Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate...Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate （NADPH） availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli （△ispB：：ddsA,△pykFA and △gapA：：gapC）,harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.展开更多
Objective:To investigate the effects of coenzyme Q10(CoQ10)supplementation on post-vitrification embryo development and gross morphology.Methods:Balb/c mouse embryos were cultured in potassium simplex optimised medium...Objective:To investigate the effects of coenzyme Q10(CoQ10)supplementation on post-vitrification embryo development and gross morphology.Methods:Balb/c mouse embryos were cultured in potassium simplex optimised medium(KSOM)with varying CoQ10 concentrations[0(control),20,40,and 60μM].The most effective CoQ10 concentration(40μM)was selected for subsequent post-vitrification morphology study.Embryos were randomly divided into four groups:Group A(non-vitrified without CoQ10),Group B(non-vitrified with CoQ10),Group C(vitrified without CoQ10),and Group D(vitrified with CoQ10),followed by vitrification at the 8-cell stage.Survival rates and development until the blastocyst stage were evaluated through morphological examinations using ASEBIR's system,distinguishing normal and abnormal embryos.Results:Supplementation of 40μM CoQ10 significantly increased blastocyst formation(95%)compared to the control group(92%),20μM(62%),and 60μM(56%)(P<0.001).Following vitrification,Group D exhibited a significant increase in blastocyst formation(92%)compared to Group C(82%)(P<0.05).Morphological assessments indicated superior embryo quality in Group B over Group D during the cleavage stage,morula,and blastocyst(P<0.05).Conclusions:CoQ10 supplementation exhibits promising potential to enhance preimplantation embryo development,increase blastocyst formation rates,and improve embryo quality post-vitrification.This offers a promising approach to mitigate oxidative stress on embryos,potentially improving overall assisted reproductive technology outcomes.展开更多
In order to increase the production efficiency of coenzyme Q10, the original strain Agrobacterium tumefaciens ATCC 4452 was mutated by means of Nitrogen ions implantation. A mutant strain, ATX 12, with high contents o...In order to increase the production efficiency of coenzyme Q10, the original strain Agrobacterium tumefaciens ATCC 4452 was mutated by means of Nitrogen ions implantation. A mutant strain, ATX 12, with high contents of coenzyme Q10 was selected. Subsequently, the conditions such as carbohydrate concentration, nitrogen source concentration, inoculum＇s size, seed age, aeration and temperature which might affect the production of CoQ10 were investigated in detail. Under optimal conditions, the maximum concentration of the intracellular CoQ10 reached 200.3 mg/L after 80 h fed-batch fermentation, about 245% increasing in CoQ10 production after ion implantation, compared to the original strain.展开更多
The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli stra...The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli strain for coenzyme Q10 production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution(multiple copy integration) and replicon-free and markerless chromosomal integration(single copy integration), respectively. A coenzyme Q10 hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution,replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway.The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass(DCM) of coenzyme Q10 when supplemented with 0.075 g·L-1of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q10 production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.展开更多
基金Supported by the National Natural Science Foundation of China（30970089 200876181 20831006） the Natural Science Foundation of Guangdong Province（9351027501000003） the Project of Science and Technology of Guangdong Province（2007A010900001）
文摘Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate （NADPH） availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli （△ispB：：ddsA,△pykFA and △gapA：：gapC）,harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.
基金supported by the Fundamental Research Grant Scheme(FRGS)[FRGS/1/2020/SKK06/UNIKL/02/1],from the Ministry of Higher Education,Malaysia.
文摘Objective:To investigate the effects of coenzyme Q10(CoQ10)supplementation on post-vitrification embryo development and gross morphology.Methods:Balb/c mouse embryos were cultured in potassium simplex optimised medium(KSOM)with varying CoQ10 concentrations[0(control),20,40,and 60μM].The most effective CoQ10 concentration(40μM)was selected for subsequent post-vitrification morphology study.Embryos were randomly divided into four groups:Group A(non-vitrified without CoQ10),Group B(non-vitrified with CoQ10),Group C(vitrified without CoQ10),and Group D(vitrified with CoQ10),followed by vitrification at the 8-cell stage.Survival rates and development until the blastocyst stage were evaluated through morphological examinations using ASEBIR's system,distinguishing normal and abnormal embryos.Results:Supplementation of 40μM CoQ10 significantly increased blastocyst formation(95%)compared to the control group(92%),20μM(62%),and 60μM(56%)(P<0.001).Following vitrification,Group D exhibited a significant increase in blastocyst formation(92%)compared to Group C(82%)(P<0.05).Morphological assessments indicated superior embryo quality in Group B over Group D during the cleavage stage,morula,and blastocyst(P<0.05).Conclusions:CoQ10 supplementation exhibits promising potential to enhance preimplantation embryo development,increase blastocyst formation rates,and improve embryo quality post-vitrification.This offers a promising approach to mitigate oxidative stress on embryos,potentially improving overall assisted reproductive technology outcomes.
基金National Natural Science Foundation of China(No.20576132)
文摘In order to increase the production efficiency of coenzyme Q10, the original strain Agrobacterium tumefaciens ATCC 4452 was mutated by means of Nitrogen ions implantation. A mutant strain, ATX 12, with high contents of coenzyme Q10 was selected. Subsequently, the conditions such as carbohydrate concentration, nitrogen source concentration, inoculum＇s size, seed age, aeration and temperature which might affect the production of CoQ10 were investigated in detail. Under optimal conditions, the maximum concentration of the intracellular CoQ10 reached 200.3 mg/L after 80 h fed-batch fermentation, about 245% increasing in CoQ10 production after ion implantation, compared to the original strain.
基金Supported by the National Natural Science Foundation of China(30970089,20876181,21276289)the Natural Science Foundation of Guangdong Province(9351027501000003,S2011010001396)
文摘The plasmid-expression system is routinely plagued by potential plasmid instability. Chromosomal integration is one powerful approach to overcome the problem. Herein we report a plasmid-free hyper-producer E.coli strain for coenzyme Q10 production. A series of integration expression vectors, pxKC3T5b and pxKT5b, were constructed for chemically inducible chromosomal evolution(multiple copy integration) and replicon-free and markerless chromosomal integration(single copy integration), respectively. A coenzyme Q10 hyper-producer Escherichia coli TBW20134 was constructed by applying chemically inducible chromosomal evolution,replicon-free and markerless chromosomal integration as well as deletion of menaquinone biosynthetic pathway.The engineered E. coli TBW20134 produced 10.7 mg per gram of dry cell mass(DCM) of coenzyme Q10 when supplemented with 0.075 g·L-1of 4-hydroxy benzoic acid; this yield is unprecedented in E. coli and close to that of the commercial producer Agrobacterium tumefaciens. With this strain, the coenzyme Q10 production capacity was very stable after 30 sequential transfers and no antibiotics were required during the fermentation process. The strategy presented may be useful as a general approach for construction of stable production strains synthesizing natural products where various copy numbers for different genes are concerned.
