1,3-Dihydroxyacetone (DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans ( G. oxydans ) fermentation, but th...1,3-Dihydroxyacetone (DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans ( G. oxydans ) fermentation, but the high concentration of glycerol in the fermentation broth inhibits cells growth. To overcome this obstacle, in this study, we overexpressed the glycerol transporter (GlpFp) by the use of promoters P tufB , P gmr , P glp1 , and P glp2 in G. oxydans 621H. The results show that the glycerol tolerances of strains overexpressing G lpF were all much better than that of the control strain. The glycerol dehydrogenase gene (G dh) was overexpressed by the promoters P tufB and P gdh , which increased the DHA titer by 12.7% compared with that of the control group. When G lpF and Gdh genes were co-overexpressed in G. oxydans 621H, the OD600 value of the engineered strains all increased, but the DHA titers decreased in di erent degrees, as compared with strains that overexpressed only G dh . This study provides a reference for future research on DHA production.展开更多
该研究以BS120作为出发菌株,通过常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术进行诱变处理,第一轮以40 mg/L 8-氮鸟嘌呤为筛选拮抗物进行筛选,得到核黄素产量和得率分别提升61.60%和58.12%的菌株BSG1。第...该研究以BS120作为出发菌株,通过常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术进行诱变处理,第一轮以40 mg/L 8-氮鸟嘌呤为筛选拮抗物进行筛选,得到核黄素产量和得率分别提升61.60%和58.12%的菌株BSG1。第二轮诱变以300 mg/L寡霉素为筛选拮抗物进行筛选,筛选获得菌株BSG3,核黄素产量和得率较BS120分别提升83.59%和78.76%。将核黄素操纵子表达质粒pMX45转入BSG3中,得到菌株BSG5,核黄素产量达到(4467.08±99.47)mg/L,得率为(42.56±1.25)mg/g葡萄糖,较BS120分别提高140.94%和120.52%,展现了良好的核黄素发酵性能和遗传稳定性。展开更多
Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the is...Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.展开更多
Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two ...Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.展开更多
The strain LWYZ003, which can restrain multiple pathogens, was screened from the sediment of the ocean and identified as Bacillus amyloliquefaciens. Large-scale fermentation and modern chromatographic separation techn...The strain LWYZ003, which can restrain multiple pathogens, was screened from the sediment of the ocean and identified as Bacillus amyloliquefaciens. Large-scale fermentation and modern chromatographic separation technologies(macroporous resin column chromatography, silica gel column chromatography, thin-layer chromatography and high-performance liquid chromatography) were used to separate antimicrobial products from the fermentation broth of marine-derived Bacillus amyloliquefaciens LWYZ003. Bioactive-guided separation was used in the term of seeking antimicrobial products from the secondary metabolites of Bacillus amyloliquefaciens LWYZ003. As a result, two natural products cycloheximide( 1) and trehalose( 2) were obtained. Their structures were elucidated by Fourier transform infrared spectroscopy, high-resolution mass spectrometry, ~1 H and ^(13) C nuclear magnetic resonance analysis. In the cylinder plate method, compound 1 exhibited stronger antimicrobial activities than compound 2 against Micrococcus luteus, and also exhibited wider antimicrobial spectrum than compound 2. In conclusion, isolation of bioactive secondary metabolites from marine Bacillus sp. has enormous potentials in finding suitable antibiotics to inhibit multiple pathogens.展开更多
In recent years,metabolic engineering has made great progress in both academic research and industrial applications.However,we have not found any articles that specifically analyze the current state of metabolic engin...In recent years,metabolic engineering has made great progress in both academic research and industrial applications.However,we have not found any articles that specifically analyze the current state of metabolic engineering in China in comparison with other countries.Here,we review the current development and future trends of global metabolic engineering,conduct an in-depth benchmarking analysis of the development situation of China’s metabolic engineering,and identify current problems as well as future trends.We searched publications in the Scopus database from 2015 to September 2020 in the field of metabolic engineering,and analyzed the output in general,including publication trends,research distribution,popular journals,hot topics and vital institutions,but also analyzed the share of citations,field-weighted citation impact,and production in collaboration with strategic countries in science and technology.This study aims to serve as a reference for later studies,offering a comprehensive view of China’s contribution to metabolic engineering,and as a tool for the elaboration of national public policy in science and technology.展开更多
基金supported by the Major Research Plan of Tianjin (16YFXTSF00460)
文摘1,3-Dihydroxyacetone (DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans ( G. oxydans ) fermentation, but the high concentration of glycerol in the fermentation broth inhibits cells growth. To overcome this obstacle, in this study, we overexpressed the glycerol transporter (GlpFp) by the use of promoters P tufB , P gmr , P glp1 , and P glp2 in G. oxydans 621H. The results show that the glycerol tolerances of strains overexpressing G lpF were all much better than that of the control strain. The glycerol dehydrogenase gene (G dh) was overexpressed by the promoters P tufB and P gdh , which increased the DHA titer by 12.7% compared with that of the control group. When G lpF and Gdh genes were co-overexpressed in G. oxydans 621H, the OD600 value of the engineered strains all increased, but the DHA titers decreased in di erent degrees, as compared with strains that overexpressed only G dh . This study provides a reference for future research on DHA production.
文摘该研究以BS120作为出发菌株,通过常压室温等离子体诱变(atmospheric and room temperature plasma,ARTP)技术进行诱变处理,第一轮以40 mg/L 8-氮鸟嘌呤为筛选拮抗物进行筛选,得到核黄素产量和得率分别提升61.60%和58.12%的菌株BSG1。第二轮诱变以300 mg/L寡霉素为筛选拮抗物进行筛选,筛选获得菌株BSG3,核黄素产量和得率较BS120分别提升83.59%和78.76%。将核黄素操纵子表达质粒pMX45转入BSG3中,得到菌株BSG5,核黄素产量达到(4467.08±99.47)mg/L,得率为(42.56±1.25)mg/g葡萄糖,较BS120分别提高140.94%和120.52%,展现了良好的核黄素发酵性能和遗传稳定性。
基金National Program on Key Basic Research Project by the Ministry of Science and Technology of China(2014CB745100)the National Natural Science Foundation of China(21676190 and 21621004)the Innovative Talents and Platform Program of Tianjin(16PTGCCX00140 and 16PTSYJC00050).
文摘Poly(ethylene terephthalate)hydrolase(PETase)from Ideonella sakaiensis exhibits a strong ability to degrade poly(ethylene terephthalate)(PET)at room temperature,and is thus regarded as a potential tool to solve the issue of polyester plastic pollution.Therefore,we explored the interaction between PETase and the substrate(a dimer of the PET monomer ethylene terephthalate,2PET),using a model of PETase and its substrate.In this study,we focused on six key residues around the substrate-binding groove in order to create novel high-efficiency PETase mutants through protein engineering.These PETase mutants were designed and tested.The enzymatic activities of the R61A,L88F,and I179F mutants,which were obtained with a rapid cell-free screening system,exhibited 1.4 fold,2.1 fold,and 2.5 fold increases,respectively,in comparison with wild-type PETase.The I179F mutant showed the highest activity,with the degradation rate of a PET film reaching 22.5 mg perμmol·L^-1 PETase per day.Thus,this study has created enhanced artificial PETase enzymes through the rational protein engineering of key hydrophobic sites,and has further illustrated the potential of biodegradable plastics.
基金the Ministry of Science and Technology of China (2014CB745100)the National Natural Science Foundation of China (21390203 and 21706186).
文摘Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.
基金supported by the National Basic Research Program of China ("973" Program) (no. 2012CB721105)the Major Research Plan of Tianjin, China (no. 16YFXTSF00460)
文摘The strain LWYZ003, which can restrain multiple pathogens, was screened from the sediment of the ocean and identified as Bacillus amyloliquefaciens. Large-scale fermentation and modern chromatographic separation technologies(macroporous resin column chromatography, silica gel column chromatography, thin-layer chromatography and high-performance liquid chromatography) were used to separate antimicrobial products from the fermentation broth of marine-derived Bacillus amyloliquefaciens LWYZ003. Bioactive-guided separation was used in the term of seeking antimicrobial products from the secondary metabolites of Bacillus amyloliquefaciens LWYZ003. As a result, two natural products cycloheximide( 1) and trehalose( 2) were obtained. Their structures were elucidated by Fourier transform infrared spectroscopy, high-resolution mass spectrometry, ~1 H and ^(13) C nuclear magnetic resonance analysis. In the cylinder plate method, compound 1 exhibited stronger antimicrobial activities than compound 2 against Micrococcus luteus, and also exhibited wider antimicrobial spectrum than compound 2. In conclusion, isolation of bioactive secondary metabolites from marine Bacillus sp. has enormous potentials in finding suitable antibiotics to inhibit multiple pathogens.
基金The research was financially supported by the National Natural Science Foundation of China(NSFC-21776209,NSFC-21621004,NSFC-21776208)Natural Science Foundation of Tianjin(No.19JCYBJC21100).
文摘In recent years,metabolic engineering has made great progress in both academic research and industrial applications.However,we have not found any articles that specifically analyze the current state of metabolic engineering in China in comparison with other countries.Here,we review the current development and future trends of global metabolic engineering,conduct an in-depth benchmarking analysis of the development situation of China’s metabolic engineering,and identify current problems as well as future trends.We searched publications in the Scopus database from 2015 to September 2020 in the field of metabolic engineering,and analyzed the output in general,including publication trends,research distribution,popular journals,hot topics and vital institutions,but also analyzed the share of citations,field-weighted citation impact,and production in collaboration with strategic countries in science and technology.This study aims to serve as a reference for later studies,offering a comprehensive view of China’s contribution to metabolic engineering,and as a tool for the elaboration of national public policy in science and technology.
