【作者】 张丹；

【导师】 梁淑芳；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2021， 博士

【摘要】 天然产物具有丰富的结构和活性多样性,是药物研发的重要来源之一。很多链霉菌源天然产物已被批准用于临床治疗耐药菌感染、病毒感染、癌症等。正是因为链霉菌天然产物展现出良好的临床应用潜力,研究者们不断发掘链霉菌源新天然产物。然而,传统的对链霉菌来源的次级代谢产物分离提取获得高活性天然产物或新活性医药产物越来越困难。因此,亟待开发出新的技术和策略来显著提升活性天然产物的产量和挖掘新结构新活性的链霉菌天然产物。合成生物学作为21世纪初新兴出现的学科,是在阐明并模拟生物合成的基本规律之上,采用标准化、去耦化和模块化达到人工设计并构建新的、有特定生理功能的生物系统,从而建立药物、功能材料或能源替代品等的生物制造途径。近年来,合成生物学在国际国内发展迅猛,理性设计通路、通过各种编辑及组装技术对设计的通路进行重构、评估及优化等合成生物学技术广泛应用于天然产物研究,其对人口健康、生物医药等领域产生深远影响。例如,青蒿素前体在酵母中的成功合成促进抗疟疾药的推广就是典型的成功范例。随着链霉菌基因组测序的不断发展,研究者们不断揭示链霉菌产生未知天然产物的巨大潜力,也开发了用于天然产物发掘的一系列技术。Clustered regularly interspaced short palindromic repeats(CRISPR)技术作为近年来前沿先进的基因编辑技术,在基因敲除、转录抑制及激活方面得到广泛应用。蛋白质组学(Proteomics)策略定量分析微生物生长代谢过程中参与蛋白质的组成及表达丰度的变化,并解析生物量和次级代谢产物生产中涉及的生物过程和代谢途径的调节,为定向设计和调控优化生物合成通路提供依据。因此,组学(Omics)技术结合基因组优化、代谢工程研究方法构建并优化链霉菌底盘细胞,为链霉菌天然产物改造和发掘奠定基础。玫瑰孢链霉菌Streptomyces roseosporus(S.roseosporus)NRRL11379发酵产生的非糖体肽合成酶(Nonribosomal peptide synthetase,NRPS)类十元环状脂肽抗生素-达托霉素(daptomycin,DAP)于2003年首次被美国FDA批准用于治疗革兰氏阳性菌引起的复杂皮肤感染和结构性皮肤感染,随后又被批准用于治疗金黄色葡萄球菌引起的菌血症和右侧心内膜炎。DAP生物合成基因簇(dpt)共约127.8 kb,其中包括三个主要的NRPS合成基因(dpt A,dpt BC,dpt D),其上游的dpt E和dpt F基因分别编码acetyl-Co A连接酶和酰基载体蛋白,负责脂肪酸的激活并将其加载至N端的L-Trp上。而其下游的dpt I、dpt J基因则负责DAP非天然氨基酸Kyn、m Glu的产生。除此之外,DAP基因簇内还包括3个转录调控基因(dpt R1、dpt R2、dpt R3)和3个DAP转运、抗性基因(dpt M、dpt N、dpt P)。S.roseosporus因其产生结构独特、抗菌活性好的DAP而被众多研究者关注。截至目前,DAP是该菌中研究最深入的天然活性化合物。已有研究通过异源表达dpt、替换dpt中的同源性模块、转录调控、翻译后修饰等方面进行研究并试图提高DAP的产量。除了DAP,研究者们还从玫瑰孢链霉菌发现了抗菌活性较好的新产物auroramycin、arylomycin、napsamycin及抗肿瘤活性潜力的polycyclic tetramate macrolactam。除此之外,玫瑰孢链霉菌还有很多未被鉴定和未激活的未知基因簇。此外,玫瑰孢链霉菌的生长调控机制、次级代谢通路及分子作用网络并未研究清楚,对该菌的代谢背景了解甚少,进而阻碍了该菌中天然产物产量提升和有效发掘。为了更高效地提高玫瑰孢链霉菌中有价值的活性天然产物的产量,发掘玫瑰孢链霉菌中的新活性化合物,本研究课题从基因编辑的角度出发,通过构建适于链霉菌的新型基因编辑系统来提高化合物生物合成基因簇中核心基因的表达水平;并从代谢背景出发,通过蛋白质组学分析揭示该菌在不同生长期前体和辅因子代谢通路的变化情况,分析菌体在不同生长期初级代谢与次级代谢的关联点-前体和辅因子的水平,筛选并鉴定验证辅因子代谢相关的关键基因对次级代谢产物产生的影响,并采用CRISPR系统优化基因组并激活辅因子代谢的关键基因,从而构建高水平辅因子的玫瑰孢链霉菌底盘细胞,为提高有价值的天然产物产量或发掘链霉菌源新天然产物奠定基础。首先,我们通过体外PCR扩增、同源重组等分子生物学技术,构建了4个由不同启动子kas Op*、rps Lp(XC)、erm Ep*或诱导系统Potr*启动Fn Cas12a的链霉菌基因组编辑新工具-CRISPR-Fn Cas12a系统,即p YL-kas Op*-Fn Cas12a、p YL-rps Lp(XC)-Fn Cas12a、p YL-erm Ep*-Fn Cas12a和p YL-Potr*-Fn Cas12a(以下分别简称p CF-kas O、p CF-XC、p CF-erm E、p CF-Potr)。四个系统的接合转移效率随着Fn Cas12a的启动子的减弱而增加,其效率由低到高为p CF-kas O<p CF-XC<p CF-erm E<p CF-Potr。通过在S.coelicolor和S.lividans两个宿主中比较4个系统的基因编辑效率发现,Fn Cas12a的启动子越强,其编辑效率越高。其中,p CF-kas O的编辑效率最高,达100%。随后通过相关性分析发现,Fn Cas12a的转录水平与其基因编辑水平呈正相关。除此之外,当Fn Cas12a的启动子为强启动子时,direct repeat(DR)长度越长,Fn Cas12a的编辑效率越高。我们还在接合转移效率较低的Streptomyces griseus中成功应用CRISPR-Fn Cas12a进行基因敲除;接着应用CRISPR-Fn Cas12a敲除了玫瑰孢链霉菌中编码DAP的长达127.8kb的基因组大片段;最后应用CRISPR-Fn Cas12a激活DAP基因簇中的核心基因dpt A、dpt BC、dpt D,提高了DAP的产量。其次,我们对S.roseosporus NRRL11379原始野生型菌株(S.roseosporus NRRL11379 wild type,SRWT)和经过诱变筛选的能产生DAP且发酵产物效价更高的19号玫瑰孢链霉菌突变株(S.roseosporus NRRL11379 mutant 19,SRWT)进行定量蛋白质组分析,以鉴定筛选菌体组成蛋白的差异和DAP生物合成涉及的关键初级、次级代谢通路。以鉴定筛选菌体组成蛋白的差异和次级代谢产物生物合成涉及的关键初级、次级代谢通路。分别提取SRWT和SRMT19的菌体总蛋白,进行质谱级胰蛋白酶酶切,用stage tips方法对酶切后的肽段进行除盐,通过液相色谱-质谱/质谱(Liquid chromatography-tandem mass spectrometry,LC-MS/MS)鉴定蛋白,基于这种无标记质谱定量(Label-free quantification,LFQ)比较两组菌体组成蛋白丰度的差异。我们鉴定了529个在SRMT19中上调的蛋白,335个下调的蛋白(差异倍数在2倍及以上,p<0.05)。KEGG分析显示,SRMT19中碳代谢、次级代谢产物代谢、脂质代谢、转运过程4个代谢通路被显著激活。碳代谢作为宿主的基本代谢,为次级代谢产物的合成提供前体、辅因子和能量。随后,我们基于RT-q PCR分析比较SRWT和SRMT19的转录组。结果显示,TCA循环、丙酮酸代谢都在SRMT19的对数期早期(48 h)被显著激活,尤其是丙酮酸脱氢酶复合物,其转录水平分别是SRWT的2.80、3.18、1.30倍。丙酮酸代谢不仅产生一系列辅酶A,更产生大量的NADH,为次级代谢产物合成提供前体的同时,提供还原力。随后,我们通过生化反应检测SRWT和SRMT19的NAD~+、NADH及ATP辅因子水平,发现SRMT19在对数期(72h)积累了大量ATP。为了验证丙酮酸脱氢酶对辅因子产量的影响,我们基于p SET152K过表达质粒及p CF-kas O系统对丙酮酸脱氢酶复合物进行过表达和敲除,并结合生化分子实验,检测辅因子的变化。结果显示,丙酮酸脱氢酶复合物的过表达将ATP的水平分别提高至转入空载质粒的野生型的12.26倍;相反,我们在ATP水平较高的突变株SRMT19菌中敲除丙酮酸脱氢酶复合物后,其ATP的水平显著降低至SRMT19的0.42倍。为了验证丙酮酸代谢对次级代谢产物产生的影响,我们在玫瑰孢链霉菌野生型中过表达丙酮酸脱氢酶复合物并检测其发酵产物效价,结果显示,发酵产物的效价提高至转入空载质粒的野生型的2.53倍。随后,为了进一步验证丙酮酸脱氢酶对PKS类化合物产生的影响,我们选择用有颜色指示的可快速检测的化合物-放线菌紫素(Actinorhodin,ACT)来评估。我们在S.lividans中过表达丙酮酸脱氢酶复合物,也促进了次级代谢产物ACT的积累。丙酮酸代谢促进ATP产生的通路很多,例如丙酮酸脱氢生成乙酰辅酶A,再进入TCA产生ATP。为了探究链霉菌中丙酮酸代谢产生ATP的其它通路,我们对蛋白质组数据和转录组数据进行了综合再分析,发现一系列NADH醌氧化还原酶亚基在SRMT19中被显著激活。NADH醌氧化还原酶能利用氧化还原将NADH氧化成NAD~+和H~+,并将H~+转移到膜上,进而对能量的产生有重要贡献。基于基因过表达验证,发现04370和04379基因(分别编码NADH醌氧化还原酶B亚基和K亚基)通过催化NADH的再氧化,显著促进NAD~+的积累,同时将H~+传递给ADP生成大量ATP,为次级代谢产物合成提供能量,进而促进次级代谢产物ACT的产生。NADH醌氧化还原酶亚基的过表达不仅能氧化NADH生成NAD~+,还能通过积累的产物NAD~+再进入丙酮酸代谢通路而进一步激活丙酮酸代谢。丙酮酸代谢和NADH醌氧化还原酶激活所产生的NAD~+和NADH循环,为玫瑰孢链霉菌次级代谢产物合成提供了辅因子和能量。最后,我们在玫瑰孢链霉菌中过表达NADH醌氧化还原酶亚基B和K并检测其发酵产物效价。结果显示,K亚基过表达后,其发酵产物的效价提高至16.65 U/m L,是野生型(4.83 U/m L)的3.44倍。天然产物的发现往往依赖于其生物合成途径在异源宿主的高效表达。异源宿主为其它沉默基因簇提供优良代谢背景从而激活沉默基因簇,提高其编码的天然产物的产量。为了构建理想的链霉菌底盘细胞,我们优化基因组和代谢网络,从而构建高水平辅因子的玫瑰孢链霉菌底盘细胞。首先,我们基于生物信息学工具anti SMASH在线网站和Mauve软件分析玫瑰孢链霉菌中非必须基因簇,并利用p CF-kas O系统对筛选出的7个非必须基因簇进行敲除从而构建S.roseosporusΔ7(SRΔ7)菌株。随后,再用p CF-kas O系统在丙酮酸脱氢酶复合物基因03488前插入强启动子kas Op*构建SRΔ7KP菌株,进而激活丙酮酸代谢,来提高辅因子水平。RT-q PCR检测结果显示SRΔ7KP菌中03486、03487、03488基因(编码丙酮酸脱氢酶复合物)的转录水平分别是7.63±3.10、2.53±0.46、22.89±3.48,与野生型对应基因的转录水平1.48±0.16、0.25±0.07、2.00±0.10相比,提高了4.16、9.25和10.42倍。随后,通过生化实验检测,SRΔ7KP的辅因子NADH水平为0.42±0.05 pmol/mg,是野生型(0.11±0.01 pmol/mg)的3.72倍。辅因子的高浓度积累,为玫瑰孢链霉菌次级代谢产物的合成提供了辅因子和能量,进而促进次级代谢产物的产生。综上所述,本研究对构建的4种不同链霉菌基因编辑系统CRISPR-Fn Cas12a的编辑效率进行分析比较,揭示了影响该系统编辑效率的因素,并获得高效基因编辑系统,为后续代谢优化和底盘细胞构建提供有效工具。随后应用该系统联合LFQ定量蛋白质组学策略解析并验证玫瑰孢链霉菌初级代谢与次级代谢的联系,并验证了玫瑰孢链霉菌初级代谢中的丙酮酸脱氢酶复合物和NADH醌氧化还原酶亚基的激活为次级代谢积累辅因子进而促进次级代谢产物的产生,为后续优化底盘细胞的代谢网络提供重要信号通路。最后基于CRISPR-Fn Cas12a优化玫瑰孢链霉菌基因组和丙酮酸代谢,构建高水平辅因子的链霉菌底盘细胞。本文的研究结果不仅直接揭示了参与玫瑰孢链霉菌辅因子代谢的关键基因对辅因子和次级代谢产物生物合成的贡献,也提供高效的链霉菌基因编辑工具及改造次级代谢产物的底盘细胞,为提高天然产物产量和优化生物合成途径提供新的表达底盘细胞。更多还原

【Abstract】 Natural products have become one of the significant sources of drug research due to their rich structures and various biological activities.A sea of natural products produced by Streptomyces strains have been approved for treatment of drug-resistant bacteria or virus infections,cancer and so on.However,it is challenging to obtain those natural products with high bioactivities or drugs with novel bioactivities by traditional separation and extraction.Thus,it is urgent to develop new technologies to significantly increase the production of bioactive natural products and discover the Streptomyces natural product with novel structures or bioactivities.As an emerging discipline at the beginning of the 21st century,on the basis of clarifying the basic law of biosynthesis,synthetic biology aims to achieve artificial design and construction of new biological systems with specific physiological functions using standardization,decoupling and modularization,so as to establish the biological manufacturing pathways of drugs,functional materials or energy substitutes.In recent years,synthetic biology has been developed rapidly in nationally or internationally.The synthetic biology technologies,including rational design of pathways,reconstruction,evaluation and optimization of designed pathways through various editing and assembly technologies,are widely applied in natural product research.These technologies induce far-reaching impacts on population health,biological medicine and other fields.For example,the successful synthesis of artemisinin precursor in yeast promotes the large-scale application of antimalarial drugs.With the continuous development of Streptomyces genome sequencing,researchers have revealed the great potential of Streptomyces to produce unknown natural products,and have developed a series of technologies for discovering natural products.The clustered regularly interspaced short palindromic repeats(CRISPR)technology is an advanced genome editing approach in recent years,which has been widely used in gene deletion,transcriptional repression and activation.Quantitative proteomics can sensitively analyze the changes of protein composition and abundance in the process of microbial growth and metabolism,and it also dissects biological processes and metabolic pathways those are involved in biomass or secondary metabolism.All these analyses provide references for the directional design and optimization of biosynthetic pathways.Therefore,the construction and optimization of Streptomyces chassis based on omics technology combined with genomic optimization and metabolic engineering will provide the foundation for modification and discovery of Streptomyces natural products.The daptomycin(DAP)is a ten-membered ring cyclic lipopeptide,which is produced by Streptomyces roseosporus(S.roseosporus)NRRL11379 via nonribosomal peptide synthetase(NRPS)biosynthesis process.DAP had been approved by US FDA for the treatment of complex and structural skin infections caused by Gram-positive bacteria in 2003,and then it was approved for the treatment of bacteremia and right endocarditis caused by Staphylococcus aureus.The 127.8-kb DAP biosynthetic gene cluster(dpt)includes three central NRPS genes,dpt A,dpt BC and dpt D.The dpt E and dpt F are located upstream of the NRPS genes,and encode acyl-Co A ligase and acyl carrier protein respectively.The two proteins are responsible for the activation and coupling of the fatty acids to the N-terminus of L-Trp.The genes dpt I and dpt J,just downstream of the NRPS genes,involve in the generation of non-natural amino acids Kyn and m Glu.Additionally,there are three regulatory genes(dpt R1,dpt R2 and dpt R3)and three DAP transport or resistance genes(dpt M,dpt N,dpt P)within DAP biosynthetic gene cluster.S.roseosporus has been widely studied due to its product DAP with specific structure and significant antibacterial activity.Up to now,DAP is the most studied product in S.roseosporus.Researchers have tried to improve DAP production by heterologous expression of dpt,substituting the modules in dpt with homologous modules,transcriptional regulation and post-translational modification and so on.Except DAP,there are some other new antibacterial products discovered in S.roseosporus,such as auroramycin,arylomycin and napsamycin,and polycyclic tetramate macrolactam with antitumor potential.In addition,there are several unidentified or silent gene clusters.Moreover,the mechanisms for growth regulation,secondary metabolic pathways and molecular networks don’t completely understood in S.roseosporus,which hinders the production improvement and efficient discovery of natural products.Herein,we have constructed a new gene editing CRISPR system to increase the transcription levels of core genes of biosynthetic gene clusters in S.roseosporus.Based on the quantitative proteomics identifications,we revealed the altered primary metabolism and secondary metabolism pathways,and measured the cofactor levels which was a link between primary metabolism and secondary metabolism.Next,we screened and identified the key genes that contributed to accumulation of natural products.By using CRISPR system,we constructed a S.roseosporus chassis cell system with high level of cofactor via optimizing genome and activating key genes involved in cofactor generation.The S.roseosporus chassis cell provides foundations for improving production of valuable natural products or discovering novel natural products.Firstly,we have constructed 4 CRISPR-Fn Cas12a systems for genome editing in Streptomyces based on PCR amplification and homology-direct recombination in vitro.The Fn Cas12a in these CRISPR-Fn Cas12a systems,including p YL-kas Op*-Fn Cas12a(abbreviated as p CF-kas O),p YL-rps Lp(XC)-Fn Cas12a(abbreviated as p CF-XC),p YL-erm Ep*-Fn Cas12a(abbreviated as p CF-erm E)and p YL-Potr*-Fn Cas12a(abbreviated as p CF-Potr),are controlled by constitutive promoters kas Op*,rps Lp(XC),erm Ep*and inducible system Potr*,respectively.The transformation frequencies of CRISPR-Fn Cas12a systems are increased with the weaking strength of promoters,and the transformation frequencies of CRISPR-Fn Cas12a systems from low to high are p CF-kas O<p CF-XC<p CF-erm E<p CF-Potr.By comparing the editing efficiencies of the 4 systems in S.coelicolor and S.lividans,we found that the stronger the promoter,the higher the editing efficiency.The highest editing efficiency is 100%completed by p CF-kas O.The following correlation analysis showed the transcription level of Fn Cas12a was positively correlated with its editing efficiency.Besides,the length and terminator of cr RNA influenced the editing efficiency of Fn Cas12a.When Fn Cas12a was driven by strong promoter,the longer direct repeat(DR),the higher editing efficiency of Fn Cas12a.We also achieved gene deletion using CRISPR-Fn Cas12a in Streptomyces griseus.CRISPR-Fn Cas12a was also successfully applied to delete 127.8-kb DNA fragment encoding DAP and activate core genes dpt A,dpt BC,dpt D followed by improving DAP production in S.roseosporus.Next,we screened a DAP-producing mutant S.roseosporus,which generated fermentation products with higher efficacy than wild type.Then,we quantitatively compared the proteome of wild-type S.roseosporus NRRL11379(SRWT)and mutant S.roseosporus(SRMT19)to identify and screen the differential proteins and the primary and secondary metabolic pathways related to secondary metabolite biosynthesis.We isolated total proteins of SRWT and SRMT19,followed by digesting proteins with trypsin and desalting peptides using stage tips method.The peptides were then uploaded to identify proteins by liquid chromatography-tandem mass spectrometry(LC-MS/MS).Based on this label-free quantification(LFQ)technology,we compared the difference of protein abundance between the two groups.As a result,we identified 529 upregulated proteins and 335 downregulated proteins(fold change≥2,p<0.05)in SRMT19 group.KEGG analyses showed four pathways,including carbohydrate metabolism,transport,metabolism of secondary metabolite and lipid metabolism,were dramatically activated in SRMT19.As a basic metabolism,carbohydrate metabolism provided precursors,cofactors and energy for the biosynthesis of secondary metabolites.We also analyzed the transcriptomic differences between SRWT and SRMT19 by RT-q PCR.The results showed tricarboxylic acid cycle(TCA)cycle and pyruvate metabolism were significantly activated in early exponential phase with growth for 48 h.Specially,the transcription levels of the genes for pyruvate dehydrogenase complex in SRMT19 group showed up to 2.80,3.18 and 1.30 times of these in SRWT group.The pyruvate metabolism generates not only acetyl-Co A,but also NADH,which provides precursors and reducing power for the biosynthesis of secondary metabolites.We further measured the levels of cofactors of SRWT and SRMT19,including NAD~+,NADH and ATP.We found SRMT19 accumulated lots of ATP in late exponential phase(72 h).To verify the effects of pyruvate metabolism on yields of cofactors,we overexpressed or deleted pyruvate dehydrogenase complex using an overexpressing plasmid p SET152K or a deletion system p CF-kas O in S.roseosporus,and then measured the yields of cofactors.The results showed ATP level was increased to 12.26 times of SRWT after overexpressing pyruvate dehydrogenase complex in SRWT which introduced empty vector.In contrary,the ATP level was reduced to 0.42 times of SRMT19 after deleting pyruvate dehydrogenase complex in SRMT19.To verify the effects of pyruvate metabolism on generation of secondary metabolites,we measured the efficacy of fermentation products after overexpressing pyruvate dehydrogenase complex in wild-type S.roseosporus.The results showed that after overexpressing pyruvate dehydrogenase complex,the efficacy of fermentation products increased to2.53 times of that produced by wild type which introduced empty vector.Moreover,to further verify the effects of pyruvate dehydrogenase complex on PKS-like natural products,we detected the accumulation of actinorhodin(ACT),which can be fast detected for its color change.Thus,we overexpressed pyruvate dehydrogenase complex in S.lividans,which led to an accumulation of secondary metabolite ACT.Several pyruvate metabolism-involved pathways will generate ATP.For example,pyruvate dehydrogenation produces acetyl-Co A,which enters TCA to produce ATP.To optimize pathways in which pyruvate metabolism generates ATP in Streptomyces,we reanalyzed the proteomic and transcriptomic data,and found a series of NADH-quinone oxidoreductase submits were activated in SRMT19.NADH-quinone oxidoreductase oxidizes NADH to generate NAD~+and H~+via redox,and then transfers H~+to the membrane,which has an important contribution to energy production.We confirmed 04370 and 04379 genes(encoding NADH-quinone oxidoreductase submits B and K,respectively)promoted accumulation of NAD~+by catalyzing the reoxidation of NADH,and then transferred H~+to ADP to generate a large amount of ATP,which provided energy for the biosynthesis of secondary metabolites and further promoted generation of ACT.Overexpression of NADH-quinone oxidoreductase subunit can not only oxidize NADH to produce NAD~+,but also further activate pyruvate metabolism by entering the accumulated NAD~+into pyruvate metabolism pathway.The NAD~+and NADH cycles generated by pyruvate metabolism and NADH-quinone oxidoreductase provided cofactors and energy for the biosynthesis of secondary metabolites.In the last,we overexpressed NADH-quinone oxidoreductase submits B and K in S.roseosporus.After overexpressing NADH-quinone oxidoreductase submit K,the efficacy of fermentation products increased to 16.65 U/m L,which was 3.44 times of wild type(4.83 U/m L).Discovery of natural products often relies on efficient expression of their biosynthetic pathways in heterologous hosts.These heterologous hosts provide a nice metabolic background and contribute to activate the biosynthetic gene clusters and then improve the production of their encoding natural products.To construct an ideal chassis cell system of S.roseosporus,we optimized the genome and metabolism network,and then constructed a S.roseosporus chassis cell with high-level cofactors.Firstly,we analyzed the nonessential gene clusters of S.roseosporus using online bioinformatics tools anti SMASH online website and mauve software,and used p CF-kas O system to delete seven nonessential gene clusters to construct S.roseosporusΔ7(SRΔ7)strain.Then,the strong promoter kas Op*was inserted in front of pyruvate dehydrogenase gene 03488 by p CF-kas O system to construct SRΔ7KP strain.RT-q PCR analysis showed that the transcription levels of 03486,03487 and 03488 genes(encoding pyruvate dehydrogenase complex)of SRΔ7KP were 7.63±3.10,2.53±0.46 and 22.89±3.48,respectively,which were increased4.16,9.25,10.42 times compared to the transcription levels of corresponding genes of wild type(1.48±0.16,0.25±0.07,2.00±0.10),respectively.Subsequently,biochemical results showed that the NADH level of SRΔ7KP was 0.42±0.05pmol/mg,which was 3.72 times of wild type(0.11±0.01 pmol/mg).The accumulation of cofactor provides source and energy for the biosynthesis of secondary metabolites,and then promotes the production of secondary metabolites in S.roseosporus.In summary,we compared the editing efficienies of 4 different CRISPR-Fn Cas12a systems and revealed the factors affecting the editing efficiencies in Streptomces.We obtained a high-efficient gene editing system whcin provided a powerful tool for following metabolism optimization and chassis cell construction.Furthermore,we combined CRISPR tool and LFQ proteomics strategy to analyze and verify the correlations between primary and secondary metabolism in S.roseosporus.We also verified that overexpression of pyruvate dehydrogenase complex and NADH quinone oxidoreductase subunit accumulated cofactors for the secondary metabolism and further stimulated the generation of secondasry metabolites in Streptomces.This work provided important pathways for following chassis cell construction.Finally,we optimized the genome and cofactor generation of S.roseosporus by CRISPR-Fn Cas12a system,and then constructed a S.roseosporus chassis cell with high levels of cofactors.Our studies directly revealed the contribution of cofactor metabolism related key genes to the production of cofactor and secondary metabolites in S.roseosporus.On the other hand,we also provided efficient genome editing tools and a new Streptomyces chassis cell system for improving production of natural products and optimizing biosynthesis pathways.更多还原