Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic...Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic linkage.Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a bioassay.Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of tunicamycins.Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond formation.Hence,a seven-step novel pathway is proposed for tunicamycin biosynthesis.Moreover,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827.These data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.展开更多
Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas.To improve the fermentation titer of tylosin in the hyperproduci...Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas.To improve the fermentation titer of tylosin in the hyperproducing Streptomyces xinghaiensis strain TL01,we sequenced its whole genome and identified the biosynthetic gene cluster therein.Overexpression of the tylosin efflux gene tlrC,the cluster-situated S-adenosyl methionine(SAM)synthetase gene metK_(cs),the SAM biosynthetic genes adoK_(cs)-metFcs,or the pathway-specific activator gene tylR enhanced tylosin production by 18%,12%,11%,and 11%in the respective engineered strains TLPH08-2,TLPH09,TLPH10,and TLPH12.Co-overexpression of metK_(cs)and adoK_(cs)-metFcs as two transcripts increased tylosin production by 22%in the resultant strain TLPH11 compared to that in TL01.Furthermore,combinational overexpression of tlrC,metK_(cs),adoK_(cs)-metFcs,and tylR as four transcripts increased tylosin production by 23%(10.93g/L)in the resultant strain TLPH17 compared to that in TL01.However,a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2.Transcription analyses indicated that the expression of tlrC and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11.Based on this observation,the five genes were rearranged into one or two operons to coordinate their overexpression,yielding two engineered strains TLPH23 and TLPH24,and leading to further enhancement of tylosin production over TLPH17.In particular,the production of TLPH23 reached 11.35 g/L.These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.展开更多
A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which ...A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.展开更多
The maremycin biosynthetic gene cluster has been identified in Streptomyces sp. B9173. Comparative metabolic profiling with knockout mutant strains led to the identification of new products correlated to the maremycin...The maremycin biosynthetic gene cluster has been identified in Streptomyces sp. B9173. Comparative metabolic profiling with knockout mutant strains led to the identification of new products correlated to the maremycin biosynthesis, in particular the"demethyl"-maremycins with an unexpected D-tryptophan unit. A biosynthetic pathway for the maremycins is proposed and plausible reasoning for tryptophan epimerization in the demethylmaremycin biosynthesis is also provided.展开更多
Flavin-dependent halogenases (FDHs) are well known to introduce carbon halide bonds (mainly C–Cl and C–Br) into natural products with the assistance of a partner protein flavin reductase to generate reduced flavin (...Flavin-dependent halogenases (FDHs) are well known to introduce carbon halide bonds (mainly C–Cl and C–Br) into natural products with the assistance of a partner protein flavin reductase to generate reduced flavin (FADH_(2)or FMNH_(2)).Compared with the common chloride-and bromide-containing natural products (approximately 5,000 compounds),iodinated natural products(approximately 100 compounds) are very limited.Specific iodinases have also rarely been identified in nature to date.This study discovered a novel relationship between iodination and flavin reductases for the first time.Through mechanistic studies,it was identified that peroxide (H_(2)O_(2)) was released from the uncoupling reaction of flavin reductases and then reacted with iodide ions(I^(-)) to produce hypoiodous acid (IOH) for the final iodination.Furthermore,this study also unintentionally verified that the recently reported flavin-dependent iodinase Vir X1 from the marine virus and its two homologs (MBG and NCV) did not catalyze iodination in the in vitro biochemical system but likely belonged to a new phylogenetic clade in the tryptophan halogenase superfamily.As a consequence,actual flavin-dependent iodinases in nature remain to be discovered by the scientific community in the future.展开更多
基金This work was supported by the National Basic Research Program(973 Program)the National Programs for High Technology Research Development Program(863 Program)from the Ministry of Science and Technology,the National Science Foundation of China,the Ministry of Education,the Science and Technology Commission of Shanghai Municipality,and Shanghai Leading Academic Discipline Project B203.
文摘Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic linkage.Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a bioassay.Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of tunicamycins.Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond formation.Hence,a seven-step novel pathway is proposed for tunicamycin biosynthesis.Moreover,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827.These data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.
基金the National Key Research and Development Program of China(grant no.2022YFC210540303)the“Major Project”of Haihe Laboratory of Synthetic Biology(22HHSWSS00001).
文摘Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas.To improve the fermentation titer of tylosin in the hyperproducing Streptomyces xinghaiensis strain TL01,we sequenced its whole genome and identified the biosynthetic gene cluster therein.Overexpression of the tylosin efflux gene tlrC,the cluster-situated S-adenosyl methionine(SAM)synthetase gene metK_(cs),the SAM biosynthetic genes adoK_(cs)-metFcs,or the pathway-specific activator gene tylR enhanced tylosin production by 18%,12%,11%,and 11%in the respective engineered strains TLPH08-2,TLPH09,TLPH10,and TLPH12.Co-overexpression of metK_(cs)and adoK_(cs)-metFcs as two transcripts increased tylosin production by 22%in the resultant strain TLPH11 compared to that in TL01.Furthermore,combinational overexpression of tlrC,metK_(cs),adoK_(cs)-metFcs,and tylR as four transcripts increased tylosin production by 23%(10.93g/L)in the resultant strain TLPH17 compared to that in TL01.However,a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2.Transcription analyses indicated that the expression of tlrC and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11.Based on this observation,the five genes were rearranged into one or two operons to coordinate their overexpression,yielding two engineered strains TLPH23 and TLPH24,and leading to further enhancement of tylosin production over TLPH17.In particular,the production of TLPH23 reached 11.35 g/L.These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.
基金supported in part by grants from the National Key Research and Development Program of China(2018YFA0901900)the National Natural Science Foundation of China(22137009)the China Postdoctoral Science Foundation(2020M671271).
文摘A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.
基金supported by the National Natural Science Foundation of China (NNSFC, 31425001 and 31070057 for SL)the Leopoldina Fellowship Program (German National Academy of Sciences Leopoldina, LPDS 2013-12 for NLB)
文摘The maremycin biosynthetic gene cluster has been identified in Streptomyces sp. B9173. Comparative metabolic profiling with knockout mutant strains led to the identification of new products correlated to the maremycin biosynthesis, in particular the"demethyl"-maremycins with an unexpected D-tryptophan unit. A biosynthetic pathway for the maremycins is proposed and plausible reasoning for tryptophan epimerization in the demethylmaremycin biosynthesis is also provided.
基金the National Natural Science Foundation of China(21632007,21661140002 for S.Lin81903525 for Y.Zhang)+1 种基金Research Fund for High-level Talents of Xinxiang Medical University(300-505272)Open Funding Project of State Key Laboratory of Microbial Metabolism,Shanghai Jiao Tong University(MMLKF2011)。
文摘Flavin-dependent halogenases (FDHs) are well known to introduce carbon halide bonds (mainly C–Cl and C–Br) into natural products with the assistance of a partner protein flavin reductase to generate reduced flavin (FADH_(2)or FMNH_(2)).Compared with the common chloride-and bromide-containing natural products (approximately 5,000 compounds),iodinated natural products(approximately 100 compounds) are very limited.Specific iodinases have also rarely been identified in nature to date.This study discovered a novel relationship between iodination and flavin reductases for the first time.Through mechanistic studies,it was identified that peroxide (H_(2)O_(2)) was released from the uncoupling reaction of flavin reductases and then reacted with iodide ions(I^(-)) to produce hypoiodous acid (IOH) for the final iodination.Furthermore,this study also unintentionally verified that the recently reported flavin-dependent iodinase Vir X1 from the marine virus and its two homologs (MBG and NCV) did not catalyze iodination in the in vitro biochemical system but likely belonged to a new phylogenetic clade in the tryptophan halogenase superfamily.As a consequence,actual flavin-dependent iodinases in nature remain to be discovered by the scientific community in the future.
