Spinosyns,including spinosad and spinetoram,act on the insect central nervous system,gradually paralyzing or destroying the target insect.Spinosad resistance is associated with loss-of-function mutations in the nicoti...Spinosyns,including spinosad and spinetoram,act on the insect central nervous system,gradually paralyzing or destroying the target insect.Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor(nAChR)α6 subunit in a number of agricultural pests.Using gene editing,nAChRα6 has been verified as a target for spinosyns in five insect species.Recently,a point mutation(G275E)in exon 9 of nAChRα6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta.To date,no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests.In this study,the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene-editing technology.Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain.The results of bioassays showed that the 275E strain was highly resistant to spinosad(230-fold)and spinetoram(792-fold)compared to the unedited background strain,directly confirming that the G275E mutation of the nAChRα6 subunit confers high levels of spinosyn resistance in S.exigua.Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive.This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChRα6 of S.exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.展开更多
Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our in...Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16,located upstream of spinosad biosynthetic genes,in spinosad biosynthesis.Through reverse transcription PCR(RT-PCR)and 5′-rapid amplification of cDNA ends(5′-Race),we unveiled that the spinosad biosynthetic gene cluster(BGC)contains six transcription units and seven promoters.Electrophoretic mobility shift assays(EMSAs)demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC,indicating its involvement in regulating spinosad biosynthesis.Notably,deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L,accompanied by decreased transcription levels of spinosad biosynthetic genes,confirming its positive regulatory function.Additionally,isothermal titration calorimetry(ITC)and EMSA confirmed that spinosyn A,the main product of the spinosad BGC,served as an effector of ORF-L16.Specifically,it decreased the binding affinity between ORF-L16 and spinosad BGC promoters,thus exerting negative feedback regulation on spinosad biosynthesis.This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S.spinosa.展开更多
Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-...Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain,and poor adaptability of the heterologous strain to the production of spinosyn.In this study,we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad(spinosyn A and spinosyn D)based on our previous work.We systematically investigated the result of overexpressing polyketide synthase genes(spnA,B,C,D,E)using a constitutive promoter on the spinosad titer in S.albus J1074.The supply of polyketide synthase precursors was then increased to further improve spinosad production.Finally,increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of~70 mg/L,which is the highest titer of spinosad achieved in heterologous Streptomyces species.This research provides useful strategies for efficient heterologous production of natural products.展开更多
基金funded by the National Natural Science Foundation of China(32001941 and 31972303)the China Postdoctoral Science Foundation(2020M683586)the Research Fund for the Doctoral Program of Northwest A&F University(Z1090219195).
文摘Spinosyns,including spinosad and spinetoram,act on the insect central nervous system,gradually paralyzing or destroying the target insect.Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor(nAChR)α6 subunit in a number of agricultural pests.Using gene editing,nAChRα6 has been verified as a target for spinosyns in five insect species.Recently,a point mutation(G275E)in exon 9 of nAChRα6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta.To date,no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests.In this study,the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)gene-editing technology.Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain.The results of bioassays showed that the 275E strain was highly resistant to spinosad(230-fold)and spinetoram(792-fold)compared to the unedited background strain,directly confirming that the G275E mutation of the nAChRα6 subunit confers high levels of spinosyn resistance in S.exigua.Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive.This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChRα6 of S.exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.
基金supported by the National Key R&D Program of China(grant number 2018YFA0900400)the National Natural Science Foundation of China(grant number 32100053)the Young Elite Scientists Sponsorship Program by China Association for Science and Technology(grant number YESS20210068).
文摘Spinosad,a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa,has significant market potential.Despite its effectiveness,the regulatory mechanisms of spinosad biosynthesis remain unclear.Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16,located upstream of spinosad biosynthetic genes,in spinosad biosynthesis.Through reverse transcription PCR(RT-PCR)and 5′-rapid amplification of cDNA ends(5′-Race),we unveiled that the spinosad biosynthetic gene cluster(BGC)contains six transcription units and seven promoters.Electrophoretic mobility shift assays(EMSAs)demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC,indicating its involvement in regulating spinosad biosynthesis.Notably,deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L,accompanied by decreased transcription levels of spinosad biosynthetic genes,confirming its positive regulatory function.Additionally,isothermal titration calorimetry(ITC)and EMSA confirmed that spinosyn A,the main product of the spinosad BGC,served as an effector of ORF-L16.Specifically,it decreased the binding affinity between ORF-L16 and spinosad BGC promoters,thus exerting negative feedback regulation on spinosad biosynthesis.This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S.spinosa.
基金This work was supported by the National Key R&D Program of China[grant number 2018YFA0900400]the National Natural Science Foundation of China[grant number 31670090],and J1 Biotech Co.,Ltd.
文摘Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain,and poor adaptability of the heterologous strain to the production of spinosyn.In this study,we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad(spinosyn A and spinosyn D)based on our previous work.We systematically investigated the result of overexpressing polyketide synthase genes(spnA,B,C,D,E)using a constitutive promoter on the spinosad titer in S.albus J1074.The supply of polyketide synthase precursors was then increased to further improve spinosad production.Finally,increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of~70 mg/L,which is the highest titer of spinosad achieved in heterologous Streptomyces species.This research provides useful strategies for efficient heterologous production of natural products.
