Co-culture engineering is an emerging approach for microbial biosynthesis of a variety of biochemicals.In this study,E.coli-E.coli co-cultures were developed for heterologous biosynthesis of the natural product naring...Co-culture engineering is an emerging approach for microbial biosynthesis of a variety of biochemicals.In this study,E.coli-E.coli co-cultures were developed for heterologous biosynthesis of the natural product naringenin.The co-cultures were composed of two independent E.coli strains dedicated to functional expression of different portions of the biosynthetic pathway,respectively.The co-culture biosynthesis was optimized by investigating the effect of carbon source,E.coli strain selection,timing of IPTG induction and the inoculation ratio between the co-culture strains.Compared with the monoculture strategy,the utilization of the designed co-cultures significantly improved the naringenin production,largely due to the reduction of metabolic stress,employment of proper hosts for improving pathway enzyme activities,and flexible adjustment of the relative biosynthetic strength between the coculture strains.The findings of this study extend the applicability of co-culture engineering in complex natural product biosynthesis.展开更多
Background:Heterologous synthesis presents a promising new approach for accessing the active ingredients of traditional Chinese medicine(TCM),contributing to the conservation of natural medicinal resources.Compound Da...Background:Heterologous synthesis presents a promising new approach for accessing the active ingredients of traditional Chinese medicine(TCM),contributing to the conservation of natural medicinal resources.Compound Danshen preparation,a widely used TCM formulation,is designed to treat coronary heart disease and angina pectoris.It is primarily composed of Salviae Miltiorrhizae Radix et Rhizoma(Danshen),Notoginseng Radix et Rhizoma(Sanqi),and borneol(Bingpian).Danshen primarily yields tanshinones and phenolic acids,while Sanqi produces notoginsenosides.Borneol serves as an auxiliary agent to promote mental clarity,dissipate heat,and relieve pain.Objectives:The objective is to employ heterologous synthesis in a single yeast strain to produce the active ingredients of Compound Danshen preparation,and these include notoginsenosides,tanshinones,and borneol.Methods:Firstly,the“plug-and-play”terpene synthase screening framework in Saccharomyces cerevisiae was used for the identification of a novel,highly efficient bornyl diphosphate synthase.Furthermore,by leveraging the Compound Danshen preparation as a basis to concurrently reconstruct the biosynthetic pathways for the notoginsenoside precursor protopanaxadiol(a triterpene),the tanshinone precursor miltiradiene(a diterpene),and borneol(a monoterpene)within a single yeast strain.Results:This engineered strain,termed Compound Danshen Yeast 1.0,successfully produced protopanaxadiol at 62.34 mg/L,miltiradiene at 15.38 mg/L,and borneol at 2.54 mg/L in shake-flask fermentation.Conclusions:This research signifies the inaugural cross-species andmulticomponent synthetic biology endeavor that enables the synthesis of active ingredients in engineered cells,setting the stage for the industrial manufacture of TCM compounds.展开更多
Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces s...Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.展开更多
Natural products have had a major impact upon quality of life,with antibiotics as a classic example of having a transformative impact upon human health.In this contribution,we will highlight both historic and emerging...Natural products have had a major impact upon quality of life,with antibiotics as a classic example of having a transformative impact upon human health.In this contribution,we will highlight both historic and emerging methods of natural product bio-manufacturing.Traditional methods of natural product production relied upon native cellular host systems.In this context,pragmatic and effective methodologies were established to enable widespread access to natural products.In reviewing such strategies,we will also highlight the development of heterologous natural product biosynthesis,which relies instead on a surrogate host system theoretically capable of advanced production potential.In comparing native and heterologous systems,we will comment on the base organisms used for natural product biosynthesis and how the properties of such cellular hosts dictate scaled engineering practices to facilitate compound distribution.In concluding the article,we will examine novel efforts in production practices that entirely eliminate the constraints of cellular production hosts.That is,cell free production efforts will be introduced and reviewed for the purpose of complex natural product biosynthesis.Included in this final analysis will be research efforts made on our part to test the cell free biosynthesis of the complex polyketide antibiotic natural product erythromycin.展开更多
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
Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the1970 s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in ...Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the1970 s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials.But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxn D. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.展开更多
Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of th...Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.展开更多
基金The authors gratefully acknowledge Professor Matthews Koffas(Rensselaer Polytechnic Institute)and Professor Gregory Stephanopoulos(Massachusetts Institute of Technology)for the generous gift of plasmids pOM-PhCHS-MsCHI,pCDF-trc-RgTAL^syn-Pc4CL^syn and pCA1 in support of this study.This work is supported by startup research funds from Rutgers,The State University of New Jersey.Zhenghong Li is grateful for the Ph.D.fellowship from the China Scholarship Council.
文摘Co-culture engineering is an emerging approach for microbial biosynthesis of a variety of biochemicals.In this study,E.coli-E.coli co-cultures were developed for heterologous biosynthesis of the natural product naringenin.The co-cultures were composed of two independent E.coli strains dedicated to functional expression of different portions of the biosynthetic pathway,respectively.The co-culture biosynthesis was optimized by investigating the effect of carbon source,E.coli strain selection,timing of IPTG induction and the inoculation ratio between the co-culture strains.Compared with the monoculture strategy,the utilization of the designed co-cultures significantly improved the naringenin production,largely due to the reduction of metabolic stress,employment of proper hosts for improving pathway enzyme activities,and flexible adjustment of the relative biosynthetic strength between the coculture strains.The findings of this study extend the applicability of co-culture engineering in complex natural product biosynthesis.
基金supported by National Key Research and Development Program of China(2020YFA0908000)Prof.Zhubo Dai was supported by Youth Innovation Promotion Association of CAS(2015138)+1 种基金Haihe Laboratory Disruptive Innovation Talent Program(22HHSWSS00026)supported by the Key Project at Central Government Level:The ability establishment of sustainable use for valuable Chinese medicine resources(2060302)。
文摘Background:Heterologous synthesis presents a promising new approach for accessing the active ingredients of traditional Chinese medicine(TCM),contributing to the conservation of natural medicinal resources.Compound Danshen preparation,a widely used TCM formulation,is designed to treat coronary heart disease and angina pectoris.It is primarily composed of Salviae Miltiorrhizae Radix et Rhizoma(Danshen),Notoginseng Radix et Rhizoma(Sanqi),and borneol(Bingpian).Danshen primarily yields tanshinones and phenolic acids,while Sanqi produces notoginsenosides.Borneol serves as an auxiliary agent to promote mental clarity,dissipate heat,and relieve pain.Objectives:The objective is to employ heterologous synthesis in a single yeast strain to produce the active ingredients of Compound Danshen preparation,and these include notoginsenosides,tanshinones,and borneol.Methods:Firstly,the“plug-and-play”terpene synthase screening framework in Saccharomyces cerevisiae was used for the identification of a novel,highly efficient bornyl diphosphate synthase.Furthermore,by leveraging the Compound Danshen preparation as a basis to concurrently reconstruct the biosynthetic pathways for the notoginsenoside precursor protopanaxadiol(a triterpene),the tanshinone precursor miltiradiene(a diterpene),and borneol(a monoterpene)within a single yeast strain.Results:This engineered strain,termed Compound Danshen Yeast 1.0,successfully produced protopanaxadiol at 62.34 mg/L,miltiradiene at 15.38 mg/L,and borneol at 2.54 mg/L in shake-flask fermentation.Conclusions:This research signifies the inaugural cross-species andmulticomponent synthetic biology endeavor that enables the synthesis of active ingredients in engineered cells,setting the stage for the industrial manufacture of TCM compounds.
基金supported in part by the National Natural Science Foundation of China (31170037)Ministry of Science and Technology of China (2013CB734003)the China Postdoctoral Science Foundation (2013M530755)
文摘Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.
基金The authors recognize support from the University at Buffalo Blue Sky Initiative for funding related to natural product heterologous biosynthesis and the NIH(AI126367).
文摘Natural products have had a major impact upon quality of life,with antibiotics as a classic example of having a transformative impact upon human health.In this contribution,we will highlight both historic and emerging methods of natural product bio-manufacturing.Traditional methods of natural product production relied upon native cellular host systems.In this context,pragmatic and effective methodologies were established to enable widespread access to natural products.In reviewing such strategies,we will also highlight the development of heterologous natural product biosynthesis,which relies instead on a surrogate host system theoretically capable of advanced production potential.In comparing native and heterologous systems,we will comment on the base organisms used for natural product biosynthesis and how the properties of such cellular hosts dictate scaled engineering practices to facilitate compound distribution.In concluding the article,we will examine novel efforts in production practices that entirely eliminate the constraints of cellular production hosts.That is,cell free production efforts will be introduced and reviewed for the purpose of complex natural product biosynthesis.Included in this final analysis will be research efforts made on our part to test the cell free biosynthesis of the complex polyketide antibiotic natural product erythromycin.
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supported by the National Natural Science Foundation of China (81621064, 81603006, 81402836 and 31170042)the National Mega-Project for Innovative Drugs (2015ZX09102007016 and 2017ZX09101003-006-011)the CAMS Initiative for Innovative Medicine (2016-I2M-3–012)
文摘Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the1970 s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials.But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxn D. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.
文摘Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.
