library from a deep-sea sediment of east Pacific Nodule Province. FEMS Microbiol Ecol. 2007, 62(3): 233-241.
[51]Zhang Y, Muyerers JPP, Testa G, et al. DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol. 2000, 18: 1314-1317.
[52]Reeves CD, Chung LM, Liu YQ, et al. A New Substrate Specificity for Acyl Transferase Domains of the Ascomycin Polyketide Synthase in Streptomyces hygroscopicus. J Biol Chem, 2002, (11)277: 9155-9159.
[53]Lau J, Cane DE, Khosla C. Substrate specificity of the loading didomain of the erythromycin polyketide synthase. Biochemistry. 2000, 39(34): 10514-10520.
[54]Kim BS, Sherman DH, Reynolds KA. An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases. Protein Eng Des Sel. 2004, (17)3: 277-284.
[55]Kittendorf JD, Sherman DH. Developing tools for engineering hybrid polyketide synthetic pathways. Curr Opin Biotechnol. 2006, 17(6): 597-605.
[56]Thomas I, Martin CJ, Wilkinson CJ, et al. Skipping in a hybrid polyketide synthase-Evidence for ACP-to-ACP chain transfer. Chem Biol. 2002, 9(7): 781-787.
[57]Gokhale RS, Sankaranarayanan R, Mohanty D. Versatility of polyketide synthases in generating metabolic diversity. Curr Opin Struct Biol. 2007, 17(6): 736-743.
[58]Weissman KJ. Single amino acid substitutions alter the efficiency of docking in modular polyketide biosynthesis. Chem Bio Chem. 2006, 7: 1334-1342.
[59]Arthur CJ, Szafranska AE, Long J, et al. The malonyl transferase activity of type II polyketide synthase acyt carrier proteins.Chem Biol. 2006, 13(6): 587-596.
[60]Pfeifer BA, Admiraal SJ, Gramajo H, et al. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science. 2001, 291:1790-1792.
[61]McDaniel R, Ebert-Khosla S, Hopwood DA, et al. Engineered biosynthesis of novel polyketides. Science. 1993,262(5139): 1546-1550.
[62]Ziermann R, Betlach MC. Recombinant polyketide synthesis in Streptomyces: engineering of improved host strains. Biotechniques. 1999,26(1): 106-110.
[63]Kealey JT, Liu L, Santi DV, et al. Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA. 1998, 95(2): 505-509.
[64]Kao CM, Katz L, Khosla C. Engineered biosynthesis of a complete macrolactone in a heterologous host. Science. 1994, 265(5171): 509.
[65]Wang Y, Boghigian BA, Pfeifer BA. Improving heterologous polyketide production in Escherichia coli by overexpression of an S-adenosylmethionine synthetase gene. Appl Microbiol Biotechnol. 2007, 77: 367-373.
[66]Kennedy J, Auclair K, Kendrew SG, et al. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science. 1999, 284(5418): 1368-72.
[67]Reynolds KA. Combinatorial biosynthesis: Lesson learned from nature. Proc. Natl. Acad. Sci. USA. 1998,95: 12744-12746.
[68]Hutchinson CR. Polyketide and non-ribosomal peptide synthases: falling together by coming apart. Proc Natl Acad Sci USA. 2003, 100(6): 3010-3012.
[69]Zotchev SB, Stepanchikova AV, Sergeyko AP, et al. Rational design of macrolides by virtual screening of combinatorial libraries generated through in silico manipulation of polyketide synthases. J Med Chem. 2006, 49(6): 2077-2087.
[70]Pfeifer BA, Khosla C. Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev. 2001,65(1): 106-118.
[71] Watanabe K, Wang CC, Boddy CN, et al. Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases. J Biol Chem. 2003, 278(43): 42020-42026.
[72]McDaniel R, Welch M, Hutchinson CR. Genetic Approaches to Polyketide Antibiotics. Chem Rev. 2005, 105: 543-558.
[73]Katz L, McDaniel R. Novel macrolides through genetic engineering. Med Res Rev. 1999, 19(6): 543-558.
[74]Ruan XR, Pereda A, Stassi D L, et al. Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel "unnatural" natural products. Proc Natl Acad Sci USA. 1999, 96(5): 1846-1851.
[75]Liu L, Thamchaipenet A, Fu H, et al. Biosynthesis of 2-nor-6-deoxyerythronolide B by rationally designed domain substitution. J Am Chem Soc. 1997, 119(43): 10553-10554.
[76]Petkovic H, Lill RE, Sheridan RM, et al. A novel erythromycin, 6-desmethylerythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase. J Antibiot (Tokyo). 2003, 56(6): 543-551.
[77]Oliynyk M, Brown MJ, Cortes J, et al. A hybrid modular polyketide synthase obtained by domain swapping. Chem Biol. 1996, 3(10): 833-839.
[78]Reeves CD, Murli S, Ashley GW, et al. Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry. 2001,40(51): 15464-15470.
[79]Del Vecchio F, Petkovic H, Kendrew SG, et al. Active-site residue, domain and module swaps in modular polyketide synthases. J Ind Microbiol Biotechnol. 2003, 30(8): 489-494.
[80]Kumar P, Koppisch AT, Cane DE, et al. Enhancing the modularity of the modular polyketide synthases: transacylation in modular polyketide synthases catalyzed by malonyl-CoA: ACP transacylase. J Am Chem Soc. 2003, 125(47): 14307-14312.
[81]Corte's J, Haydock SF, Roberts GA, et al. An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature. 1990, 348(6297): 176-178.
[82]Rajgarhia VB, Priestley ND, Strohl WR. The product of dpsC confers starter unit fidelity upon the daunorubicin polyketide synthase of Streptomyces sp. strain C5. Metab Eng. 2001, 3(1): 49-63.
[83]Lopez JV. Naturally mosaic operons for secondary metabolite biosynthesis: variability and putative horizontal transfer of discrete catalytic domains of the epothilone polyketide synthase locus. Mol Genet Genomics. 2003, 270(5): 420-431.
[84]Rodriguez E, McDaniel R. Combinatorial biosynthesis of antimicrobials and other natural products. Curr Opin Microbiol. 2001, 4(5): 526-534.
[85]Dorrestein PC, Kelleher NL. Dissecting non-ribosomal and polyketide biosynthetic machineries using electrospray ionization Fourier-Transform mass spectrometry. Nat Prod Rep. 2006, 23(6): 893-918.
[86]Abe I, Watanabe T, Lou W, et al. Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase. FEBS J. 2006, 273(1): 208-218.
[87]Barrios-Llerena ME, Burja AM, Wright PC. Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites. J Ind Microbiol Biotechnol. 2007, 34(6): 443-456.
[88]Beck BJ, Aldrich CC, Fecik RA, et al. Substrate recognition and channeling of monomodules from the pikromycin polyketide synthase. J Am Chem Soc. 2003, 125(41): 12551-12557.
[89]Beja O, Koonin EV, Aravind L, et al. Comparative genomic analysis of archaeal genotypic variants in a single population, and in two different oceanic provinces. Appl Environ Microbiol. 2002, 68: 335-345.
[90]Beja O, Suzuki MT, Koonin EV, et al. Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environ Microbiol. 2000, 2: 516-529.
[91]Beja O, Spudich EN, Spudich JL, et al. Proteorhodopsin phototrophy in the ocean. Nature. 2001,411:786-789.
[92]Beja O. To BAC or not to BAC: marine ecogenomics. Curr Opin Biotechnol. 2004, 15(3): 187-190.
[93]Bisang C, Long PF, Cortes J, et al. A chain initiation factor common to both modular and aromatic polyketide synthases. Nature. 1999, 401: 502-505.
[94]Castonguay R, He W, Chen AY, et al. Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase. J Am Chem Soc. 2007, 129(44): 13758-13769.
[95]Cheng YQ, Tang GL, Shen B. Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA, 2003, (100) 6: 3149-3154
[96]Chin YW, Balunas MJ, Chai HB, et al. Drug Discovery From Natural Sources. J AAPS. 2006, 8(2): 28
[97]Donadio S, Monciardini P, Sosio M. Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics. Nat Prod Rep. 2007, 24(5): 1073-1109.
[98]Fritzler JM, Zhu G. Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase. Int J Parasitol. 2007, 37: 307-316
[99]Funa N, Ohnishi Y, Ebizuka Y, et al. Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus. J Biol Chem. 2002, 277(7): 4628-4635.
[100]Hans M, Hornung A, Dziarnowski A, et al. Mechanistic analysis of acyltransferase domain exchange in polyketide synthase modules. J Am Chem Soc. 2003, 125(18): 5366-5374.
[101]Homburg S, Oswald E, Hacker J, et al. Expression analysis of the colibactin gene cluster coding for a novel polyketide in Escherichia coli. FEMS Microbiol Lett. 2007, 275(2): 255-262.
[102]Ikeda H, Nonomiya T, Usami M, et al. Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci USA. 1999, 96(17): 9509-9514.
[103]John U, Beszteri B, Derelle E, et al. Novel Insights into Evolution of Protistan Polyketide Synthases through Phylogenomic Analysis. Protist. 2008, 159(1): 21-30.
[104]Khatun S, Waugh AC, Redpath MB, et al. Distribution of polyketide synthase genes in bacterial populations. J Antibiot (Tokyo). 2002, 55(1): 107-108.
[105]Kroken S, Glass NL, Taylor JW, et al. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc Natl Acad Sci U S A. 2003, 100(26): 15670-15675.
[106]Kurosaki F, Mitsuma S, Arisawa M. Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme, by free coenzyme A. Phytochemistry. 2002, 61(6): 597-604.
[107]Kwon HJ, Smith WC, Xiang L, et al. Cloning and heterologous expression of the macrotetrolide biosynthetic gene cluster revealed a novel polyketide synthase that lacks an acyl carrier protein. J Am Chem Soc. 2001, 123(14): 3385-3386.
[108]Ma Y, Smith LH, Cox RJ, et al. Catalytic relationships between type I and type II iterative polyketide synthases: The Aspergillus parasiticus norsolorinic acid synthase. Chembiochem. 2006,7(12): 1951-1958.
[109]Mayer KM, Ford J, Macpherson GR, et al. Exploring the diversity of marine-derived fungal polyketide synthases. Can J Microbiol. 2007, 53(2): 291-302.
[110]McDaniel R, Ebert-Khosla S, Fu H, et al. Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature. 1995, 375: 549-554.
[111]Mikkelsen J, Hojrup P, Rasmussen MM, et al. Amino acid sequence around the active-site serine residue in the acyltransferase domain of goat mammary fatty acid synthetase. Biochem J. 1985, 227(1): 21-27.
[112]Moldenhauer J, Chen XH, Borriss R, et al. Biosynthesis of the antibiotic bacillaene, the product of a giant polyketide synthase complex of the trans-AT family. Angew Chem Int Ed Engl. 2007, 46(43): 8195-8197.
[113]Nicholson TP, Winfield C, Westcott J, et al. First in vitro directed biosynthesis of new compounds by a minimal type II polyketide synthase: evidence for the mechanism of chain length determination. Chem Commun (Camb). 2003, (6): 686-687.
[114]O'Hare HM, Baerga-Ortiz A, Popovic B, et al. High-throughput mutagenesis to evaluate models of stereochemical control in ketoreductase domains from the erythromycin polyketide synthase. Chem Biol. 2006, 13(3): 287-296.
[115]Piel J, Hui D, Wen G, et al. Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc Natl Acad Sci USA. 2004, 101(46): 16222-16227.
[116]Sherman DH, Smith JL. Clearing the skies over modular polyketide synthases. Chem Biol. 2006, 1(8): 505-9.
[117]Smith S, Tsai SC. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep. 2007, 24(5): 1041-1072.
[118]Stacevic A, Jaspars M, Cullum J, et al. Predicting the Nature and Timing of Epimerisation on a Modular Polyketide Synthase. ChemBioChem. 2007, 8(1): 28-31.
[119]Strohl WR. The role of natural products in a modern drug discovery program. Drug Discov Today. 2000, 5(2): 39-41.
[120]Strohl, W. R. Industrial antibiotics: today and the future. In W. R. Strohl (ed.), Biotechnology of antibiotics, 2nd ed, 1997. Marcel Dekker, NewYork, N.Y.
[121]Tae H, Kong EB, Park K, et al. ASMPKS: an analysis system for modular polyketide synthases. BMC Bioinformatics. 2007, 8: 327.
[122]Tsai SC, Miercke LJ, Krucinski J, et al. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc Natl Acad Sci USA. 2001, 98(26): 14808-14813.
[123]Vecchio FD, Petkovic H, Kendrew SG, et al. Active-site residue, domain and module swaps in modular polyketide synthases. J Ind Microbiol Biotechnol. 2003, 30(8): 489-494.
[124]Watanabe CM, Townsend CA. Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B(1). Chem Biol. 2002, 9(9): 981-988.
[125]Wawrik B, Kerkhof L, Zylstra GJ, et al. Identification of Unique Type II Polyketide Synthase Genes in Soil. App Environ Microbiol, 2005, (71)5: 2232-2238
[126]Weissman KJ. The structural basis for docking in modular polyketide biosynthesis. Chembiochem. 2006, 7(3): 485-494.
[127]Wu J, Kinoshita K, Khosla C, et al. Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase. Biochemistry. 2004,43(51): 16301-16310.
[128]Wu N, Tsuji SY, Cane DE, et al. Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J Am Chem Soc. 2001, 123(27): 6465-6474.
[129]Yadav G, Gokhale RS, Mohanty D. Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. J Mol Biol. 2003, 328(2): 335-363.
[130]Yin Y, Lu H, Khosla C, et al. Expression and kinetic analysis of the substrate specificity of modules 5 and 6 of the picromycin/methymycin polyketide synthase. J Am Chem Soc. 2003, 125(19): 5671-5676.
[131]Zha W, Rubin-Pitel SB, Zhao H. Characterization of the substrate specificity of PhlD, a type III poiyketide synthase from Pseudomonas fluorescens. J Biol Chem. 2006, 281(42): 32036-47.
[132]Zhang H, White-Phillip JA, Melancon CE 3rd, et al. Elucidation of the kijanimicin gene cluster: insights into the biosynthesis of spirotetronate antibiotics and nitrosugars. J Am Chem Soc. 2007, 129(47): 14670-14683.
[133]Zhang WJ, Ames BD, Tsai SC, et al. Engineered Biosynthesis of a Novel Amidated Poiyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Poiyketide Synthase. App Environ Microbiol. 2006, (72) 4: 2573-2580
[134]Zhang X, Chen Z, Li M, et al. Construction of ivermectin producer by domain swaps of avermectin poiyketide synthase in Streptomyces avermitilis. Appl Microbiol Biotechnol. 2006, 72(5): 986-994.
[135]Zhou P, Florova G, Reynolds KA. Poiyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis. Chem Biol. 1999, 6: 577-584.
[136]Zhu X, Yu F, Li XC, et al. Production of dihydroisocoumarins in Fusarium verticillioides by swapping ketosynthase domain of the fungal iterative poiyketide synthase Fumlp with that of lovastatin diketide synthase. J Am Chem Soc. 2007, 129(1): 36-37.