A halophilic bacterium,named Halomonas sp.NEC-1 was isolated from the Nyer Co Salt Lake on the Xizang Plateau,SW China.The strain exhibited a broad pH tolerance range of 5-11,distinguishing it from Halomonas alkaliphi...A halophilic bacterium,named Halomonas sp.NEC-1 was isolated from the Nyer Co Salt Lake on the Xizang Plateau,SW China.The strain exhibited a broad pH tolerance range of 5-11,distinguishing it from Halomonas alkaliphila despite a 99% genetic similarity.Optimal growth conditions,determined through orthogonal experiments,were found to be 37℃,100-g/L salinity,and an initial pH of 6,resulting in a maximum OD_(600) of 7.98±0.06.Halomonas sp.NEC-1 produced 545.43±25.10 mg/L of ectoine under optimal conditions of 75-g/L salinity,40-g/L sodium glutamate,and an initial pH of 6.This production increased to 1388.81±3.69 mg/L after five rounds of hypo-osmotic shocks.During the shocks,ectoine productivity remained stable at approximately 16.29±0.04 to 17.28±0.48 mg/(L·h),representing a 43.40%-52.11% increase compared to the rate without any shock(11.36±1.05 mg/(L·h)).Additionally,the expression of the ectABC gene cluster,related to ectoine synthesis,significantly increased following the shocks,enhancing ectoine production.The ectoine extract demonstrated notable protective effects on Escherichia coli and plasmid DNA.After 10 min of exposure at 60℃,the colony count of E.coli treated with ectoine extract increased by 342% compared to treatment with distilled water.Furthermore,the ectoine extract protected plasmid DNA from 2,2′-Azobis(2-methylpropionamidine)dihydrochloride-induced damage.This study highlights Halomonas sp.NEC-1 is a promising strain for ectoine production and underscores the potential of microbial resources in salt lakes from Xizang region.展开更多
Photoprotection effect of the hyaluronic acid and ectoine composition was studied by evaluated its effects on inhibiting oxidative stress and MMP-1 production,ROS scavenging ability and promoting collagen synthesis in...Photoprotection effect of the hyaluronic acid and ectoine composition was studied by evaluated its effects on inhibiting oxidative stress and MMP-1 production,ROS scavenging ability and promoting collagen synthesis in skin cells under blue light irradiation.The result show that the composition is non-toxic to keratinocytes within a concentration of 5%.And the sample can significantly inhibit the increase of reactive oxygen species(ROS)caused by blue light irradiation at a concentration of 2%,and can also repaire the damaged skin by scavenge oxygen free radicals.At the same time,the composition can inhibit the increase of MMP-1 caused by blue light irradiation,so as to have the effect of inhibiting the decomposition of collagen and promoting the secretion of Collagen I.展开更多
Previously,we have developed an Escherichia coli strain MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 in which the titer of ectoine reached 30.37 g/L with a yield of 0.13 g/g glucose after 36 h fed-batch fermentation.In t...Previously,we have developed an Escherichia coli strain MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 in which the titer of ectoine reached 30.37 g/L with a yield of 0.13 g/g glucose after 36 h fed-batch fermentation.In this study,this strain was further modified to improve the production of ectoine.Genes pflB encoding the pyruvate formatelyase,poxB encoding the pyruvate oxidase,adhE encoding the alcohol dehydrogenase,and aroG encoding the 3-deoxy-7-phosphoheptulonate syn-thase were deleted from MWZ003,resulting in the strain MWL007.Comparing with the control MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3,ectoine production in MWL007/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 increased 21%with a yield of 0.43 g/g glucose.The gene mscS encoding the small conductance mechanosensitive channel MscS was further deleted in MWL007,resulting in the strain MWL009.Comparing with the control MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3,ectoine production in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 increased 28%with a yield of 0.46 g/g glucose.After the fermentation conditions were optimized,ectoine production in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 further increased and the yield reached 0.63 g/g glucose.After 60 h fed-batch fermentation,the titer of ectoine in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 reached 34.27 g/L with the yield of 0.34 g/g glucose.These results indicate that ectoine production in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 can be improved by accumulation of the key precursors.展开更多
DNA scaffold that enhances the spatial proximity of enzymes and the local concentration of intermediates,is promising tools in optimizing heterologous metabolic pathways for target product biosynthesis.Here,we display...DNA scaffold that enhances the spatial proximity of enzymes and the local concentration of intermediates,is promising tools in optimizing heterologous metabolic pathways for target product biosynthesis.Here,we display the utility of a DNA scaffold system for the production of ectoine in E.coli MWZ003.Three fused enzymes EctA-ZFa,EctB-ZFb,and EctC-ZFc were firstly constructed by fusing enzymes of ectoine synthesis pathway with corresponding zinc finger domains.The copy number of the plasmid-expressing fusions was adapted by substitution of different replicons.Furthermore,a series of modifications were carried out on the DNA scaffold system through optimizing the spacer between enzyme binding sites,the binding direction of fusion enzymes,the repeating unit of DNA scaffolds,the stoichiometric ratio of enzyme binding sites,and the expression level of the rate-limiting enzyme.The optimized DNA scaffold system in the plasmid pFV30 involving use of pMB1 replicon,reverse binding,11-bp spacer,4 repeating units,stoichiometric ratio(1:2:2),and enhanced expres-sion of EctB-ZFb increased the ectoine titer and yield,respectively,to 22.79 g/L and 0.65 g/g glucose with increase by 92%compared with that of the control strain.The post-translational strategy based on DNA scaffold was efficient in promoting heterologous synthesis of ectoine,which could also be used in combination with other genetic engineering tools.展开更多
基金Supported by the Key Science and Technology Program of Xizang Autonomous Region(No.XZ202301ZY0012N)the Key Fisheries Resources and Environmental Survey Project in the Southwest Region(No.CJW2023034)the National Natural Science Foundation of China(No.42306106)。
文摘A halophilic bacterium,named Halomonas sp.NEC-1 was isolated from the Nyer Co Salt Lake on the Xizang Plateau,SW China.The strain exhibited a broad pH tolerance range of 5-11,distinguishing it from Halomonas alkaliphila despite a 99% genetic similarity.Optimal growth conditions,determined through orthogonal experiments,were found to be 37℃,100-g/L salinity,and an initial pH of 6,resulting in a maximum OD_(600) of 7.98±0.06.Halomonas sp.NEC-1 produced 545.43±25.10 mg/L of ectoine under optimal conditions of 75-g/L salinity,40-g/L sodium glutamate,and an initial pH of 6.This production increased to 1388.81±3.69 mg/L after five rounds of hypo-osmotic shocks.During the shocks,ectoine productivity remained stable at approximately 16.29±0.04 to 17.28±0.48 mg/(L·h),representing a 43.40%-52.11% increase compared to the rate without any shock(11.36±1.05 mg/(L·h)).Additionally,the expression of the ectABC gene cluster,related to ectoine synthesis,significantly increased following the shocks,enhancing ectoine production.The ectoine extract demonstrated notable protective effects on Escherichia coli and plasmid DNA.After 10 min of exposure at 60℃,the colony count of E.coli treated with ectoine extract increased by 342% compared to treatment with distilled water.Furthermore,the ectoine extract protected plasmid DNA from 2,2′-Azobis(2-methylpropionamidine)dihydrochloride-induced damage.This study highlights Halomonas sp.NEC-1 is a promising strain for ectoine production and underscores the potential of microbial resources in salt lakes from Xizang region.
文摘Photoprotection effect of the hyaluronic acid and ectoine composition was studied by evaluated its effects on inhibiting oxidative stress and MMP-1 production,ROS scavenging ability and promoting collagen synthesis in skin cells under blue light irradiation.The result show that the composition is non-toxic to keratinocytes within a concentration of 5%.And the sample can significantly inhibit the increase of reactive oxygen species(ROS)caused by blue light irradiation at a concentration of 2%,and can also repaire the damaged skin by scavenge oxygen free radicals.At the same time,the composition can inhibit the increase of MMP-1 caused by blue light irradiation,so as to have the effect of inhibiting the decomposition of collagen and promoting the secretion of Collagen I.
基金supported by the National Key Research and Development Program of China(2018YFA0900300).
文摘Previously,we have developed an Escherichia coli strain MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 in which the titer of ectoine reached 30.37 g/L with a yield of 0.13 g/g glucose after 36 h fed-batch fermentation.In this study,this strain was further modified to improve the production of ectoine.Genes pflB encoding the pyruvate formatelyase,poxB encoding the pyruvate oxidase,adhE encoding the alcohol dehydrogenase,and aroG encoding the 3-deoxy-7-phosphoheptulonate syn-thase were deleted from MWZ003,resulting in the strain MWL007.Comparing with the control MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3,ectoine production in MWL007/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 increased 21%with a yield of 0.43 g/g glucose.The gene mscS encoding the small conductance mechanosensitive channel MscS was further deleted in MWL007,resulting in the strain MWL009.Comparing with the control MWZ003/pFT28-ectABC-EclysC^(*)-aspDH-ppc3,ectoine production in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 increased 28%with a yield of 0.46 g/g glucose.After the fermentation conditions were optimized,ectoine production in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 further increased and the yield reached 0.63 g/g glucose.After 60 h fed-batch fermentation,the titer of ectoine in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 reached 34.27 g/L with the yield of 0.34 g/g glucose.These results indicate that ectoine production in MWL009/pFT28-ectABC-EclysC^(*)-aspDH-ppc3 can be improved by accumulation of the key precursors.
基金supported by the National Key Research and Development Program of China(2018YFA0900300).
文摘DNA scaffold that enhances the spatial proximity of enzymes and the local concentration of intermediates,is promising tools in optimizing heterologous metabolic pathways for target product biosynthesis.Here,we display the utility of a DNA scaffold system for the production of ectoine in E.coli MWZ003.Three fused enzymes EctA-ZFa,EctB-ZFb,and EctC-ZFc were firstly constructed by fusing enzymes of ectoine synthesis pathway with corresponding zinc finger domains.The copy number of the plasmid-expressing fusions was adapted by substitution of different replicons.Furthermore,a series of modifications were carried out on the DNA scaffold system through optimizing the spacer between enzyme binding sites,the binding direction of fusion enzymes,the repeating unit of DNA scaffolds,the stoichiometric ratio of enzyme binding sites,and the expression level of the rate-limiting enzyme.The optimized DNA scaffold system in the plasmid pFV30 involving use of pMB1 replicon,reverse binding,11-bp spacer,4 repeating units,stoichiometric ratio(1:2:2),and enhanced expres-sion of EctB-ZFb increased the ectoine titer and yield,respectively,to 22.79 g/L and 0.65 g/g glucose with increase by 92%compared with that of the control strain.The post-translational strategy based on DNA scaffold was efficient in promoting heterologous synthesis of ectoine,which could also be used in combination with other genetic engineering tools.
