The chemical synthesis of Guanine arabinoside （ara-G） is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine ara...The chemical synthesis of Guanine arabinoside （ara-G） is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside （ara-DA） was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L^-1 uracil arabinoside as arabinose donor, 10 mmol·L^- 1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L^-1 phosphate buffer, and reacted at 60℃ for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.展开更多
Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP cou...Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.展开更多
Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell g...Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth.The lactose-induced UPase was stable at 50°C.Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L 1uridine and 5-fluorouracil in phosphate buffer（pH 7.0）catalyzed at 50°C for 1.5 h and the yield of 5-fluorouridine was higher than 68%.Under the optimum reaction conditions for production of 5-fluorouridine,5-methyluridine and azauridine were synthesized from uridine by pUDP,the yield was 61.7%and 47.2%respectively.Deoxynucleosides were also synthesized by pUDP,but the yield was only about 20%.展开更多
Itaconic acid(ITA),an effective alternative fossil fuel,derives from the bypass pathway of the tricarboxylic acid(TCA)cycle.Therefore,the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seri...Itaconic acid(ITA),an effective alternative fossil fuel,derives from the bypass pathway of the tricarboxylic acid(TCA)cycle.Therefore,the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA.The optimization of flux distribution between biomass and production has the potential to the productivity of ITA.Based on the previously constructed strain Escherichia coli MG1655Δ1-SAS-3(ITA titer:1.87 g/L),a CRISPRi-mediated self-inducible system(CiMS),which contained a responsive module based on the ITA biosensor YpItcR/Pccl and a regulative CRISPRi-mediated interferential module,was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA.First,a higher ITA-yielding strain,Δ4-Prmd-SAS-3(ITA titer:3.20 g/L),derived fromΔ1-SAS-3,was constructed by replacing the promoter PJ23100,for the expression of ITA synthesis genes,with Prmd and knocking out the three bypass genes poxB,pflB,and ldhA.Subsequently,the CiMS was used to inhibit the expression of key genes icd,pykA,and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage.The constructed strainΔ4-Prmd-SAS-3 under the dynamic regulation of the CiMS,showed a 23%increase in the ITA titer,which reached 3.93 g/L.This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.展开更多
CoproporphyrinⅢ(CPⅢ),a natural porphyrin derivative,has extensive applications in the biomedical and material industries.S.cerevisiae has previously been engineered to highly accumulate the CPⅢprecursor 5-aminolevu...CoproporphyrinⅢ(CPⅢ),a natural porphyrin derivative,has extensive applications in the biomedical and material industries.S.cerevisiae has previously been engineered to highly accumulate the CPⅢprecursor 5-aminolevulinic acid(ALA)through the C4 pathway.In this study,a combination of cytoplasmic metabolic engineering and mitochondrial compartmentalization was used to enhance CPⅢproduction in S.cerevisiae.By integrating pathway genes into the chromosome,the CPⅢtiter gradually increased to 32.5±0.5 mg/L in shake flask cultivation.Nevertheless,increasing the copy number of pathway genes did not consistently enhance CPⅢsynthesis.Hence,the partial synthesis pathway was compartmentalized in mitochondria to evaluate its effectiveness in increasing CPⅢproduction.Subsequently,by superimposing the mitochondrial compartmentalization strategy on cytoplasmic metabolic engineered strains,the CPⅢtiter was increased to 64.3±1.9 mg/L.Furthermore,augmenting antioxidant pathway genes to reduce reactive oxygen species(ROS)levels effectively improved the growth of engineered strains,resulting in a further increase in the CPⅢtiter to 82.9±1.4 mg/L.Fed-batch fermentations in a 5 L bioreactor achieved a titer of 402.8±9.3 mg/L for CPⅢ.This study provides a new perspective on engineered yeast for the microbial production of porphyrins.展开更多
基金Supported by the Innovation Fund for Technology Based Firms from Ministry of Science and Technology of China(07C26213101283)
文摘The chemical synthesis of Guanine arabinoside （ara-G） is extremely complex, time-consuming, and seriously polluted. A two-step enzymatic synthesis process was developed to acquire ara-G easily. 2,6-Diaminopurine arabinoside （ara-DA） was first synthesized with purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase produced by Enterobacter aerogenes DGW-07. The conversion yield of ara-DA could reach above 90% when the reaction liquid contained 30 mmol·L^-1 uracil arabinoside as arabinose donor, 10 mmol·L^- 1 2,6-diaminopurine as arabinose acceptor in pH 7.0 20 mmol·L^-1 phosphate buffer, and reacted at 60℃ for 48h. Then, ara-DA was effectively transformed into ara-G with adenylate deaminase produced by Aspergillus oryzae DAW-01. The total process had no complex separation and purification.
基金Project (No. 07C26213101283) supported by the Innovation Fundfor Technology Based Firms from the Ministry of Science andTechnology of China
文摘Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5′-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.
基金Supported by"Production,Education&Research"item of Shanghai Baoshan(08-H-4)
文摘Recombinant Escherichia coli pUDP,which overexpressed uridine phosphorylase（UPase）,was constructed.0.5 mmol·L 1lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth.The lactose-induced UPase was stable at 50°C.Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol·L 1uridine and 5-fluorouracil in phosphate buffer（pH 7.0）catalyzed at 50°C for 1.5 h and the yield of 5-fluorouridine was higher than 68%.Under the optimum reaction conditions for production of 5-fluorouridine,5-methyluridine and azauridine were synthesized from uridine by pUDP,the yield was 61.7%and 47.2%respectively.Deoxynucleosides were also synthesized by pUDP,but the yield was only about 20%.
基金supported by the National Natural Science Foundation of China(No.21778018)Research Program of State Key Laboratory of Bioreactor Engineering and the grant from the National Key Research and Development Program of China(2021YFC2100300)the Chinese Plastic Surgery Foundation(No.2020M671021).
文摘Itaconic acid(ITA),an effective alternative fossil fuel,derives from the bypass pathway of the tricarboxylic acid(TCA)cycle.Therefore,the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA.The optimization of flux distribution between biomass and production has the potential to the productivity of ITA.Based on the previously constructed strain Escherichia coli MG1655Δ1-SAS-3(ITA titer:1.87 g/L),a CRISPRi-mediated self-inducible system(CiMS),which contained a responsive module based on the ITA biosensor YpItcR/Pccl and a regulative CRISPRi-mediated interferential module,was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA.First,a higher ITA-yielding strain,Δ4-Prmd-SAS-3(ITA titer:3.20 g/L),derived fromΔ1-SAS-3,was constructed by replacing the promoter PJ23100,for the expression of ITA synthesis genes,with Prmd and knocking out the three bypass genes poxB,pflB,and ldhA.Subsequently,the CiMS was used to inhibit the expression of key genes icd,pykA,and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage.The constructed strainΔ4-Prmd-SAS-3 under the dynamic regulation of the CiMS,showed a 23%increase in the ITA titer,which reached 3.93 g/L.This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.
基金supported by the grant from the National Key Research and Development Program of China(2021YFC2100300)2023 Double World-class Project Key Program“Intelligent Biomanufacturing”.
文摘CoproporphyrinⅢ(CPⅢ),a natural porphyrin derivative,has extensive applications in the biomedical and material industries.S.cerevisiae has previously been engineered to highly accumulate the CPⅢprecursor 5-aminolevulinic acid(ALA)through the C4 pathway.In this study,a combination of cytoplasmic metabolic engineering and mitochondrial compartmentalization was used to enhance CPⅢproduction in S.cerevisiae.By integrating pathway genes into the chromosome,the CPⅢtiter gradually increased to 32.5±0.5 mg/L in shake flask cultivation.Nevertheless,increasing the copy number of pathway genes did not consistently enhance CPⅢsynthesis.Hence,the partial synthesis pathway was compartmentalized in mitochondria to evaluate its effectiveness in increasing CPⅢproduction.Subsequently,by superimposing the mitochondrial compartmentalization strategy on cytoplasmic metabolic engineered strains,the CPⅢtiter was increased to 64.3±1.9 mg/L.Furthermore,augmenting antioxidant pathway genes to reduce reactive oxygen species(ROS)levels effectively improved the growth of engineered strains,resulting in a further increase in the CPⅢtiter to 82.9±1.4 mg/L.Fed-batch fermentations in a 5 L bioreactor achieved a titer of 402.8±9.3 mg/L for CPⅢ.This study provides a new perspective on engineered yeast for the microbial production of porphyrins.
