Construction Of Streptomyces Roseosporus Chassis Cell With High Level Of Cofactors Based On CRISPR System And Proteomics Dissection Strategy

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.