【作者】 刘焱；

【导师】 王海龙；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2025， 博士

【摘要】 聚酮合酶由若干功能模块组成,是细菌中最大的蛋白质,负责多杀菌素、阿维菌素等多种重要药物的生物合成。相邻聚酮合酶蛋白间通过对接结构域介导的蛋白质相互作用形成多酶复合体,执行聚酮骨架的合成。聚酮药物高效合成对我国医药、农药的自主创新和开发应用至关重要,当前聚酮合酶工程改造集中在改造调控因子、增加前体供应、敲除竞争途径等方面,对催化核心聚酮合酶的工程改造鲜有涉及。因此,对聚酮合酶的研究将进一步促进聚酮化合物生物合成的工程化。编码聚酮合酶的基因通常大于10 kb并组成操纵子,它们的mRNAs比普通基因的mRNAs长,mRNA越长被截断的风险越大。截断的mRNAs翻译成不含末端对接结构域和C端催化结构域的无功能蛋白,导致细胞资源浪费。目前尚未有研究评估截断的mRNAs对聚酮合酶基因翻译和聚酮化合物生物合成的影响。高效杀虫剂丁烯基多杀菌素的发酵产量有待提高以实现工业生产。本研究将编码蛋白分子量456 kDa三模块聚酮合酶的13 kb 丁烯基多杀菌素busA基因,拆分成三个较小的独立翻译基因,每个基因编码一个单模块聚酮合酶。在白色链霉菌(Streptomyces albus)J1074中野生型和拆分型busA基因的表达揭示截断的mRNAs构成了大部分(>93%)的聚酮合酶mRNAs。基于该发现,本研究开发了一种拯救截断聚酮合酶mRNA翻译的策略。通过在模块编码序列之间插入CDD-TGA-RBS-ATG-NDD编码序列将多模块聚酮合酶蛋白拆分成独立翻译的单模块聚酮合酶亚基,使超大聚酮合酶基因截断的mRNA翻译成功能性聚酮合酶亚基,这些亚基通过末端对接结构域介导的蛋白质相互作用形成功能性聚酮合酶复合体,并使更靠近N端的聚酮合酶亚基浓度显著升高,从而使携带拆分型聚酮合酶基因的细菌宿主的聚酮化合物生物合成效率远高于携带天然聚酮合酶基因的细菌宿主。基于该策略,本研究将丁烯基多杀菌素产量提高13倍,并通过优化聚酮合酶模块组成和下游基因表达,使其产量进一步提高30倍。还将阿维菌素基因簇中负责编码蛋白分子量666kDa四模块聚酮合酶的19kbaveA2基因,拆分成2个较小的独立翻译基因,每个基因负责编码操纵子中的双模块聚酮合酶,阿维菌素聚酮合酶生物合成效率提高5倍。对接结构域互作亲和力影响聚酮合成效率,目前还没有系统测试不同来源对接结构域对聚酮合成效率影响的报道。本研究开发的聚酮合酶对接结构域替换策略,将盐霉素、红霉素、雷帕霉素的12对对接结构域分别替换到多杀菌素聚酮合酶SpnA蛋白的C端和SpnB蛋白的N端构建重组基因簇;利用高效液相色谱-质谱联用(HPLC-MS)检测多杀菌素产量,通过与原始基因簇比较,以不同对接结构域引起的产量增加或减少倍数作为对接结构域相对亲和力,建立了对接结构域元件库。实验结果显示不同对接结构域介导的蛋白互作强弱不同,而且替换互作更强的对接结构域可以提高合成效率。将多杀菌素聚酮合酶对接结构域DDspnAB替换成盐霉素聚酮合酶对接结构域DDslnA78时,多杀菌素产量增加到1.5倍;将盐霉素聚酮合酶对接结构域DDslnA12替换成多杀菌素聚酮合酶对接结构域DDSpnDE时,盐霉素产量增加到2.3倍。并发现当把多杀菌素聚酮合酶1b型对接结构域DDspnAB替换成盐霉素聚酮合酶1a型对接结构域DDslnA78时,多杀菌素产量增加1.5倍,说明不同类型对接结构域可以相互替换并有增强相邻聚酮合酶相互作用的潜力。此外,本研究通过序列比对解析对接结构域形成疏水界面和盐桥的保守氨基酸,对雷帕霉素聚酮合酶对接结构域的CDDrapB(α3)进行S64D无痕定点突变,使其与NDDrapC(α4)的K92形成盐桥,将它们替换到多杀菌素SpnA蛋白C端和SpnB蛋白N端,进一步使多杀菌素产量提高到未突变基因簇菌株的6.0倍。成千上万成对的对接结构域已被成功注释,构成潜力巨大的交互元件库。为了高通量表征对接结构域亲和力,本研究分别在链霉菌和大肠杆菌中构建了基于靛蓝和虾青素生物合成的对接结构域亲和力显色报告系统,以靛蓝和虾青素相对产量表征对接结构域相对亲和力,利用聚酮合酶对接结构域对靛蓝生物合成的关键酶GlnA/IndC和虾青素生物合成的关键酶Idi/CrtE进行双酶组装。使用盐霉素聚酮合酶对接结构域DDslnA23组装靛蓝生物合成途径的GlnA和IndC时,靛蓝产量是未组装菌株的2.1倍;使用多杀菌素聚酮合酶对接结构域DDSpnDE组装虾青素生物合成途径的Idi和CrtE时,虾青素产量是未组装菌株的2.0倍。结果表明利用对接结构域组装生物合成途径关键酶可以增强蛋白互作从而提高生物合成效率。相比于通过化合物产量来测试对接结构域亲和力,靛蓝和虾青素显色报告基因测试系统操作更方便而且成本更低,有利于对多种对接结构域的亲和力进行高通量表征。综上所述,本研究通过拆分模块化聚酮合酶及替换互作更强的对接结构域提高了聚酮生物合成,利用化合物产量检测方法系统测试不同对接结构域亲和力建立对接结构域元件库,并通过组装靛蓝和虾青素生物合成途径关键酶构建了对接结构域亲和力显色报告系统,为基于模块组成和蛋白质相互作用的聚酮合酶工程改造提供了理论支持和策略依据。更多还原

【Abstract】 Polyketide synthases(PKSs)consist of several functional modules and represent the largest proteins in bacteria,being responsible for the biosynthesis of numerous significant drugs such as spinosad and avermectin.Adjacent PKS proteins within PKSs form multi-enzyme complexes through protein-protein interactions mediated by docking domains,thereby determining the synthesis of polyketide skeletons.The efficient synthesis of polyketide drugs is crucial for the independent innovation,development,and application of medicines and pesticides in China.Currently,the engineering modification of PKSs mainly focuses on modifying regulatory factors,increasing precursor supplies,and eliminating competing pathways.However,engineering modification studies focused on the catalytic core PKSs are scarce.Hence,research on PKS genes will further facilitate the engineering of polyketide biosynthesis.Genes encoding PKSs are typically larger than 10 kb and form operons;consequently,their mRNAs are longer than those of ordinary genes.The longer the mRNA is,the more likely it is to be truncated.Truncated mRNAs translate into nonfunctional proteins lacking the terminal docking domain and C-terminal catalytic domain,leading to the waste of cellular resources.Up to now,there is no study to evaluate the effect of truncated mRNA on PKS gene translation and polyketide biosynthesis.To investigate the effect of truncated mRNAs on polyketide biosynthesis,this study selected the biosynthetic gene cluster of the highly effective insecticide butenyl-spinosyn,whose fermentation yield requires enhancement for industrial production.In this study,the 13 kb busA gene,which encodes a three-module PKS with a molecular weight of 456 kDa,was split into three smaller independent translation genes,each responsible for encoding one module of the PKS.The expression of wildtype and split busA genes in Streptomyces albus J1074 revealed that truncated mRNAs constituted the majority(>93%)of the PKS mRNAs.Based on these findings,this study developed a strategy to rescue the translation of truncated PKS mRNAs by inserting CDD-TGA-RBS-ATG-NDD coding sequences between the module coding sequences to dissect multi-module PKS proteins into independently translated single-module PKS subunits.This approach was applied to the super-large PKS genes,enabling the translation of truncated mRNAs into functional PKS subunits.The PKS subunits form functional PKS complexes through protein-protein interactions mediated by terminal docking domains,and significantly increase the concentration of PKS subunits closer to the N-terminus.This approach ultimately resulted in a much higher polyketide biosynthesis efficiency in bacterial hosts carrying the split PKS genes compared to those carrying the natural PKS genes.Based on this approach,the butenyl-spinosyn yield was increased by 13 times and further enhanced by 30 times through the optimization of downstream gene expression.In addition,the 19 kb aveA2 gene encoding a four-module PKS with a molecular weight of 666 kDa in the avermectin PKS gene cluster was also split into two smaller independent translation genes,each encoding two modules of the PKS,and the avermectin PKS biosynthesis efficiency was increased by 5 times.The interaction affinity of docking domains impacts the efficiency of polyketide synthesis.Up to now,there is no report on systematically testing the influence of docking domains from different sources on the efficiency of polyketide synthesis.In this study,a PKS docking domain replacement strategy was developed.Twelve pairs of PKS docking domains from salinomycin,erythromycin and rapamycin PKS were respectively replaced at the C-terminal of SpnA protein and the N-terminal of SpnB protein of the spinosad PKS to construct recombinant gene clusters.Then,Utilizing high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS)to detect the production of spinosad,the relative affinity of docking domains was determined by comparing the yield increase or decrease caused by different docking domains relative to the original gene cluster,and a library of docking domain components was established.The results indicated that the protein interactions mediated by different docking domains varied in strength,and replacing stronger docking domains could improve the synthesis efficiency of polyketides.When the spinosad PKS docking domain DDspnAB was replaced with the salinomycin PKS docking domain DDslnA78,the spinosad production increased by 1.5 times.In addition,when the salinomycin PKS docking domain DDslnA12 was replaced with the spinosad PKS docking domain DDspnDE,the salinomycin production increased by 2.3 times.Additionally,this study analyzed the conserved amino acids that form hydrophobic interfaces and salt bridges in the docking domains through sequence alignment.A seamless site-directed mutation was performed on the CDDrapB(α3)of the rapamycin polyketide synthase docking domain,changing S64 to D64,to form a salt bridge with K92 of NDDrapC(α4).These mutations were then replaced at the C-terminus of the spinosad SpnA protein and the N-terminus of the SpnB protein,further increase the production of spinosad to 6.0 times that of the unmutated gene cluster strain.Thousands of pairs of docking domains have been successfully annotated,forming a vast library of potential interaction modules.To characterize the affinity of docking domains on a high-throughput scale,this study constructed a colorimetric reporter system for the affinity of docking domains based on the biosynthesis of indigo and astaxanthin in Streptomyces and Escherichia coli,respectively.The relative yields of indigo and astaxanthin were used to characterize the relative affinity of the docking domains,and polyketide synthase docking domains were utilized to assemble the key enzymes GlnA/IndC for indigo biosynthesis and Idi/CrtE for astaxanthin biosynthesis.When using the salinomycin PKS docking domain DDslnA23 to assemble the key enzymes GlnA and IndC in the indigo biosynthetic pathway,the production of indigo was 2.1 times that of the unassembled strain;when using the spinosad PKS docking domain DDspnDE to assemble the key enzymes Idi and CrtE in the astaxanthin biosynthetic pathway,the production of astaxanthin was 2.0 times that of the unassembled strain.The results indicate that assembling key enzymes in biosynthetic pathways using docking domains can enhance protein-protein interactions,thereby improving biosynthetic efficiency.Compared to the previous chapter’s method of testing docking domain affinity through compound production,the indigo and astaxanthin colorimetric reporting gene test system is more cost-effective and conducive to high-throughput characterization of the affinity of various docking domains.This study enhanced polyketide biosynthesis by splitting modular polyketide synthases and replacing them with stronger interacting docking domains.systematically tested the affinity of different docking domains using compound yield detection methods to establish a docking domain element library,and constructed a docking domain affinity colorimetric reporter system by assembling key enzymes in indigo and astaxanthin biosynthetic pathways,providing theoretical support and strategic basis for PKS engineering based on module composition and protein-protein interactions.更多还原