An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes.In this study,firstly,a genetic transformation system...An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes.In this study,firstly,a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527,a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi.Some experimental parameters involved in this procedure were optimized,including the conjugative media,ratio of donor to recipient,heat shock temperature,and incubation time of mixed culture.Under the optimal conditions,a maximal conjugation frequency of 3.05^10-5 per recipie nt was obtai ned.Subseque ntly,based on the above developed and optimized tran sformati on system,the synthetic promoters SPL-21 and SPL-57,a native promoter potrB,and a constitutive promoter permE commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S.rimosus M527.Among the four tested promoters,SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in p-glucuronidase(GUS)activity compared with the control promoter permE.Promoter SPL-57 showed activity comparable to that of permE.Promoter potrB,which showed the lowest activity,showed a 50%decrease in GUS activity compared with the control permE.The transformation system developed in this study and the tested promotors provide a basis for the further modification of S.rimosus M527.展开更多
otrA resembles elongation factor G （EF-G） and is considered to be an oxytetracycline （OTC）-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and ot...otrA resembles elongation factor G （EF-G） and is considered to be an oxytetracycline （OTC）-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coeficolor M145 by intergenedc conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M14-~OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin （Act） production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction （RT-PCR） analysis revealed that the transcrip- tional level of pathway-specific regulatory gene acUl-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.展开更多
Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics,enzyme inhibi-tors,and other small molecules with potential physiological a...Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics,enzyme inhibi-tors,and other small molecules with potential physiological activity(Niu et al.,2016;Song et al.,2019;Yin et al.,2019).Secondary metabolites are produced by a multi-step reaction of a primary metabolite as a precursor(Liu et al.,2013;Li et al.,2021).Therefore,it is of great research significance to increase the overall synthesis level of antibiotics by increasing the amount of synthesis of precursors.展开更多
WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 ...WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.展开更多
基金Project supported by the National Natural Science Foundation of China(Nos.31772213 and 31972320)the Excellent Youth Fund of Zhejiang Province,China(No.LR17C140002)
文摘An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes.In this study,firstly,a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527,a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi.Some experimental parameters involved in this procedure were optimized,including the conjugative media,ratio of donor to recipient,heat shock temperature,and incubation time of mixed culture.Under the optimal conditions,a maximal conjugation frequency of 3.05^10-5 per recipie nt was obtai ned.Subseque ntly,based on the above developed and optimized tran sformati on system,the synthetic promoters SPL-21 and SPL-57,a native promoter potrB,and a constitutive promoter permE commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S.rimosus M527.Among the four tested promoters,SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in p-glucuronidase(GUS)activity compared with the control promoter permE.Promoter SPL-57 showed activity comparable to that of permE.Promoter potrB,which showed the lowest activity,showed a 50%decrease in GUS activity compared with the control permE.The transformation system developed in this study and the tested promotors provide a basis for the further modification of S.rimosus M527.
基金Project supported by the National Natural Science Foundation of China(No.31772213)the Excellent Youth Fund of Zhejiang Province,China(No.LR17C140002)
文摘otrA resembles elongation factor G （EF-G） and is considered to be an oxytetracycline （OTC）-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coeficolor M145 by intergenedc conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M14-~OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin （Act） production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction （RT-PCR） analysis revealed that the transcrip- tional level of pathway-specific regulatory gene acUl-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.
基金supported by the Excellent Youth Fund of Zhejiang Province,China(No.LR17C140002)。
文摘Streptomyces are famous for their ability to synthesize a large number of bioactive compounds as secondary metabolites containing antibiotics,enzyme inhibi-tors,and other small molecules with potential physiological activity(Niu et al.,2016;Song et al.,2019;Yin et al.,2019).Secondary metabolites are produced by a multi-step reaction of a primary metabolite as a precursor(Liu et al.,2013;Li et al.,2021).Therefore,it is of great research significance to increase the overall synthesis level of antibiotics by increasing the amount of synthesis of precursors.
基金supported by the BBSRC(MIBTP studentship to Daniel J.Leng)the Monash Warwick Alliance(Seed Fund Award to Manuela Tosin and Max J.Cryle)+6 种基金the University of Warwick(Career Support Award to Manuela Tosin)Monash University,EMBL Australia,the Australian Research Council(Discovery Project DP210101752 to Max J.Cryle)the National Health and Medical Research Council(APP1140619 to Max J.Cryle)the Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science(CE200100012)funded by the Australian Governmentfunded by the National Natural Science Foundation of China(82104044 to Songya Zhang)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-PTJS-003-07)。
文摘WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.
