海洋微生物Ⅰ型PKS基因资源的筛选与鉴定
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摘要
海洋是一个极其复杂的生态大系统,孕育了丰富的微生物基因资源。使用传统的微生物分离培养方法,建立海洋微生物种质基因库十分必要,但无疑杯水车薪,因为占环境微生物总量99%的属于未获得培养的微生物,对这些微生物种群的研究只能另辟蹊径。依靠目前成熟的基因工程技术,从海洋生境中直接分离、克隆和表达有用的海洋微生物功能基因,是非常实际的选择。其中,合成红霉素、雷帕霉素和埃博霉素等具有医学治疗价值的次级代谢产物的Ⅰ型聚酮合酶(polyketide synthase,PKS)基因很适合作为宏基因组功能基因筛选目标。
目前国际通用的技术路线是直接提取某种环境中的总DNA,构建环境宏基因组文库,使用杂交或PCR方法从中筛选含Ⅰ型PKS基因的阳性克隆,再对其进行测序得到目的基因序列。目前最典型的工作就是从土壤宏基因组文库中筛选Ⅰ型PKS基因。在海洋宏基因组研究领域,目前已经在可产生已知聚酮类化合物的海绵、总草苔虫等海洋无脊椎动物的共生微生物宏基因组文库中筛选到了一些Ⅰ型PKS基因。
本研究以能生物合成聚酮类化合物的Ⅰ型聚酮合酶(polyketide synthase, PKS)基因作为研究对象,设计了一套针对Ⅰ型PKS基因中保守序列的简并引物,对我国东海海域的沿岸土壤、海水、海洋沉积物等不同生境以及可产生聚酮类化合物的海洋细菌进行Ⅰ型PKS基因的筛选鉴定以及功能分析,希望能获得一批Ⅰ型PKS基因片段,为今后开发海洋来源的新型聚酮类抗肿瘤药物打下工作基础。
一、东海不同生境总基因组DNA的提取
采集我国东海洋山港沿岸土壤样本、海水和海底沉积物等不同生境样本以及各种微生物样本,针对不同样本使用了不同方法以及商品化试剂盒提取其总DNA,试用16 s rDNA通用引物以及分光光度法进行质量鉴定,比较其提取得率和纯度,从中选取最适合该样本的DNA提取方法用于下一步研究。
二、简并引物设计以及有效性验证
从GenBank中查找已知PKS基因的保守序列,使用网上CODEHOP简并引物设计服务,结合Primer Premier 5和DNAMAN等生物学软件,已经设计出针对Ⅰ型PKS基因保守区域KS片段的简并引物一套:其中正义引物针对PQQR模序,共32条,序列为: 5- CGC TCC ATG GAY CCS CAR CA -3’反义引物则针对HGTGT模序,共24条,序列为: 5-- GTS CCS GTS SCR TGS SHY TCS A -3’
在含Ⅰ型PKS基因的游动束丝放线菌(Actinosynnema pretiosum,产生大环内酰胺抗生素Ansamitocin)和Strentomyces avermitili(s产生阿维菌素Avermectin)上验证了其有效性,从两种放线菌的基因组DNA中均扩增出了长约700 bp的目的片断,将其连接到PMD-18 T载体上测序,测序结果经BLASTN比对,完全正确。
三、东海洋山港沿岸土壤、海水以及海底沉积物样本中Ⅰ型PKS筛选鉴定与功能分析
试用Mobio公司的基于玻璃珠与蛋白酶K消化方法的土壤基因组提取试剂盒,从东海沿岸土壤中和海水中提取了高质量的基因组DNA,使用前面设计验证过的兼并引物进行PCR扩增。反应体系:2.5μl 10×buffer,4μl dNTP(2.5 mmol/L),正义引物与反义引物各0.25μl(40μmmol/L),1μl模板DNA,0.25μl Taq Ex(5U/μl,Takara),双蒸水补足反应体系。反应条件:94℃预变性5 min;94℃,1 min;63℃,1 min;72℃,2 min;35个循环;72℃延伸10 min。将PCR阳性条带胶回收后,克隆到PMD18-T载体上,转化大肠杆菌BL21,将筛选得到的阳性克隆子送样到上海英骏生物技术有限公司用3730测序仪进行序列测定。将得到的目标片段序列提交GenBANK数据库,采用网上BLAST程序进行序列的同源性分析。使用CLUSTAL X软件和PHYLIP软件包进行序列比对和功能分析。
从不同生境中获得了23条I型PKS基因的KS序列片段,其中土壤来源的19个,海水来源的4个,长度从630 bp到690 bp不等。GenBANK序列号为:DQ640993 , DQ640997 , DQ641926 , DQ641927 ; DQ673137-DQ673152 ;EF554859-EF554861。
将其与已知的PKS酮缩合酶基因片段进行BLAST比对,所获得的23条的核苷酸序列BLAST得分大部分在40-150之间,与Genbank中已有序列同源性很低,提示其来源于新物种;而所推断的氨基酸序列保守,BLAST比对直接提示其归属于PKS的KS片段,负责聚酮类化合物生物合成过程中的酮缩合反应。用CLUSTAL X和PHYLIP软件对所获序列与GenBank中已知功能的KS片段进行分子进化分析和功能比对,结果发现:发现DQ640997;DQ673137,DQ673138;DQ673140;DQ673142;DQ673144;DQ673145;DQ673149;DQ673151核心保守序列为VQTACSTS,结合其上游的N(DE)KD序列排布方式,提示这些KS片段可能来源于PKS/NRPS杂和基因簇,参与杂和的聚酮/非核糖体多肽类化合物的合成。
四、产Macrolactins的海洋细菌X-2中I型PKS基因的筛选鉴定与功能分析
Macrolactins是近年来从海洋微生物中分离的一群具有抗菌、抗病毒、抗肿瘤以及抑制胆固醇合成活性的大环内酯类化合物。其生物合成基因簇迄今仍未有报道。
本室从东海海泥中分离到一株海洋细菌X-2,可产生多种Macrolactins。从Macrolactins的化学结构分析,其属于典型的大环内酯类化合物,其生物合成基因簇应为典型的Ⅰ型PKS基因簇。利用现有对于Ⅰ型PKS基因簇的认识,可对Macrolactins的生物合成基因簇的构成作出预测,本研究构建了该细菌的fosmid基因组文库,以扩增出的KS结构域的DNA片段(GenBank accessing number: EF486351)做探针筛选文库,克隆并鉴定了该细菌中可能为Macrolactins生物合成负责的部分Ⅰ型PKS基因。
总之,本研究使用简并引物扩增的方法,设计了一套针对Ⅰ型PKS基因中保守序列的简并引物,对我国东海海域的沿岸土壤、海水、海洋沉积物等不同生境以及产聚酮类化合物的海洋细菌中的Ⅰ型PKS基因的进行了初步的筛选鉴定和功能分析工作,获得了相当数量的新型Ⅰ型PKS基因,进行了初步的功能分析。研究结果从一个侧面揭示了PKS基因在海洋不同生境和微生物中的广泛分布状况,已经获得的序列不仅可作为探针,用于后期海洋宏基因组文库中PKS阳性克隆的筛选,还可直接用于后期新型聚酮类化合物的异源表达及生物合成研究。本研究最终将为我国海洋微生物功能基因资源开发利用和人民身体健康作出巨大贡献。
The biosphere is dominated by microorganisms, which produce numerous secondary metabolites with various biological activities. The efforts to discover new bioactive molecules from microbes have lasted one century. However, the number of novel compounds discovered in recent years has not increased in proportion to the progress in culture-based screening methods, because only a small fraction of all microbes can be cultured by traditional methods. The knowledge about the“underexplored majority”is still poor.
To avoid culture limit, modern biological technology approaches have provide direct access to explore genes or gene clusters that are responsible for the synthesis of microbial secondary metabolites. Polyketide synthase (PKS) gene clusters are suitable to be the screening target. PKSs synthesize polyketides, a large family of secondary metabolites that include many clinically important drugs such as erythromycin (antibacterial), epothilone (antitumor), soraphen (antifungal), rapamycin (immunosuppressant) and lovastatin (anti-hypercholesterolemic).
In this study, a set of degenerate oligonucleotide primers, designed for amplification KSs domains, had been employed to identify KS gene fragments from soil and seawater DNA samples. Our purpose was to develop a culture-independent method to directly access PKSⅠgene diversity in Chinese soil and seawater, because the related knowledge was incomplete. This would open up the possibilities of using the results for DNA fingerprints of secondary metabolites and as the basis for a search for attractive antibiotic biosynthesis genes that could be used in the heterogenous expression and combinatorial biosynthesis. Furthermore, the amplified KS gene fragments can also be used as homologous hybridization probes to detect the clones harbored PKS gene clusters in the recombinant metagenomic libraries that would be constructed in the following researches.
Part 1. DNA extractions
DNA extractions were carried out by using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc.). Compared with an adaptation of the procedure described by Zhou et al, the MO BIO kit produced DNA with a higher level of purity that can be used for the following PCR directly.
DNA extractions of the soil and seawater samples were performed following the manufacturers’instructions. The DNA yield were calculated from the A260. The DNA were loaded on a 1.0% agarose gels and ethidium bromide staining to determine size and concentration. All extracted DNA were stored at -20℃until use.
Part 2. Design of the degenerate PCR primers
With the help of The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) and manually correction, a set of degenerate PCR primers were designed from conserved regions of KS domains of bacterial PKSⅠgenes.
The forward primers KSF (5’-CGC TCC ATG GAY CCS CAR CA-3’) were based on the conserved motif SDPQQR. The reverse primers KSR (5’-GTC CCG GTG CCR TGS SHY TCS A -3’) were based on the conserved motif HGTGT.
The specificity of the primer set had been confirmed by testing with a collection of polyketide-producing strain (Streptomyces rimosus 8229, Streptomyces coelicolor ATCC101478, Streptomyces avermitlis, ATCC 31271) prior to the following PCR reactions with the environmental samples. The specific fragments amplified with KSF-KSR were about 700 bp in length.
Part 3. Isolation of 23 ketosynthase fragments from soil and sea water In this study, the degenerate PCR primers were employed to amplified the soil and seawater DNA samples.
The 25μl PCR mixture consisting of 2.5μl 10×Ex Taq Buffer (Mg2+ Plus), 2μl dNTP Mixture( 2.5 mmol/L each), 0.125μl TaKaRa Ex Taq (5U/μl), 1.5μl DMSO(dimethyl sulfoxide), 0.25μl each primer (20μmol/L), 1μl template DNA (0.03 g/L), and 17.375μl ddH2O.
The initial denaturation step at 94℃for 5 minutes was followed by 35 cycles of DNA denaturation at 94℃for 1 minute, primer annealing at 65℃for 1 minute, and DNA strand extension at 72℃for 1 minute, and a final extension step at 72℃for 10 minutes.
The result PCR products about 700 bp were purified on agarose gels (mini-DNA rapid purification kit, BioDev-Tech) and then cloned into PMD18-T Vector according to the manufacturer’s instructions (TaKaRa). positive recombinants were then submitted for sequencing using an ABI3730 DNA Sequencer (USA) with M13 primer at the Invitrogen Biotechnology Company.
DNA sequences obtained from the PCR product had been translated into amino acid sequences using the Primer Premier 5 software after cutting off the vector and primer regions. Sequence analyses were performed using the BLASTP programs provided by the National Center for Biotechnology Information (NCBI).
After excluding the identical clones, 23 unique nucleotide fragments (ranged from 630 bp to 690 bp after cutting off the primer and vector sequences) were obtained. Among them 19 clones were amplified from soil (DQ640993, DQ640997, DQ641926, DQ641927, and DQ673137～DQ673151) and 4 from the seawater (DQ673151 and EF554859～EF554861).
The GC content of the resulting 23 KS gene fragments ranged from 49% to 73% with an average value 66.7%. The alignment of the predicted protein sequences revealed that all the nucleotide sequences encoded the highly conserved region corresponding to the active site of the beta-ketoacyl synthetase consensus region of PKS I genes, these KS gene fragments showed 45% to 85% identities to the known PKS I amino acid sequences in the GenBank database.
Among the 23 KS sequences obtained in present study, 9 sequences displayed a unique pattern N(DE)KD, 22 amino acids upstream from the cysteine active site in the KS domain and the conserved pattern VDTACSSS was replaced by VQTACSTS.
These two patterns were shown to identify KS domains belonging to hybrid NRPS/PKS systems, which require specialized KS domains capable of using the peptidyl substrate of the NRPS donor site. On the other hand, the remained 15 KS sequences (the 4 KS sequences derived from the sea water included) showed typical conserved patterns of KS domains. No KSQ like fragment was found from all the 23 KS fragments.
Part 4. Isolation and functional analysis of the PKS genes in the marine bacteria X-2 which produce Macrolactins
Macrolactins are 24-membered macrolides produced by unidentified marine bacterium, Actinomadura sp. and Bacillus sp., which exhibit both antibacterial and antitumor activities in vitro. In addition, macrolactins were shown to inhibit mammalian herpes simplex virus and HIV replication. Although cloning of the macrolactins biosynthetic genes has not been reported, its chemical structure suggests that it is assembled by a modular PKS system.
The environmental strain X-2 which was isolated from the sediment of the East China Sea producing Macrolatin A, B and O. KS domains cloned from the X-2 showed 98% identities to the known pks2 gene cluster in environmental strain Bacillus amyloliquefaciens FZB42. The result showed that the pks2 gene cluster may be play a important role in the biosynthesis of Macrolatins.
Data presented in this study showed that the PCR method using degenerate primer to isolate the secondary metabolites biosynthesis gene fragments from the environmental samples and strains were practically effective. This study will provide the foundation for the biosynthesis and heterogeneous expression of polyketides and contribute to the exploitation of microbial genetic resources belong to the“underexplored majority”.
目前国际通用的技术路线是直接提取某种环境中的总DNA,构建环境宏基因组文库,使用杂交或PCR方法从中筛选含Ⅰ型PKS基因的阳性克隆,再对其进行测序得到目的基因序列。目前最典型的工作就是从土壤宏基因组文库中筛选Ⅰ型PKS基因。在海洋宏基因组研究领域,目前已经在可产生已知聚酮类化合物的海绵、总草苔虫等海洋无脊椎动物的共生微生物宏基因组文库中筛选到了一些Ⅰ型PKS基因。
本研究以能生物合成聚酮类化合物的Ⅰ型聚酮合酶(polyketide synthase, PKS)基因作为研究对象,设计了一套针对Ⅰ型PKS基因中保守序列的简并引物,对我国东海海域的沿岸土壤、海水、海洋沉积物等不同生境以及可产生聚酮类化合物的海洋细菌进行Ⅰ型PKS基因的筛选鉴定以及功能分析,希望能获得一批Ⅰ型PKS基因片段,为今后开发海洋来源的新型聚酮类抗肿瘤药物打下工作基础。
一、东海不同生境总基因组DNA的提取
采集我国东海洋山港沿岸土壤样本、海水和海底沉积物等不同生境样本以及各种微生物样本,针对不同样本使用了不同方法以及商品化试剂盒提取其总DNA,试用16 s rDNA通用引物以及分光光度法进行质量鉴定,比较其提取得率和纯度,从中选取最适合该样本的DNA提取方法用于下一步研究。
二、简并引物设计以及有效性验证
从GenBank中查找已知PKS基因的保守序列,使用网上CODEHOP简并引物设计服务,结合Primer Premier 5和DNAMAN等生物学软件,已经设计出针对Ⅰ型PKS基因保守区域KS片段的简并引物一套:其中正义引物针对PQQR模序,共32条,序列为: 5- CGC TCC ATG GAY CCS CAR CA -3’反义引物则针对HGTGT模序,共24条,序列为: 5-- GTS CCS GTS SCR TGS SHY TCS A -3’
在含Ⅰ型PKS基因的游动束丝放线菌(Actinosynnema pretiosum,产生大环内酰胺抗生素Ansamitocin)和Strentomyces avermitili(s产生阿维菌素Avermectin)上验证了其有效性,从两种放线菌的基因组DNA中均扩增出了长约700 bp的目的片断,将其连接到PMD-18 T载体上测序,测序结果经BLASTN比对,完全正确。
三、东海洋山港沿岸土壤、海水以及海底沉积物样本中Ⅰ型PKS筛选鉴定与功能分析
试用Mobio公司的基于玻璃珠与蛋白酶K消化方法的土壤基因组提取试剂盒,从东海沿岸土壤中和海水中提取了高质量的基因组DNA,使用前面设计验证过的兼并引物进行PCR扩增。反应体系:2.5μl 10×buffer,4μl dNTP(2.5 mmol/L),正义引物与反义引物各0.25μl(40μmmol/L),1μl模板DNA,0.25μl Taq Ex(5U/μl,Takara),双蒸水补足反应体系。反应条件:94℃预变性5 min;94℃,1 min;63℃,1 min;72℃,2 min;35个循环;72℃延伸10 min。将PCR阳性条带胶回收后,克隆到PMD18-T载体上,转化大肠杆菌BL21,将筛选得到的阳性克隆子送样到上海英骏生物技术有限公司用3730测序仪进行序列测定。将得到的目标片段序列提交GenBANK数据库,采用网上BLAST程序进行序列的同源性分析。使用CLUSTAL X软件和PHYLIP软件包进行序列比对和功能分析。
从不同生境中获得了23条I型PKS基因的KS序列片段,其中土壤来源的19个,海水来源的4个,长度从630 bp到690 bp不等。GenBANK序列号为:DQ640993 , DQ640997 , DQ641926 , DQ641927 ; DQ673137-DQ673152 ;EF554859-EF554861。
将其与已知的PKS酮缩合酶基因片段进行BLAST比对,所获得的23条的核苷酸序列BLAST得分大部分在40-150之间,与Genbank中已有序列同源性很低,提示其来源于新物种;而所推断的氨基酸序列保守,BLAST比对直接提示其归属于PKS的KS片段,负责聚酮类化合物生物合成过程中的酮缩合反应。用CLUSTAL X和PHYLIP软件对所获序列与GenBank中已知功能的KS片段进行分子进化分析和功能比对,结果发现:发现DQ640997;DQ673137,DQ673138;DQ673140;DQ673142;DQ673144;DQ673145;DQ673149;DQ673151核心保守序列为VQTACSTS,结合其上游的N(DE)KD序列排布方式,提示这些KS片段可能来源于PKS/NRPS杂和基因簇,参与杂和的聚酮/非核糖体多肽类化合物的合成。
四、产Macrolactins的海洋细菌X-2中I型PKS基因的筛选鉴定与功能分析
Macrolactins是近年来从海洋微生物中分离的一群具有抗菌、抗病毒、抗肿瘤以及抑制胆固醇合成活性的大环内酯类化合物。其生物合成基因簇迄今仍未有报道。
本室从东海海泥中分离到一株海洋细菌X-2,可产生多种Macrolactins。从Macrolactins的化学结构分析,其属于典型的大环内酯类化合物,其生物合成基因簇应为典型的Ⅰ型PKS基因簇。利用现有对于Ⅰ型PKS基因簇的认识,可对Macrolactins的生物合成基因簇的构成作出预测,本研究构建了该细菌的fosmid基因组文库,以扩增出的KS结构域的DNA片段(GenBank accessing number: EF486351)做探针筛选文库,克隆并鉴定了该细菌中可能为Macrolactins生物合成负责的部分Ⅰ型PKS基因。
总之,本研究使用简并引物扩增的方法,设计了一套针对Ⅰ型PKS基因中保守序列的简并引物,对我国东海海域的沿岸土壤、海水、海洋沉积物等不同生境以及产聚酮类化合物的海洋细菌中的Ⅰ型PKS基因的进行了初步的筛选鉴定和功能分析工作,获得了相当数量的新型Ⅰ型PKS基因,进行了初步的功能分析。研究结果从一个侧面揭示了PKS基因在海洋不同生境和微生物中的广泛分布状况,已经获得的序列不仅可作为探针,用于后期海洋宏基因组文库中PKS阳性克隆的筛选,还可直接用于后期新型聚酮类化合物的异源表达及生物合成研究。本研究最终将为我国海洋微生物功能基因资源开发利用和人民身体健康作出巨大贡献。
The biosphere is dominated by microorganisms, which produce numerous secondary metabolites with various biological activities. The efforts to discover new bioactive molecules from microbes have lasted one century. However, the number of novel compounds discovered in recent years has not increased in proportion to the progress in culture-based screening methods, because only a small fraction of all microbes can be cultured by traditional methods. The knowledge about the“underexplored majority”is still poor.
To avoid culture limit, modern biological technology approaches have provide direct access to explore genes or gene clusters that are responsible for the synthesis of microbial secondary metabolites. Polyketide synthase (PKS) gene clusters are suitable to be the screening target. PKSs synthesize polyketides, a large family of secondary metabolites that include many clinically important drugs such as erythromycin (antibacterial), epothilone (antitumor), soraphen (antifungal), rapamycin (immunosuppressant) and lovastatin (anti-hypercholesterolemic).
In this study, a set of degenerate oligonucleotide primers, designed for amplification KSs domains, had been employed to identify KS gene fragments from soil and seawater DNA samples. Our purpose was to develop a culture-independent method to directly access PKSⅠgene diversity in Chinese soil and seawater, because the related knowledge was incomplete. This would open up the possibilities of using the results for DNA fingerprints of secondary metabolites and as the basis for a search for attractive antibiotic biosynthesis genes that could be used in the heterogenous expression and combinatorial biosynthesis. Furthermore, the amplified KS gene fragments can also be used as homologous hybridization probes to detect the clones harbored PKS gene clusters in the recombinant metagenomic libraries that would be constructed in the following researches.
Part 1. DNA extractions
DNA extractions were carried out by using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc.). Compared with an adaptation of the procedure described by Zhou et al, the MO BIO kit produced DNA with a higher level of purity that can be used for the following PCR directly.
DNA extractions of the soil and seawater samples were performed following the manufacturers’instructions. The DNA yield were calculated from the A260. The DNA were loaded on a 1.0% agarose gels and ethidium bromide staining to determine size and concentration. All extracted DNA were stored at -20℃until use.
Part 2. Design of the degenerate PCR primers
With the help of The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) and manually correction, a set of degenerate PCR primers were designed from conserved regions of KS domains of bacterial PKSⅠgenes.
The forward primers KSF (5’-CGC TCC ATG GAY CCS CAR CA-3’) were based on the conserved motif SDPQQR. The reverse primers KSR (5’-GTC CCG GTG CCR TGS SHY TCS A -3’) were based on the conserved motif HGTGT.
The specificity of the primer set had been confirmed by testing with a collection of polyketide-producing strain (Streptomyces rimosus 8229, Streptomyces coelicolor ATCC101478, Streptomyces avermitlis, ATCC 31271) prior to the following PCR reactions with the environmental samples. The specific fragments amplified with KSF-KSR were about 700 bp in length.
Part 3. Isolation of 23 ketosynthase fragments from soil and sea water In this study, the degenerate PCR primers were employed to amplified the soil and seawater DNA samples.
The 25μl PCR mixture consisting of 2.5μl 10×Ex Taq Buffer (Mg2+ Plus), 2μl dNTP Mixture( 2.5 mmol/L each), 0.125μl TaKaRa Ex Taq (5U/μl), 1.5μl DMSO(dimethyl sulfoxide), 0.25μl each primer (20μmol/L), 1μl template DNA (0.03 g/L), and 17.375μl ddH2O.
The initial denaturation step at 94℃for 5 minutes was followed by 35 cycles of DNA denaturation at 94℃for 1 minute, primer annealing at 65℃for 1 minute, and DNA strand extension at 72℃for 1 minute, and a final extension step at 72℃for 10 minutes.
The result PCR products about 700 bp were purified on agarose gels (mini-DNA rapid purification kit, BioDev-Tech) and then cloned into PMD18-T Vector according to the manufacturer’s instructions (TaKaRa). positive recombinants were then submitted for sequencing using an ABI3730 DNA Sequencer (USA) with M13 primer at the Invitrogen Biotechnology Company.
DNA sequences obtained from the PCR product had been translated into amino acid sequences using the Primer Premier 5 software after cutting off the vector and primer regions. Sequence analyses were performed using the BLASTP programs provided by the National Center for Biotechnology Information (NCBI).
After excluding the identical clones, 23 unique nucleotide fragments (ranged from 630 bp to 690 bp after cutting off the primer and vector sequences) were obtained. Among them 19 clones were amplified from soil (DQ640993, DQ640997, DQ641926, DQ641927, and DQ673137～DQ673151) and 4 from the seawater (DQ673151 and EF554859～EF554861).
The GC content of the resulting 23 KS gene fragments ranged from 49% to 73% with an average value 66.7%. The alignment of the predicted protein sequences revealed that all the nucleotide sequences encoded the highly conserved region corresponding to the active site of the beta-ketoacyl synthetase consensus region of PKS I genes, these KS gene fragments showed 45% to 85% identities to the known PKS I amino acid sequences in the GenBank database.
Among the 23 KS sequences obtained in present study, 9 sequences displayed a unique pattern N(DE)KD, 22 amino acids upstream from the cysteine active site in the KS domain and the conserved pattern VDTACSSS was replaced by VQTACSTS.
These two patterns were shown to identify KS domains belonging to hybrid NRPS/PKS systems, which require specialized KS domains capable of using the peptidyl substrate of the NRPS donor site. On the other hand, the remained 15 KS sequences (the 4 KS sequences derived from the sea water included) showed typical conserved patterns of KS domains. No KSQ like fragment was found from all the 23 KS fragments.
Part 4. Isolation and functional analysis of the PKS genes in the marine bacteria X-2 which produce Macrolactins
Macrolactins are 24-membered macrolides produced by unidentified marine bacterium, Actinomadura sp. and Bacillus sp., which exhibit both antibacterial and antitumor activities in vitro. In addition, macrolactins were shown to inhibit mammalian herpes simplex virus and HIV replication. Although cloning of the macrolactins biosynthetic genes has not been reported, its chemical structure suggests that it is assembled by a modular PKS system.
The environmental strain X-2 which was isolated from the sediment of the East China Sea producing Macrolatin A, B and O. KS domains cloned from the X-2 showed 98% identities to the known pks2 gene cluster in environmental strain Bacillus amyloliquefaciens FZB42. The result showed that the pks2 gene cluster may be play a important role in the biosynthesis of Macrolatins.
Data presented in this study showed that the PCR method using degenerate primer to isolate the secondary metabolites biosynthesis gene fragments from the environmental samples and strains were practically effective. This study will provide the foundation for the biosynthesis and heterogeneous expression of polyketides and contribute to the exploitation of microbial genetic resources belong to the“underexplored majority”.
引文
1. Bisang C, Long PF, Cortes J, Westcott J, Crosby J, Matharu AL, Cox RJ, Simpson TJ, Staunton J,Leadlay PF (1999) A chain initiation factor common to both modular and aromatic polyketide synthases. Nature 401:502-505
2. Chen, X.-H., J. Vater, J. Piel, P. Franke, R. Scholz, K. Schneider, A. Koumoutsi, G. Hitzeroth, N. Grammel, A. W. Strittmatter, G. Gottschalk, R. D. Sussmuth, and R. Borriss. (2006). Structural and functional characterization of the three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. J. Bacteriol. 188:4024-4036.
3. Cheng YQ, Tang GL, Shen B (2003) Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA 100:3149-3154
4. Choi SW, Bai DH, Yu JH., et al. Characteristics of the squalene synthase inhibitors produced by a Streptomyces species isolated from soils. Can. J. Microbiol. 2003; 49: 663-668
5. Courtois S, Cappellano CM, Ball M, Francou FX, Normand P, Helynck G, Martinez A, Kolvek SJ, Hopke J, Osburne MS, August PR, Nalin R, Guerineau M, Jeannin P, Simonet P,Pernodet JL (2003) Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Appl Environ Microbiol 69:49-55
6. Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R,Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23:321-329
7. Edwards DJ, Gerwick WH (2004) Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase. J Am Chem Soc 126:11432-11423
8. Ehrenreich IM, Waterbury JB,Webb EA (2005) Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes. Appl Environ Microbiol 71:7401-7413
9. Firn RD, Jones CG (2000) The evolution of secondary metabolism - a unifying model. Mol Microbiol 37:989-994
10. Ginolhac A, Jarrin C, Gillet B, Robe P, Pujic P, Tuphile K, Bertrand H, VogelTM, Perriere G, Simonet P,Nalin R (2004) Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol 70:5522-5527
11. Goodyear PD, MacLaughlin-Black S,Mason IJ (1994) A reliable method for the removal of co-purifying PCR inhibitors from ancient DNA.Biotechniques 16:232-5
12. Gustafson, K.; Roman, M.; Fenical, W. The macrolactins, a novel class of antiviral and cyto-toxic macrolides from a deep-sea marine bacterium. J. Am. Chem. Soc. 1989; 111: 7519-7524.
13. Jenke-Kodama H, Sandmann A, Muller R,Dittmann E (2005) Evolutionary implications of bacterial polyketide synthases.Mol Biol Evol 22:2027-2039
14. Kim TK, Hewavitharana AK, Shaw PN,Fuerst JA (2006) Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction. Appl Environ Microbiol 72:2118-2125
15. Lim GE, Haygood MG (2004) "Candidatus Endobugula glebosa," a specific bacterial symbiont of the marine bryozoan Bugula simplex. Appl Environ Microbiol 70:4921-4929
16. Lopanik NB, Targett NM,Lindquist N (2006) Isolation of two polyketide synthase gene fragments from the uncultured microbial symbiont of the marine bryozoan Bugula neritina.Appl Environ Microbiol 72:7941-7944
17. Malpartida F, Hopwood DA (1984) Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host.Nature JT - Nature 309:462-464
18. Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA,Santi DV (2005) Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 23:1171-1176
19. Moffitt MC, Neilan BA (2003) Evolutionary affiliations within the superfamily of ketosynthases reflect complex pathway associations. J Mol Evol 56:446-457
20. Nagao T, Adachi K, Sakai M, et al. Novel macrolactins as antibiotic lactones from a marine bacterium. J Antibiot (Tokyo). 2001; 54:333-339.
21. Newman DJ, Cragg GM (2006) Natural products from marine invertebrates and microbes as modulators of antitumor targets.Curr Drug Targets 7:279-304
22. Piel J (2002) A polyketide synthase-peptide synthetase gene cluster from an
23. uncultured bacterial symbiont of Paederus beetles. Proc Natl Acad Sci USA 99:14002-14007
24. Piel J, Hui D, Wen G, Butzke D, Platzer M, Fusetani N,Matsunaga S (2004) Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei.Proc Natl Acad Sci USA 101:16222-16227
25. Proksch P, Ebel R, Edrada RA, Wray V,Steube K (2003) Bioactive natural products from marine invertebrates and associated fungi.Prog Mol Subcell Biol 37:117-42
26. Rose TM, Henikoff JG,Henikoff S (2003) CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.Nucleic Acids Res 31:3763-3766
27. Salomon CE, Magarvey NA,Sherman DH (2004) Merging the potential of microbial genetics with biological and chemical diversity: an even brighter future for marine natural product drug discovery.Nat Prod Rep 21:105-21
28. Schirmer A, Gadkari R, Reeves CD, Ibrahim F, DeLong EF,Hutchinson CR (2005) Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta. Appl Environ Microbiol 71:4840-4849
29. Shen B (2003) Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opin Chem Biol 7:285-295
30. Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM,Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci USA 103:12115-20
31. Staunton J, Weissman KJ (2001) Polyketide biosynthesis: a millennium review. Nat Prod Rep 18:380-416
32. Wawrik B, Kerkhof L, Zylstra GJ,Kukor JJ (2005) Identification of unique type II polyketide synthase genes in soil.Appl Environ Microbiol 71:2232-2238
33. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification.Appl Environ Microbiol 63:3741-51
34. Yoo JS, Zheng CJ, Lee S, et al. Macrolactin N, a new peptide deformylase inhibitor produced by Bacillus subtilis. Bioorg Med Chem Lett. 2006; 16:4889-4892.
35. Zhou J, Bruns MA,Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316-322
36. 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; 49:2077-2087.
1. Staunton J, Weissman KJ. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep., 2001; 18(4), 380–416
2. Firn RD, C. G. Jones. The evolution of secondary metabolism—a unifying model. Mol. Microbiol. 2000; 37(5):989–994.
3. Pfeifer BA, Khosla C Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev. 2001; 65(1):106-118.
4. Gerth K, Bedorf N, Ho?e G., Irschik H, Reichenbach H. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties. Journal of Antibiotics. 1996; 49(6), 560-563.
5. Hildebrand M, Waggoner LE, Liu H, Sudek S, Allen S, Anderson C, Sherman DH and M Haygood. bryA: an unusual modular polyketide synthase gene from the uncultivated bacterial symbiont of the marine bryozoan Bugula neritina. Chem Biol. 2004; 11(11):1543-52.
6. Rude MA and Khosla C. Engineered biosynthesis of polyketides in heterologous hosts. Chem. Eng. Sci. 2004; 59, 4693-4701.
7. Rodriguez E, Ward S, Fu H, Revill WP, McDaniel R, Katz L. Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae. Appl. Microbiol Biotechnol. 2004; 66(1):85-91
8. Peiru S, Menzella HG, Rodriguez E, Carney J, Gramajo H. Production of the potent antibacterial polyketide erythromycin C in Escherichia coli. Appl Environ Microbiol. 2005; 71(5):2539–2547
9. Tang L, McDaniel R. Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity. Chem Biol, 2001, 8(6):547-555
10. Dayem LC, Carney JR, Santi DV, et al. Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli. Biochemistry, 2002, 41(16): 5193-5201.
11. Lombo F, Pfeifer B, Leaf T, Ou S, Kim YS, Cane DE, Licari P, Khosla CH. Enhancing the atom economy of polyketide. biosynthetic processes through metabolic engineering. Biotechnol Prog. 2001; 17(4):612–617
12. Hu Z, Hopwood D A and Hutchinson C R. Enhanced heterologous polyketide production in Streptomyces by exploiting plasmid co-integration. J. Ind. Microbiol. Biotechnol. 2003; 30(8):516-522
13. Lum AM, Huang J, Hutchinson CR, Kao CM. Reverse. engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays. Metab Eng. 2004; 6:186–.196
14. kamoto S, Lezhava A, Hosaka T, Okamoto-Hosoya Y., Ochi, K. Enhanced Expression of S-Adenosylmethionine Synthetase Causes Overproduction of Actinorhodin in Streptomyces coelicolor A3(2). J. Bacteriol. 2003; 185(2): 601-609
15. Janice Lau,Carnie Tran,Peter Licari,et al.Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli. Journal of biotechnology. 2004,110(1):95-103.
16. Watanabe K, Rude MA, Walsh CT, Khosla C: Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli. PNAS. USA. 2003; 100(17):9774-9778
17. Pfeifer BA, Wang CC, Walsh CT, & Khosla C. Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host. Appl. Environ. Microbiol. 2003;69(11), 6698–6702
2. Chen, X.-H., J. Vater, J. Piel, P. Franke, R. Scholz, K. Schneider, A. Koumoutsi, G. Hitzeroth, N. Grammel, A. W. Strittmatter, G. Gottschalk, R. D. Sussmuth, and R. Borriss. (2006). Structural and functional characterization of the three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42. J. Bacteriol. 188:4024-4036.
3. Cheng YQ, Tang GL, Shen B (2003) Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA 100:3149-3154
4. Choi SW, Bai DH, Yu JH., et al. Characteristics of the squalene synthase inhibitors produced by a Streptomyces species isolated from soils. Can. J. Microbiol. 2003; 49: 663-668
5. Courtois S, Cappellano CM, Ball M, Francou FX, Normand P, Helynck G, Martinez A, Kolvek SJ, Hopke J, Osburne MS, August PR, Nalin R, Guerineau M, Jeannin P, Simonet P,Pernodet JL (2003) Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Appl Environ Microbiol 69:49-55
6. Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R,Wittwer P (2005) Metagenomic gene discovery: past, present and future. Trends Biotechnol 23:321-329
7. Edwards DJ, Gerwick WH (2004) Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase. J Am Chem Soc 126:11432-11423
8. Ehrenreich IM, Waterbury JB,Webb EA (2005) Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes. Appl Environ Microbiol 71:7401-7413
9. Firn RD, Jones CG (2000) The evolution of secondary metabolism - a unifying model. Mol Microbiol 37:989-994
10. Ginolhac A, Jarrin C, Gillet B, Robe P, Pujic P, Tuphile K, Bertrand H, VogelTM, Perriere G, Simonet P,Nalin R (2004) Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl Environ Microbiol 70:5522-5527
11. Goodyear PD, MacLaughlin-Black S,Mason IJ (1994) A reliable method for the removal of co-purifying PCR inhibitors from ancient DNA.Biotechniques 16:232-5
12. Gustafson, K.; Roman, M.; Fenical, W. The macrolactins, a novel class of antiviral and cyto-toxic macrolides from a deep-sea marine bacterium. J. Am. Chem. Soc. 1989; 111: 7519-7524.
13. Jenke-Kodama H, Sandmann A, Muller R,Dittmann E (2005) Evolutionary implications of bacterial polyketide synthases.Mol Biol Evol 22:2027-2039
14. Kim TK, Hewavitharana AK, Shaw PN,Fuerst JA (2006) Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction. Appl Environ Microbiol 72:2118-2125
15. Lim GE, Haygood MG (2004) "Candidatus Endobugula glebosa," a specific bacterial symbiont of the marine bryozoan Bugula simplex. Appl Environ Microbiol 70:4921-4929
16. Lopanik NB, Targett NM,Lindquist N (2006) Isolation of two polyketide synthase gene fragments from the uncultured microbial symbiont of the marine bryozoan Bugula neritina.Appl Environ Microbiol 72:7941-7944
17. Malpartida F, Hopwood DA (1984) Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host.Nature JT - Nature 309:462-464
18. Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA,Santi DV (2005) Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotechnol 23:1171-1176
19. Moffitt MC, Neilan BA (2003) Evolutionary affiliations within the superfamily of ketosynthases reflect complex pathway associations. J Mol Evol 56:446-457
20. Nagao T, Adachi K, Sakai M, et al. Novel macrolactins as antibiotic lactones from a marine bacterium. J Antibiot (Tokyo). 2001; 54:333-339.
21. Newman DJ, Cragg GM (2006) Natural products from marine invertebrates and microbes as modulators of antitumor targets.Curr Drug Targets 7:279-304
22. Piel J (2002) A polyketide synthase-peptide synthetase gene cluster from an
23. uncultured bacterial symbiont of Paederus beetles. Proc Natl Acad Sci USA 99:14002-14007
24. Piel J, Hui D, Wen G, Butzke D, Platzer M, Fusetani N,Matsunaga S (2004) Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei.Proc Natl Acad Sci USA 101:16222-16227
25. Proksch P, Ebel R, Edrada RA, Wray V,Steube K (2003) Bioactive natural products from marine invertebrates and associated fungi.Prog Mol Subcell Biol 37:117-42
26. Rose TM, Henikoff JG,Henikoff S (2003) CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design.Nucleic Acids Res 31:3763-3766
27. Salomon CE, Magarvey NA,Sherman DH (2004) Merging the potential of microbial genetics with biological and chemical diversity: an even brighter future for marine natural product drug discovery.Nat Prod Rep 21:105-21
28. Schirmer A, Gadkari R, Reeves CD, Ibrahim F, DeLong EF,Hutchinson CR (2005) Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodermia dissoluta. Appl Environ Microbiol 71:4840-4849
29. Shen B (2003) Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opin Chem Biol 7:285-295
30. Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM,Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored "rare biosphere". Proc Natl Acad Sci USA 103:12115-20
31. Staunton J, Weissman KJ (2001) Polyketide biosynthesis: a millennium review. Nat Prod Rep 18:380-416
32. Wawrik B, Kerkhof L, Zylstra GJ,Kukor JJ (2005) Identification of unique type II polyketide synthase genes in soil.Appl Environ Microbiol 71:2232-2238
33. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification.Appl Environ Microbiol 63:3741-51
34. Yoo JS, Zheng CJ, Lee S, et al. Macrolactin N, a new peptide deformylase inhibitor produced by Bacillus subtilis. Bioorg Med Chem Lett. 2006; 16:4889-4892.
35. Zhou J, Bruns MA,Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316-322
36. 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; 49:2077-2087.
1. Staunton J, Weissman KJ. Polyketide biosynthesis: a millennium review. Nat. Prod. Rep., 2001; 18(4), 380–416
2. Firn RD, C. G. Jones. The evolution of secondary metabolism—a unifying model. Mol. Microbiol. 2000; 37(5):989–994.
3. Pfeifer BA, Khosla C Biosynthesis of polyketides in heterologous hosts. Microbiol Mol Biol Rev. 2001; 65(1):106-118.
4. Gerth K, Bedorf N, Ho?e G., Irschik H, Reichenbach H. Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties. Journal of Antibiotics. 1996; 49(6), 560-563.
5. Hildebrand M, Waggoner LE, Liu H, Sudek S, Allen S, Anderson C, Sherman DH and M Haygood. bryA: an unusual modular polyketide synthase gene from the uncultivated bacterial symbiont of the marine bryozoan Bugula neritina. Chem Biol. 2004; 11(11):1543-52.
6. Rude MA and Khosla C. Engineered biosynthesis of polyketides in heterologous hosts. Chem. Eng. Sci. 2004; 59, 4693-4701.
7. Rodriguez E, Ward S, Fu H, Revill WP, McDaniel R, Katz L. Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae. Appl. Microbiol Biotechnol. 2004; 66(1):85-91
8. Peiru S, Menzella HG, Rodriguez E, Carney J, Gramajo H. Production of the potent antibacterial polyketide erythromycin C in Escherichia coli. Appl Environ Microbiol. 2005; 71(5):2539–2547
9. Tang L, McDaniel R. Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity. Chem Biol, 2001, 8(6):547-555
10. Dayem LC, Carney JR, Santi DV, et al. Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli. Biochemistry, 2002, 41(16): 5193-5201.
11. Lombo F, Pfeifer B, Leaf T, Ou S, Kim YS, Cane DE, Licari P, Khosla CH. Enhancing the atom economy of polyketide. biosynthetic processes through metabolic engineering. Biotechnol Prog. 2001; 17(4):612–617
12. Hu Z, Hopwood D A and Hutchinson C R. Enhanced heterologous polyketide production in Streptomyces by exploiting plasmid co-integration. J. Ind. Microbiol. Biotechnol. 2003; 30(8):516-522
13. Lum AM, Huang J, Hutchinson CR, Kao CM. Reverse. engineering of industrial pharmaceutical-producing actinomycete strains using DNA microarrays. Metab Eng. 2004; 6:186–.196
14. kamoto S, Lezhava A, Hosaka T, Okamoto-Hosoya Y., Ochi, K. Enhanced Expression of S-Adenosylmethionine Synthetase Causes Overproduction of Actinorhodin in Streptomyces coelicolor A3(2). J. Bacteriol. 2003; 185(2): 601-609
15. Janice Lau,Carnie Tran,Peter Licari,et al.Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli. Journal of biotechnology. 2004,110(1):95-103.
16. Watanabe K, Rude MA, Walsh CT, Khosla C: Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli. PNAS. USA. 2003; 100(17):9774-9778
17. Pfeifer BA, Wang CC, Walsh CT, & Khosla C. Biosynthesis of yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host. Appl. Environ. Microbiol. 2003;69(11), 6698–6702