Development Of New Gene Editing Technology For Heterologous Expression And Structure Derivatization Of Spinosad

Spinosad is a series of typical polyketide compounds discovered from the fermentation of soil actinomycete Saccharopolyspora spinosa.Spinosad exhibited a broad-spectrum insecticidal activity and extremely low toxicity to mammals,fishes,birds,and non-target insects.Dow Agrosciences has commercialized spinosad as a biological pesticide,widely applied it to control agriculture,garden,and grain storage pets.Spinosad was awarded the Presidential Green Chemistry Award in 1999 because of its potent insecticide activity and excellent environmental profile.In addition to spinosyn A/D,the main active ingredient of spinosad,Saccharopolyspora spinosa produced a series of spinosyns such as spinosyn J and L.Chemically modified spinosyn J and spinosyn L generated 5,6-dihydro-3'-O-ethyl-spinosyn J and 3'-O-ethyl-spinosyn L,which is known as spinetoram.It shows more excellent insecticidal activity,longer duration,and a broader spectrum than spinosad while retaining a low environmental effect on non-target species.Butenyl-spinosyns are spinosyn derivatives with different macrolide structures produced by Saccharopolyspora pogona.It is generated from natural evolution and has similar insecticidal activity and spectrum to spinetoram.Spinosad industry is still in the development stage in China.Therefore,it is urgent to develop new strategies to improve spinosad production,construct high-yield strains,establish production technology with independent intellectual,and utilize synthetic biology to create derivatives with superior insecticidal activity distinct from existing spinosyn products.As a typical polyketide compound,the chemical synthesis of spinosyns is challenging due to the complex molecular structure.Therefore,biosynthesis based on microbial fermentation is more attractive due to its potential for large-scale fermentation production.However,the yield improvement process of Saccharopolyspora spinosa is impeded by its complex culture conditions,costing generation time,and complicated genetic engineering procedures.Therefore,the heterologous expression strategy of cloning the spinosad biosynthetic gene cluster and expressing it in chassis strains with convenient genetic manipulation procedures has become vital in the research of spinosad biomanufacturing.Biosynthesis of spinosad involves 23 genes,about 74 kb in length,and distributed on the Saccharopolyspora spinosa genomic at four positions.Cloning and modifying this complex gene cluster is very challenging,and novel gene editing technologies are needed.The development of breakthrough genetic engineering technologies has significantly simplified the procedures of cloning and assembling natural product biosynthetic pathways and promoted natural product synthetic biology research.However,existing technologies still have many limitations in practical applications.DNA assembly technology has become an effective method to refactor gene cluster.However,the actinomycetes derived natural product gene cluster with high GC content and serried direct repeats.Current In vitro assembly methods such as Gibson or SLIC and TAR mediated in vivo assembly method are incompatible with high GC content DNA fragments or promoter sequences.The number of DNA fragments that can be assembled is limited.These methods are unable to meet the needs of large-scale gene cluster construction,and it's systematic modification.This study comprehensively utilized DNA recombineering,direct cloning,and DNA assembly technology rapidly cloned the complete spinosad biosynthetic gene cluster,constructed an E.coli-Streptomyces shuttle vector,and heterologous expressed spinosad in Streptomyces albus J1074,Streptomyces lividans K4-114,and Streptomyces coelicolor CH999.To improve the spinosad production,we inserted the strong constitutive promoter ermE*p upstream the first polyketide synthase gene in the spinosad gene cluster,resulting in 2.1 times yield improvement.This result indicates that enhance the transcription level of biosynthetic genes can promote the heterologous expression of spinosad.However,to improve all 23 spinosad biosynthetic genes' transcription level,reliable DNA assembly technology is needed to refactor the spinosad gene cluster.This study developed the ExoCET DNA assembly technology by combing exonuclease-mediated In vitro annealing and RecET-mediated in vivo homologous recombination capable of assembling more than 13 high GC content DNA fragments efficiently.Take advantage of ExoCET DNA assembly.According to their functions,we divided the 23 spinosyn biosynthesis genes into five operons under the control of strong constitutive promoters derived from Streptomyces albus J1074 to formed the artificial spinosad gene cluster.The Streptomyces albus J1074 harboring artificial spinosad gene cluster produced 1.12 mg/L spinosad in a 250 mL flask with 50 mL medium,which is 328 times higher than the original spinosad gene cluster.ExoCET DNA assembly technology enhances the multi-fragments co-transformation efficiency of E.coli strains by promoting parts of high GC fragments ligation through In vitro annealing of ssDNA homologous arms generated by the dsDNA exonuclease activity of T4 DNA polymerase.The DNA fragments are then cyclized with high-efficiency LLHR(Linear-linear homologous recombination)mediated by the Rec recombination system.We compared the high GC fragments assemble ability of ExoCET with the commercial Gibson assembly kit.The accuracy of ExoCET technology for the assembly of high GC DNA less than seven fragments is 100%,and can assembly 13 fragments with 60%accuracy.In comparison,Gibson can only efficiently assemble up to 5 DNA fragments,and the accuracy drops sharply as the fragments number further increases.The 7-segments assembly accuracy of Gibson has fallen to 30%,but more than seven high GC DNA fragments assembly cannot be achieved.This indicates ExoCET DNA assembly technology has significant advantages in the assembly of high GC content DNA fragments.Overexpress genes related to the rate-limiting step of natural product biosynthesis is an effective way to increase natural products' yield.The multioperon gene cluster refactor strategy proposed in this study aims to solve the insufficient expression level of heterologous genes casing by the incompatibility of transcriptional regulatory elements from distinct species.Take advantage of the high GC content DNA assembly ability of ExoCET;this strategy follows the principle of the gene overexpression driven by constitutive promoters,significantly improve target compounds' production by increasing expression efficiency of all the natural product biosynthesis genes through refactoring the operon structure of the entire biosynthetic gene cluster with several constitutive promoters.The above results were published as the co-first author(1/2)in ACS Synthetic Biology in 2019.Based on the work above,we plan to modify the spinosad artificial gene cluster for heterologous expression of butenyl-spinosyn and 3'-O-demethyl-spinosyn.However,commercial site-directed mutagenesis kits rely on PCR such as Agilent QuikChange or Takara MutanBest are limited by the effective PCR amplification length and GC content of the template,which cannot be used for site-directed mutation of large high-GC content biosynthetic gene clusters.Bioinformatics analysis shows,all 49 commercial IIs endonuclease sites already exist in the spinosad gene cluster.Golden Gate technology relies on IIs restriction endonuclease outside cleavage cannot be used for seamless modified spinosad gene cluster.In the spinosad gene cluster,the functionally similar domains are highly homologous,resulting in 68 pairs of direct repeats longer than 35 bp within PKS genes.The Red recombination system can mediate efficient recombination between direct repeats,and the counter selection system cannot exclude a large number of non-specific recombination backgrounds,resulting in the seamless site-direct modification technology based on the Red recombination system and ccdB counter selection cannot be used directly to manipulate the PKS gene.Therefore,modify the artificial spinosyn gene cluster in need of novel gene editing technology.This study developed Red Ex technology by combining DNA recombineering mediated LCHR(Linear-circle homologous recombination),ccdB counterselection,and exonuclease mediated In vitro annealing.RedEx rationally coordinates the flexibility of homologous recombination incorporated with complex DNA sequence compatibility of In vitro annealing.It is feasible to edit any site in large complex natural product gene clusters with seamless insertion,deletion,replacement,and point mutations regardless of any repeated sequences.Employed the RedEx technology,we seamlessly edited the polyketide synthase gene of artificial spinosad gene cluster by inserted the 1b module derived from busA gene of butenyl-spinosyn gene cluster into the precise site of spnA gene,resulting in the artificial butenyl-spinosyn gene cluster.The Streptomyces albus J1074 strains integrated with the artificial butenyl-spinosyn gene cluster produced butenyl-spinosyn,the activity-enhanced and spectrum-extended spinosad derivatives.We also seamlessly deleted the spnK gene encoding 3'-O-methyltransferase in the artificial spinosad gene cluster.The Streptomyces albus J1074 strains integrated this gene cluster produced spinosyn J and L,the spinetoram chemical synthesis's starting materials.The modularized and linear biosynthetic mechanism of polyketides has excellent reprogramming potential,making the reprogramming of polyketides biosynthetic pathways a practical approach to modify polyketides' structure.However,such a biosynthetic mechanism also makes the coding sequence of functionally identical domains among the polyketide modules highly homologous,making PKS genes difficult to engineer.The RedEx technology significantly decreased the operational difficulty of PKS genes,provides a new method for natural product synthetic biology research.This work was published in Nucleic Acids Research in 2019 as the co-first author(1/2).The ExoCET DNA assembly method,RedEx gene editing technology,and multioperon gene cluster refactor strategy developed in this research enriched the synthetic biology toolbox,simplified the construction and modification of natural product biosynthetic pathway,inspired synthetic biology with new methods and ideas,and promoted the high-efficiency biomanufacturing and directional structural transformation of natural products.Take advantage of these technologies.We constructed the spinosad biosynthetic pathway vector and heterologously expressed spinosad in Streptomyces.Designed and assembled the artificial spinosad gene cluster and expressed it in Streptomyces with high-efficiency,resulting in a tremendous improvement of spinosad production.Directional transformed spinosyn chemical structure through seamless editing of the artificial spinosad gene cluster by RedEx and established the high-efficiency Streptomyces heterologous expression platforms of activity-enhanced spinosyn derivatives.The high-efficiency heterologous expression strains of spinosyns constructed in this research provides excellent initial strains for traditional mutagenesis breeding and promote the study of synthetic biology strategies for derivatization or high-efficiency bio-manufacturing of spinosyns.