【作者】 孙骏；

【导师】 吴坚平；

【作者基本信息】 浙江大学 ， 化学工程与技术， 2020， 博士

【摘要】 假单胞菌是一种常见的革兰氏阴性细菌,由于其代谢途径丰富、溶剂耐受性强等优点,被认为是生产化学品、药物、生物燃料的重要潜力菌株。然而,现有的假单胞菌基因组编辑工具存在操作周期长、效率低等缺点,严重限制了对假单胞菌的开发、改造与应用。本论文在恶臭假单胞菌KT2440和虫媒假单胞菌L48中建立了 CRISPR/Cas9基因编辑系统,在此基础上拓展构建了 CRISPRi表达干扰系统、碱基编辑器系统。利用CRISPR编辑技术对假单胞菌进行改造,分别在恶臭假单胞菌KT2440和虫媒假单胞菌L48构建了没食子酸和原儿茶酸的代谢途径。主要研究结论如下:第一部分,在KT2440中建立CRISPR/Cas9系统。建立了一套高效、可循环的CRISPR/Cas9双质粒系统。利用该系统,基因敲除、基因替换、基因插入等操作可以在5天内完成,编辑效率超过了 70%。由dCas9介导的CRISPRi表达干扰系统,能够将目标基因的表达水平分别降低至28.5%、29.4%、72.1%。在此基础上,继续在KT2440中拓展构建了一个CRISPR/Cpf1系统,扩大了 CRISPR系统的靶点选择范围。上述研究为改造和开发KT2440提供了高效的基因编辑工具。第二部分,胞嘧啶碱基编辑器在多种假单胞菌中的应用。构建了一套CRISPR介导的修复模板非添加的胞嘧啶碱基编辑器pSEVA6BE,通过提前引入终止密码子,能够在KT2440、L48、铜绿假单胞菌PAO1、荧光假单胞菌Pf-5中实现基因敲除效果。改造Cas9的PAM识别机制,将pSEVA6BE的PAM识别特异性由NGG拓展到了 NG。通过改造脱氨酶的工作窗口,构建得到了 一个编辑窗口缩小的pSEVA6BE-YE1。构建了具有多元碱基编辑功能的单质粒和双质粒系统,对双位点与三位点同时编辑的效率分别为90-100%和25-35%。上述研究不仅拓展了假单胞菌中碱基编辑器的功能,并且首次将CRISPR技术应用于L48和Pf-5。第三部分,代谢工程改造KT2440合成没食子酸(GA)。敲除KT2440中没食子酸相关降解基因,构建得到了一个能够积累合成78.43 mg/L GA的KTR1。过表达3-脱氢莽草酸脱水酶与对羟基苯甲酸脱氢酶,缩短没食子酸的合成步骤,实现了合成路径的重构建。针对没食子酸合成路径相关基因,采用了单个基因位点改造、多位点组合、异源组合插入的三轮次的基因组改造策略,获得的最佳菌株KT-AG21,没食子酸产量为3589.02mg/L,相比较于KTR1,提高了 44.76倍。为了缩短甘油发酵时间,从KT-AG21中删除掉甘油利用阻遏基因(glpR),成功将发酵时间从72 h缩短至了 60 h。上述CRISPR/Cas9辅助的研究证明了构建菌株突变库并进行组合优化是筛选高产菌株的一种重要研究策略。第四部分,在L48中建立CRISPR/Cas9系统及其应用。通过启动子工程策略,构建了一个含有不同启动子表达Cas9的假单胞菌CRISPR/Cas92.0工具库。通过基因敲除实验,筛选到了多个能够在L48中编辑的CRISPR/Cas9系统。利用CRISPR/Cas9系统,构建了一个L48合成原儿茶酸(PCA)的菌株突变库。将菌株突变库与不同合成路径在双质粒共表达系统进行组合,原儿茶酸产量提高到了 5.05 g/L,产率为281 mg PCA/1g甘油。上述研究对改造CRISPR/Cas9系统进行假单胞菌新宿主的拓展具有借鉴意义。更多还原

【Abstract】 The Pseudomonas genus is a common gram-negative bacteria.Inspired by its metabolic versatility and strong solvernt tolerance,Pseudomonas is considered to be the potential platform microorganism for the production of chemicals,drugs,and biofuels.However,the existing genome editing tools of Pseudomonas are still suffered from long manipulation period and low editing efficiency,which grealty hinder the development,modification and application of Pseudomonas.In this paper,we successfully established a CRISPR/Cas9 system in Pseudomonas putida KT2440 and Pseudomonas entomophila L48.On this basis,the base editor system and CRISPRi expression interference system were extended into Pseudomonas.By the utilization of CRISPR techniques,we constructed a gallic acid(GA)-producing KT2440 mutant strain and a protocatechuic acid(PCA)-producing L48 mutant strain.The major conclusions can be divided into four parts:Part I.Establishment of CRISPR/Cas9 system in P.putida KT2440.An efficient and iterative CRISPR/Cas9 two-plasmid system was established in KT2440.Gene deletion,gene insertion and gene replacement could be achieved within 5 days,and the mutation efficiency reached>70%.The dCas9-mediated CRISPR interference system was capable of reducing the expression level of target genes to 28.5,29.4,and 72.1%of the control level.Furthermore,based on this CRISPR-Cas9 system,we also constructed a CRISPR-Cpfl system in KT2440,which expanded the target scope of CRISPR system.Our CRISPR systems would be a powerful tool for modification and exploitation of KT2440 in future studies.Part II.Application of cytosine base editor in multiple Pseudomonas species.An CRISRP-assisted template-free cytosine base editor was established in P.putida KT2440,P.entomophila L48,P.aeruginosa PAO1 and P.fluorescens Pf-5.By introducing a premature stop codon at target locus,this system could be used to generate gene knockout mutants.Transformation of the PAM recoginization mechanism via sitedirected mutations,the PAM specificity of pSEVA6BE system was expanded from NGG to NG.By altering the editing window of APOBEC1,we constructed a pSEVA6BE-YE1 system with a narrow editing window.Multiplex base editing in double-locus and triple-locus were achieved by one-plasmid and two-plasmid systems with the mutation efficiencies of 90-100%and 25-35%,respectively.Our study not only expanded the application scope of based editing system in Pseudomonas,but also extended the CRISPR techiques into P.fluorescens Pf-5 and P.entomophila L48 for the first time.Part Ⅲ.Metabolic engineering of P.putida KT2440 for synthesis of GA.A GAaccumulating strain KTR1 was generated by deletion of GA degradation genes,and the titer of GA was increased to 78.43 mg/L.To reconstruct the synthetic payhway of GA,overexpression of 3-dehydroshikimate dehydratase and p-hydroxybenzoate dehydrogenase was implemented,which shortened the synthesis steps of GA.To enhance the GA titer,stepwise genomic modifications in three rounds including single target site modification,multi-sites combination and heterologous combination insertion,were applied to modify the upstream genes related to GA synthesis pathway.The titer of GA in the optimal resulting strain KT-AG21 was increased to 3589.02 mg/L,thereby improving GA production by 44.76-fold when compared to KTR1.To shorten the cultivation period of glycerol,the glycerol utilization repressor gene(glpR)was deleted from KT-AG21,and the fermentation time was successfully shortened from 72 h to 60 h.Our study showed the combination optimization of mutant strain libraries and different genomic modifications could be an effective strategy for screening high-yield strains.Part Ⅳ.Establishment and application of CRISPR/Cas9 system in P.entomophila L48.By applying promoter engineering strategy,a CRISPR/Cas9 toolkit containing different Cas9 promoters was established in Pseudomonas.Multiple CRISPR/Cas9 systems were proved to be functionally efficient in P.entomophila L48 by the knockout experiments.By utilization of CRISPR/Cas9 system,the L48 mutant libraries for the production of protocatechuic acid(PCA)was constructed.By the combination of mutant strain libraries and different PCA synthetic pathway in a two-plasmid coexpression system,the titer of PCA was drastically increased to 5.05 g/L,and the yield reached 281 mg PCA/1 g glycerol.The establishment of CRISPR/Cas9 system in L48 has reference significance for expanding CRISPR/Cas9 system into a new Pseudomonas host.更多还原