Objective Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy,however,their extraction remains costly.Given that levopimaradiene is a key biosynthetic precursor to ginkgolides,its high-yie...Objective Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy,however,their extraction remains costly.Given that levopimaradiene is a key biosynthetic precursor to ginkgolides,its high-yield production via heterologous gene expression therefore establishes a critical foundation for scaling up their manufacture.This study primarily aims to enhance the yield of levopimaradiene in Escherichia coli(LB medium)by remodeling the unnatural isopentenol utilization pathway(IUP).Methods Plasmid construction was driven by the mechanism of homologous recombination,which utilizes recombinase to facilitate the ligation process.The expression of kinase was carried out using type 7(T7)promoter and isopropylβ-D-1-thiogalactopyranoside(IPTG)as the inducer.The quantification of levopimaradiene produced by E.coli was determined by comparison with a standard curve that we constructed.Results Combining the selection of kinases,ribosome-binding site(RBS)screening,protein directed evolution and optimization of fermentation parameters,the production of levopimaradiene in E.coli was ultimately enhanced to 2691.3 mg/L,surpassing the highest reported titers of levopimaradiene with 6-fold in E.coli to date.Additionally,the engineered E.coli was designed to collaborate with farnesyl pyrophosphate(FPP)synthase and geranylfarnesyl pyrophosphate(GFPP)synthase to efficiently produce FPP and GFPP for sesquiterpene and sesterterpene synthesis.Conclusion Our work showcases a combinatorial engineering strategy that employs an IUP-enhanced E.coli chassis for the microbial production of levopimaradiene,as well as other natural terpenoids.展开更多
Longifolene(C15H24)is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications.Traditional extraction methods from plants suffer from low titer and lengthy production...Longifolene(C15H24)is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications.Traditional extraction methods from plants suffer from low titer and lengthy production cycles,while chemical synthesis is hampered by the compound’s complex structure,leading to high costs and insufficient market supply.This study aimed to develop a microbial cell factory for enhanced longifolene production.The strategy involved integrating longifolene synthase from Pinus sylvestris(PsTPS)into Yarrowia lipolytica and employing multiple metabolic engineering approaches.Initially,key genes in the mevalonate(MVA)pathway were overexpressed to enhance longifolene precursor availability for longifolene biosynthesis.Subsequently,protein engineering techniques were applied to optimize PsTPS(tPsTPS)for improved catalytic efficiency.Furthermore,co-expression of molecular chaperones was implemented to enhance the synthesis and secretion of PsTPS.The introduction of the isopentenol utilization pathway(IUP)further augmented the supply of C5 substrate.By optimizing the culture conditions,including a reduction in culture temperature,the efflux of longifolene was increased,and the dissolved oxygen levels were enhanced to promote the growth of the strain.These collective efforts resulted culminated in the engineered strain Z03 achieving a noteworthy production level of 34.67 mg/L of longifolene in shake flasks.This study not only demonstrates the feasibility of enhancing sesquiterpene production in Y.lipolytica but also highlights the potential of microbial platforms in meeting industrial demands for complex natural products.展开更多
基金co-financed by the Young Scientists Fund of the National Natural Science Foundation of China(NSFC)(No.82003608)the Key Project of NSFC(No.81991524)+3 种基金the Innovation Projects of State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture(No.NZYSKL240109)the Interdisciplinary Project of Nanjing University of Chinese Medicine(No.ZYXJC2024-003 and No.JC202402)the Youth Project of Jiangsu Commission of Health(No.QN202405)the Jiangsu Annual Basic Science Institutions(No.25KJB350006).
文摘Objective Plant-derived terpenoid ginkgolides exhibit significant pharmacological efficacy,however,their extraction remains costly.Given that levopimaradiene is a key biosynthetic precursor to ginkgolides,its high-yield production via heterologous gene expression therefore establishes a critical foundation for scaling up their manufacture.This study primarily aims to enhance the yield of levopimaradiene in Escherichia coli(LB medium)by remodeling the unnatural isopentenol utilization pathway(IUP).Methods Plasmid construction was driven by the mechanism of homologous recombination,which utilizes recombinase to facilitate the ligation process.The expression of kinase was carried out using type 7(T7)promoter and isopropylβ-D-1-thiogalactopyranoside(IPTG)as the inducer.The quantification of levopimaradiene produced by E.coli was determined by comparison with a standard curve that we constructed.Results Combining the selection of kinases,ribosome-binding site(RBS)screening,protein directed evolution and optimization of fermentation parameters,the production of levopimaradiene in E.coli was ultimately enhanced to 2691.3 mg/L,surpassing the highest reported titers of levopimaradiene with 6-fold in E.coli to date.Additionally,the engineered E.coli was designed to collaborate with farnesyl pyrophosphate(FPP)synthase and geranylfarnesyl pyrophosphate(GFPP)synthase to efficiently produce FPP and GFPP for sesquiterpene and sesterterpene synthesis.Conclusion Our work showcases a combinatorial engineering strategy that employs an IUP-enhanced E.coli chassis for the microbial production of levopimaradiene,as well as other natural terpenoids.
基金supported by the National Natural Science Foundation of China(No.42206137,32270118)the Science and Technology Planning Project of Guangzhou(2024A04J4129)the Natural Science Foundation of Guangdong Province(No.2019B1515120062).
文摘Longifolene(C15H24)is a tricyclic sesquiterpene widely utilized in the cosmetics and fragrances due to its versatile applications.Traditional extraction methods from plants suffer from low titer and lengthy production cycles,while chemical synthesis is hampered by the compound’s complex structure,leading to high costs and insufficient market supply.This study aimed to develop a microbial cell factory for enhanced longifolene production.The strategy involved integrating longifolene synthase from Pinus sylvestris(PsTPS)into Yarrowia lipolytica and employing multiple metabolic engineering approaches.Initially,key genes in the mevalonate(MVA)pathway were overexpressed to enhance longifolene precursor availability for longifolene biosynthesis.Subsequently,protein engineering techniques were applied to optimize PsTPS(tPsTPS)for improved catalytic efficiency.Furthermore,co-expression of molecular chaperones was implemented to enhance the synthesis and secretion of PsTPS.The introduction of the isopentenol utilization pathway(IUP)further augmented the supply of C5 substrate.By optimizing the culture conditions,including a reduction in culture temperature,the efflux of longifolene was increased,and the dissolved oxygen levels were enhanced to promote the growth of the strain.These collective efforts resulted culminated in the engineered strain Z03 achieving a noteworthy production level of 34.67 mg/L of longifolene in shake flasks.This study not only demonstrates the feasibility of enhancing sesquiterpene production in Y.lipolytica but also highlights the potential of microbial platforms in meeting industrial demands for complex natural products.
