2-keto-D-gluconic acid(2-KGA)is a key precursor for synthesising vitamin C and isovitamin C.However,phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens.Gluconobac...2-keto-D-gluconic acid(2-KGA)is a key precursor for synthesising vitamin C and isovitamin C.However,phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens.Gluconobacter holds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids,and high utilisation of glucose.In this study,the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-D-gluconic acid(5-KGA)and D-gluconic acid(D-GA)in the 2-KGA producer Gluconobacter japonicus CGMCC 1.49.Knocking out the ga5dh-1 gene from a competitive pathway and overexpressing the ga2dh-A gene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81%in shake flasks.Additionally,accumulation of 5-KGA was decreased by 63.52%with the resulting G.japonicas-Δga5dh-1-ga2dh-A strain.Using an intermittent fed-batch mode in a 3 L fermenter,2-KGA reached 235.3 g L^−1 with a 91.1%glucose conversion rate.Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L^−1 h^−1,11.99%higher than in the 3 L fermenter,and D-GA and 5-KGA by-products were completely converted to 2-KGA.展开更多
芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛...芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。展开更多
基金the National Key Research and Development Program of China(2017YFC1600403)the National Natural Science Foundation of China(31830068,21822806)+2 种基金the Fundamental Research Funds for the Central Universities(JUSRP51701A)the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-08)the Distinguished Professor Project of Jiangsu Province.
文摘2-keto-D-gluconic acid(2-KGA)is a key precursor for synthesising vitamin C and isovitamin C.However,phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens.Gluconobacter holds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids,and high utilisation of glucose.In this study,the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-D-gluconic acid(5-KGA)and D-gluconic acid(D-GA)in the 2-KGA producer Gluconobacter japonicus CGMCC 1.49.Knocking out the ga5dh-1 gene from a competitive pathway and overexpressing the ga2dh-A gene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81%in shake flasks.Additionally,accumulation of 5-KGA was decreased by 63.52%with the resulting G.japonicas-Δga5dh-1-ga2dh-A strain.Using an intermittent fed-batch mode in a 3 L fermenter,2-KGA reached 235.3 g L^−1 with a 91.1%glucose conversion rate.Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L^−1 h^−1,11.99%higher than in the 3 L fermenter,and D-GA and 5-KGA by-products were completely converted to 2-KGA.
文摘芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。
