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
The title compound(E)-2-(2-(4,8,8-trimethyldecahydro-1,4-methanoazulen-9-ylidene)ethyl)benzo[d]isothiazol-3(2H)-one(Ic)was synthesized from longifolene and 1,2-benzoisothiazolinone(short as BIT)through Prins,halogenat...The title compound(E)-2-(2-(4,8,8-trimethyldecahydro-1,4-methanoazulen-9-ylidene)ethyl)benzo[d]isothiazol-3(2H)-one(Ic)was synthesized from longifolene and 1,2-benzoisothiazolinone(short as BIT)through Prins,halogenation and nitro-alkylation reaction and structurally identified by means of HRMS,IR,1H-NMR,13C-NMR and single-crystal X-ray diffraction.The crystal of compound Ic is of bi-molecular structure and belongs to orthorhombic system,P212121 space group with a=7.5715(7),b=16.8824(9),c=31.1926(14)?,V=3987.2(5)A^3,Mr=367.53,Dc=1.225 mg/m^3,Z=8,μ=0.174 mm^–1 and F(000)=1584.A total of 17045 reflections were collected,of which 7306 were unique(Rint=0.0566).The structure was refined to R=0.0967 and wR=0.1998 for 7306 observed reflections withⅠ>2σ(Ⅰ).1D chain along the a-axis is formed by two types ofπ-πinteractions between benzene rings from adjacent molecules.Especially,compound Ic shows improved solubility in nonpolar organic solvents and higher antimicrobial activity than longifolene and BIT against bacteria and fungi.The minimum inhibition concentration(MIC)of Ic against two Gram-positive bacteria(S.aureus and B.subtili),two Gram-negative bacteria(E.coli and K.pneumoniae)and three fungi(C.albicans,C.tropicalis and A.niger)are 0.242,0.242,15.6,15.6,1.95,1.95 and 1.95μg/mL,respectively.展开更多
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.
基金Supported by the Natural Science Foundation of Guangxi Zhuang Autonomous Region(No.2017GXNSFAA198027)Special Fund for Innovation-driven Devlopment of Guangxi(GUIKE AA17204087-21)National Training Program of Innovation and Entrepreneurship for Undergraduates(201510608042)。
文摘The title compound(E)-2-(2-(4,8,8-trimethyldecahydro-1,4-methanoazulen-9-ylidene)ethyl)benzo[d]isothiazol-3(2H)-one(Ic)was synthesized from longifolene and 1,2-benzoisothiazolinone(short as BIT)through Prins,halogenation and nitro-alkylation reaction and structurally identified by means of HRMS,IR,1H-NMR,13C-NMR and single-crystal X-ray diffraction.The crystal of compound Ic is of bi-molecular structure and belongs to orthorhombic system,P212121 space group with a=7.5715(7),b=16.8824(9),c=31.1926(14)?,V=3987.2(5)A^3,Mr=367.53,Dc=1.225 mg/m^3,Z=8,μ=0.174 mm^–1 and F(000)=1584.A total of 17045 reflections were collected,of which 7306 were unique(Rint=0.0566).The structure was refined to R=0.0967 and wR=0.1998 for 7306 observed reflections withⅠ>2σ(Ⅰ).1D chain along the a-axis is formed by two types ofπ-πinteractions between benzene rings from adjacent molecules.Especially,compound Ic shows improved solubility in nonpolar organic solvents and higher antimicrobial activity than longifolene and BIT against bacteria and fungi.The minimum inhibition concentration(MIC)of Ic against two Gram-positive bacteria(S.aureus and B.subtili),two Gram-negative bacteria(E.coli and K.pneumoniae)and three fungi(C.albicans,C.tropicalis and A.niger)are 0.242,0.242,15.6,15.6,1.95,1.95 and 1.95μg/mL,respectively.
