Discovery of angucyclinone polyketides from marine actinomycetes with a genomic DNA-based PCR assay targeting type Ⅱ polyketide synthase

Angucyclinones are aromatic polyketides produced by type Ⅱ polyketide synthases（PKS） and are mainly found in terrestrial actinomycetes. To discover more angucyclinones from marine actinomycetes, a genomic DNA-based PCR assay targeting type Ⅱ polyketide synthases was performed. Among the 167 marine actinomycetes strains screened, twelve strains were identified as the ＂positive＂ strains possessing type Ⅱ PKS-encoding genes based on the sequencing of PCR products. One of the 12 ＂positive＂ strains, Streptomyces sp. PKU-MA00218 was selected for the large-scale fermentation based on the HPLC and TLC analysis. Four angucyclinones, 6-deoxy-8-O-methylrabelomycin（1）, 8-O-methylrabelomycin（2）, 8-O-methyltetrangulol（3）, C-ring cleavage product of angucyclinone C（4）, were isolated and their structures were elucidated based on spectroscopic analyses. The isolation of angucyclinones 1–4 highlights the power of genome mining technologies based on biosynthetic knowledge in natural products discovery.

Phylogenetic diversity of TypeⅠpolyketide synthase genes from sediments of Ardley Island in Antarctica

The diversity of modular polyketide synthase （PKS） genes in sediments of Ardley Island in Antarctica, was studied by restriction fragment length polymorphism （RFLP） analysis. Phylogenetic analysis of 14 amino acid （AA） sequences indicates that the identified ketosynthase （KS） domains were clustered with those from diverse bacterial groups, including Cyanobacteria, γ-Proteobacteria, Actinobacteria, Firmicutes, and some unidentified microorganisms from marine sponge, bryozoan and other environmental samples. The obtained KS domains showed 43%–81% similarity at the AA level to reference sequences in GenBank. Six identified KS domains showed diverse sequences of the motif （VQTACSTS） that was used to identify the hybrid PKS/nonribosomal peptide synthetase （NRPS） enzyme complex, and formed a new branch. These results reveal a high diversity and novelty of PKS genes in antarctic sediments.

Biosynthesis and Genetic Engineering of Polyketides

Polyketides are one of the largest groups of natural products produced by bacteria, fungi, and plants. Many of these metabolites have highly complex chemical structures and very important biological activities, including antibiotic, anticancer, immunosuppressant, and anti-cholesterol activities. In the past two decades, extensive investigations have been carried out to understand the molecular mechanisms for polyketide biosynthesis. These efforts have led to the development of various rational approaches toward engineered biosynthesis of new polyketides. More recently, the research efforts have shifted to the elucidation of the three-dimentional structure of the complex enzyme machineries for polyketide biosynthesis and to the exploitation of new sources for polyketide production, such as filamentous fungi and marine microorganisms. This review summarizes our general understanding of the biosynthetic mechanisms and the progress in engineered biosynthesis of polyketides.

Editing function of type Ⅱ thioesterases in the biosynthesis of fungal polyketides

Polyketide synthases(PKSs)are megasynthases with multiple autonomously folding domains,which operate cooperatively in the PKS assemblies to synthesize specific polyketide scaffolds.Any nonreactive intermediates tethered to acyl carrier protein(ACP)domain in the PKS will block the elongation process of polyketide chains.In this study,we systematically elucidate the editing function of fungal typeⅡthioesterases(TEIIs)to hydrolyze ACP domain-bounded nonreactive acyl groups,which are uploaded by substrate promiscuous fungal phosphopantetheinyl transferase.Thereof,the TEIIs encoded in gene clusters of nonreducing PKS with reductase domain exhibit universal editing function.Besides,editing function was also found for TEIIs encoded in gene clusters of highly-reducing PKS with condensation domain.Hence,the editing TEIIs with function of recovery PKS are applied to improve the yield of the fungal polyketides in vivo.Our study provides valuable insights into the editing process of fungal PKSs,highlights the crucial role of TEIIs in enhancing polyketide production and introduces a novel metabolic engineering strategy for fungal polyketide biosynthesis by leveraging the editing function of TEIIs.

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice

Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases （PKSs） are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase （OsPKS2） in male reproduction of rice （Oryza sativa）. Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl- CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT （wild type）, ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds. However, unlike pksb/laps, in which most detected phenolics were substantially decreased, ospks2 accumu- lated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/laps, we propose that PKS proteins have functionally diversified during evolution. Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.

Engineered polyketides:Synergy between protein and host level engineering

Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities.Polyketides encompass one of the largest classes of chemically diverse natural products.With thousands of known polyketides,modular polyketide synthases(PKSs)share a particularly attractive biosynthetic logic for generating chemical diversity.The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds.In this review,we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures,and we examine future applications of PKSs in the production of medicines,fuels and other industrially relevant chemicals.

Functional characterization of key polyketide synthases by integrated metabolome and transcriptome analysis on curcuminoid biosynthesis in Curcuma wenyujin

Leaf and tuber extracts of Curcuma wenyujin contain a mixture of curcuminoids.However,the curcuminoid constituents and their molecular mechanisms are poorly understood,and the relevant curcumin synthases remain unclear.In this study,we comprehensively compared the metabolite profiles of the leaf and tuber tissues of C.wenyujin.A total of 11 curcuminoid metabolites were identified and exhibited differentially changed contents in the leaf and tuber tissues.An integrated analysis of metabolomic and transcriptomic data revealed the proposed biosynthesis pathway of curcuminoid.Two candidate type III polyketide synthases(PKSs)were identified in the metabolically engineering yeasts,indicating that CwPKS1 and CwPKS2 maintained substrate and product specificities.Especially,CwPKS1 is the first type III PKS identified to synthesize hydrogenated derivatives of curcuminoid,dihydrocurcumin and tetrehydrocurcumin.Interestingly,the substitution of the glycine at position 219 with aspartic acid(G219D mutant)resulted in the complete inactivation of CwPKS1.Our results provide the first comparative metabolome analysis of C.wenyujin and functionally identified type III PKSs,giving valuable information for curcuminoids biosynthesis.

Site directed mutagenesis as a precision tool to enable synthetic biology with engineered modular polyketide synthases

Modular polyketide synthases(PKSs)are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds,from new pharmaceuticals to high value commodity and specialty chemicals.Their colinear biosynthetic logic has been viewed as a promising platform for synthetic biology for decades.Due to this colinearity,domain swapping has long been used as a strategy to introduce molecular diversity.However,domain swapping often fails because it perturbs critical protein-protein interactions within the PKS.With our increased level of structural elucidation of PKSs,using judicious targeted mutations of individual residues is a more precise way to introduce molecular diversity with less potential for global disruption of the protein architecture.Here we review examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity,affect protein stability and interdomain communication,and promote more complex catalytic reactivity.

Fragment antigen binding domains (F_(ab)s) as tools to study assembly-line polyketide synthases

The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antigen binding domains(F_(ab)s)as protein crystallization chaperones is discussed.F_(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA.The ability to bind three F_(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins.We describe a high-throughput workflow for identifying F_(ab)s to aid the crystallization of a protein of interest(POI)by leveraging phage display technologies and differential scanning fluorimetry(DSF).This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases(PKSs),which harbor flexible domains and assume transient conformations.PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds.Despite years of research studying these megasynthases,their overall topology has remained elusive.One F ab in particular,1B2,has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic(cryoEM)analyses of multiple modules from distinct assembly-line PKSs.Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM,thereby enabling the visualization of intact PKS modules at near-atomic(3–5Å)resolution.The identification of PKS-binding F_(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

聚酮化合物非天然延伸单元的生物合成与结构改造应用

聚酮天然产物包括10000多种具有广泛生物活性的分子,是获批临床药物中最著名的类别之一。已知活性先导化合物通常需要经过结构修饰改良其吸收、分布、代谢和排泄等特性,从而促进成药开发,但针对聚酮化合物的结构修饰极具挑战,需要应对聚酮骨架中大量的立体中心以及多个惰性碳原子,导致化学合成手段难以对聚酮骨架进行精准和高效的衍生化,因此,通过合成生物学方法实现其结构优化就成为了研究者们关注的热点。自然界中,绝大多数聚酮化合物主要由简单的乙酸盐和丙酸盐结构单元通过聚酮合酶组装而成,而少数存在的具有特殊结构单元的聚酮案例给了研究者以灵感——通过设置和引入非天然结构单元从而有选择性地高效改造聚酮结构。聚酮骨架的生物合成有赖于一个起始单元与多个延伸单元的组装,因此,通过人工设计延伸单元向聚酮引入预期结构被认为是精准高效改造聚酮的有力突破点。本文在此总结了近十年来报道的聚酮非天然延伸单元的三种重要的酶促合成方法,通过挖掘新颖的延伸单元合成酶并探索其底物宽泛性,或利用酶工程手段改造延伸单元合成酶的底物催化范围,获得了大量自然界不存在的延伸单元。此外,本文还归纳了利用非天然延伸单元对聚酮结构进行改造的案例,借助聚酮的天然合成途径或利用改造的合成途径达到预期目的。最后,作者讨论了该研究领域内存在的一些制约因素以及可优化的研究方向,包括聚酮合酶对非天然延伸单元的兼容性问题、非天然延伸单元的前体供给等。近年来,利用非天然延伸单元改造聚酮结构的研究兴趣和热度日益高涨,本文绘制了一份基于延伸单元改造聚酮结构研究的简明清晰的图谱,期望为加速聚酮类药物的高效开发打下坚实基础。

海洋链霉菌酮合成酶结构域基因的克隆及分析

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

细菌聚酮合酶间的杂合方式及聚酮化合物生物合成逻辑

聚酮化合物(polyketide)是一类来源广泛、结构多样的活性天然产物,聚酮合酶(polyketide synthase,PKS)负责聚酮骨架的生物合成。细菌次级代谢中PKS广泛存在,不同类型的PKS在组成和生物合成机制上各不相同,从而产生截然不同的聚酮骨架。根据细菌PKS功能和生物合成途径的不同,可以将其分为Ⅰ型、Ⅱ型和Ⅲ型。PKS通常能与其他生物合成酶系杂合以产生结构更为复杂的天然产物。同时,不同类型PKS之间也可以形成多种内部杂合,产生更多样的聚酮骨架。本文总结和比较PKS间的内部杂合,包括Ⅰ型PKS内部杂合、Ⅰ型/Ⅱ型PKS杂合以及Ⅰ型/Ⅲ型PKS杂合,归纳各种杂合基因簇的形成方式及其杂合特征。通过比较杂合聚酮化合物的生物合成机制并讨论杂合聚酮工程化改造的进展,展望了多种潜在的聚酮杂合模式,合理假设存在合成过程相反的Ⅰ型/Ⅱ型PKS杂合模式,或随着化合物的挖掘发现迄今未报道的Ⅱ型/Ⅲ型PKS杂合模式等,指出可以充分和全面地利用细菌基因组信息,通过酶和基因的生物勘探,发现更多更特殊的PKS杂合化合物等一系列针对新颖聚酮化合物进行基因组挖掘的方向,同时也提出了工程化改造trans-AT PKS在cis-AT模块中实现不同寻常的骨架修饰等多种PKS的工程化改造设想,为后续PKS内部杂合基因簇挖掘和表征提供一些新思路。

Phylogenetically diverse endozoic fungi in the South China Sea sponges and their potential in synthesizing bioactive natural products suggested by PKS gene and cytotoxic activity analysis

Sponges are well documented to harbor large amounts of microbes.Though it is known that spongederived fungi are important sources for marine natural products,the phylogenetic diversity and biological function of sponge-associated fungi remain largely unknown.In this study,the diversity of culturable endozoic fungi in sponges from the South China Sea was revealed based on the ITS phylogenetic analysis.Meanwhile the fungal potential for producing bioactive natural products was estimated according to the detection of Beta-ketosynthase in the polyketide synthase(PKS)gene cluster and cytotoxic activity bioassay.As a result,diverse fungi including 14 genera(Aspergillus,Penicillium,Scolecobasidium,Eurotium,Alternaria,Fusarium,Hypocreales,Yarrowia,Candida,Hypoxylon,Sporidiobolus,Schizophyllum,Bjerkandera,and Trichosporon)in ten orders(Xylariales,Moniliales,Pleosporales,Saccharomycetales,Hypocreales,Eurotiales,Sporidiobolales,Agaricales,Aphyllophorales and Tremellales)of phyla Ascomycota and Basidiomycota were isolated with Aspergillus as the predominant component in the culturable fungal community.Particularly,genera Schizophyllum,Sporidiobolus,and Bjerkandera in phylum Basidiomycota and genus Yarrowia in phylum Ascomycota were isolated from marine sponges for the first time.PKS genes were detected in 12 isolates suggesting their potential for synthesizing PKS compounds.Among the 12 isolates with PKS genes,9 isolates displayed strong in vitro cytotoxic activity(e.g.IC50<50μg/ml)against human cancer cell lines A549,Bel-7402,A-375 and MRC-5.This study demonstrates the phylogenetically diverse endozoic fungi in South China Sea sponges,and highlights the potential of spongeassociated fungi in producing biologically active natural products.

聚酮合酶的催化机制及聚酮类化合物的组合生物合成

聚酮类化合物(polyketides,PKs)是一类来源广泛、数目庞大的天然产物。聚酮合酶(polyketide synthase,PKS)负责催化PKs的生物合成,是具有模块化结构的多功能复合酶,由一系列对称或非对称的二聚体模块组成。模块按照其功能的不同,可分为加载模块、延伸模块、卸载模块。各个模块中含有多个催化域,不同的催化域在聚酮链延伸过程中扮演着不同角色。模块以首尾相连的方式排列,通过非结构多肽接头或离散的对接结构域彼此连接,形成装配线来生产PKs。PKSs按照结构域和催化机制的不同,可以分为合成大环内酯类抗生素的I型PKSs(由迭代化PKSs和模块化PKSs组成)、合成芳香族聚酮类化合物的II型PKSs(也被叫做迭代类PKSs)和合成类黄酮化合物的III型PKSs。利用组合生物合成方法,如对不同聚酮物质或同一物质的不同结构域或模块进行交换,以及特异性地插入、替代、缺失关键基因,或者通过点突变等操作,可以形成重组PKSs,进而改变聚酮类化合物的结构。选择不同的起始和延伸单元,引入不同类型的PKSs后修饰酶以及对PKSs后修饰酶基因进行改造等方法,均可用于合成非天然PKs。本文概述了3大类型聚酮合酶的结构、催化机制和组合生物合成近期研究进展,为今后合理设计产量高、效价高、化学性质稳定的新型聚酮类化合物提供参考依据。

聚酮合酶基因岛在肺炎克雷伯菌临床分离株中的流行研究

目的 研究肺炎克雷伯菌(Klebsiella pneumoniae)的聚酮合酶(PKS)基因岛的临床分布情况,探讨PKS基因岛与肺炎克雷伯菌感染的关系。方法 收集234株肺炎克雷伯菌,根据有、无PKS基因岛分为PKS+组和PKS-组。采用聚合酶链式反应(PCR)检测PKS基因岛,并结合肺炎克雷伯菌感染患者的临床资料,分析其在临床样本中的分布情况。结果 234例感染者中,PKS+组感染者占37.6%(88/234)。菌株主要来源于痰液(70.1%,164/234)、血液(7.3%,17/234)、腹水(5.6%,13/234)和脓液(4.3%,10/234)。PKS+菌株感染者中患有肝脓肿的比例较PKS-菌株感染者多[(13.6%,12/88)vs(1.4%,2/146),P<0.001]。药敏实验发现,PKS+菌株在氨曲南、亚胺培南、美罗培南、阿米卡星、妥布霉素、环丙沙星、左氧氟沙星和复方新诺明的药物敏感性更高(P<0.001)。结论 PKS+感染者数量有进一步上升的趋势,肝病患者更易感染PKS+肺炎克雷伯菌,而PKS+菌株对多种抗生素的敏感性较PKS-菌株高。应高度重视PKS基因在肺炎克雷伯菌流行的可能性,并进一步发掘PKS基因在肺炎克雷伯菌感染中的诊疗价值。

产生芳香环聚酮类天然产物放线菌的分子筛选研究

目的建立一套简便、快速、高效的芳香环聚酮类化合物产生菌基因筛选体系,为聚酮类药物筛选搭建一个新的技术平台;认识PKS-II基因在不同环境放线菌群中的分布规律,为新药筛选中微生物资源的定向分离提供依据。方法通过对芳香环聚酮类化合物生物合成途径所用的II型聚酮合酶氨基酸和核苷酸序列的同源性比较分析,设计一对简并引物且以微波炉法提取的基因组DNA为模板扩增KS/CLF功能域约0.8kb目的基因片段,依据放线菌基因组中II型PKS合酶基因的存在与否标定可能的芳香环聚酮类天然产物产生菌。结果利用建立的芳香环聚酮类化合物产生菌基因筛选体系对99株嗜碱放线菌、100株链霉菌、47株嗜盐放线菌和776株一般放线菌进行了筛选,研究表明链霉菌、嗜碱放线菌、嗜盐放线菌和一般放线菌II型聚酮合酶基因的阳性率分别为54%、29.3%、25.5%和24.1%。结论组合放线菌基因组DNA快速提取技术和PKSII基因简并引物PCR扩增技术建立了快速、简便、高效的芳香环聚酮类化合物产生菌基因筛选体系并对不同环境条件的放线菌菌群的PKSII基因分布进行了研究。结果表明放线菌PKSII聚酮合酶分布具有广泛性;而且同时表明链霉菌是芳香环聚酮类化合物的最丰富来源;在极端环境放线菌中,嗜盐放线菌的PKSII基因在中度嗜盐放线菌分布的频率远高?

玉米大斑病菌聚酮体合成酶基因StPKS功能分析

【目的】利用RNA干扰技术探讨玉米大斑病菌聚酮体合成酶基因StPKS的功能。【方法】将玉米大斑病菌StPKS基因片段连接到pSilent-1载体中,构建StPKS的RNA干扰载体pSilent-StPKS1-2。利用聚乙二醇(PEG)介导的方法将之转入玉米大斑病菌野生型菌株01-23的原生质体中,通过潮霉素筛选得到转化子,采用半定量RT-PCR方法分析转化子中StPKS的表达情况,显微观察转化子与野生型菌丝形态的差异。【结果】本研究构建了StPKS RNA干扰载体并进行了成功转化,经潮霉素抗性筛选和PCR验证最终获得了9个阳性转化子,其中5个转化子菌落颜色变浅。对转化子进行半定量PCR分析发现,5株转化子的StPKS表达量均有所下降;黑色素产量显著降低;菌丝呈不规则状,发生了膨大、变形、分枝等现象。【结论】StPKS在DHN黑色素合成途径中起作用,其表达量下降会减少黑色素的产生。

七叶一枝花内生真菌Penicillium sp.(NO.24)次级代谢产物研究

从神农架地区濒危药用植物七叶一枝花叶片中一株内生真菌Penicillium sp.中分离纯化得到七个聚酮类化合物和一个简单的生物碱类的次级代谢产物,其结构经核磁共振,质谱等方法确定,分别为Citrinin H1(1),dehydroisopenicillide(2),penicillide(3),7,9-二烯-十九碳酸单甘油酯(4),7-烯-十九碳酸单甘油酯(5),Silvaticol(6),5-hydroxy-2-pyridinemethanol(7),2,4,6-辛三烯酸(8);运用MTT法评价了八个次级代谢产物的体外抑制肝癌细胞的活性,结果表明化合物1,2,3,7具有较好体外抑制肝癌细胞的活性,其IC50分别为8.5,12.5,15.0,18.2μg.mL-1。

大环内酯类化合物产生菌的基因筛选及其代谢产物研究

应用Ⅰ型聚酮合酶(PKS-I)基因筛选体系从32株海洋放线菌中寻找大环内酯类化合物的潜在产生菌,通过DNA序列相似性比对从5株阳性菌株中筛选获得了游动放线菌Actinoplanes sp.FIM060065。采用HPLC制备色谱从该菌株发酵液中分离纯化得到一个大环内酯类化合物FW060065,紫外、质谱以及核磁共振波谱分析表明它与台勾霉素B同质,药敏实验表明其对艰难梭菌、消化链球菌、双歧杆菌等革兰氏阳性厌氧菌显示出较强的抗菌能力。本研究通过基因筛选获得了台勾霉素的产生菌及其相应的代谢产物,为Ⅰ型聚酮合酶基因与大环内酯类化合物之间的联系提供了证据。

聚酮类化合物生物合成基因簇与药物筛选

由微生物和植物产生的聚酮类化合物的数量极其庞大,是一大类结构多样化和生物活性多样性的天然产物,已经成为新药的重要来源。介绍了3种类型聚酮类化合物生物合成基因簇的特点,即以模块形式存在的Ⅰ型聚酮合酶,包含一套可重复使用结构域的Ⅱ型聚酮合酶以及不需要ACP参与,以植物中的查耳酮合酶为代表的Ⅲ型聚酮合酶。同时,还介绍了基于3种类型聚酮类化合物生物合成基因的特点,利用分子生物学方法构建筛选探针,进行当前药物基因筛选的进展。