| Non-radioactive Assay to Determine Product Profile of Short-chain Isoprenyl Diphosphate Synthases
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Author:
Date:
2021-01-05
[Abstract] Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), geranylgeranyl diphosphate (GGPP; C20), and geranylfarnesyl diphosphate (GFPP; C25) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C5) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C5), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed ...
[摘要] [Abstrac吨]类异戊二烯代表最大的一类代谢物在结构和功能惊人多样性。它们参与保护植物免受病原体或草食动物侵害，或参与吸引传粉媒介。（;çGPP类异戊二烯是从牻牛儿基二磷酸衍生的10 ），法呢基二磷酸（FPP;Ç 15 ），香叶基香叶基二磷酸（GGPP;Ç 20 ），和geranylfarnesyl二磷酸（GFPP; C ^ 25 ），它们又通过的顺序缩合形成异戊烯基二甲基磷酸酯（IPP; C 5 ）与烯丙基受体，例如二磷酸二甲基烯丙酯（DMAPP; C 5），GPP，FPP或GGPP）由异戊二烯基二磷酸合酶（IDS）催化的反应。用于确定异戊二烯基二磷酸酯产物的IDS酶测定法通常是使用放射性标记的底物进行的，并且所形成的产物是通过使用昂贵的仪器（例如磷光成像仪，radio-GC或radio-HPLC）来鉴定的。尽管已经报道了一种用于测量粗植物提取物中IDS活性的非放射性测定方法，但它需要使用色谱结合串联质谱（LC / MS-MS）的复杂方法。在这里，我们描述了用于确定使用非放射性标记的IPP及其共同烯丙基底物DMAPP，GPP的IDS分析产物非放射性和简单廉价的测定法，和FPP。在测定中生成的异戊二烯基二磷酸产物的检测非常高效，并且在浓度大于40 µM的IPP和DMAPP / GPP / ...
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| In vivo Quantification of Alkanes in Escherichia coli
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Author:
Date:
2020-04-20
[Abstract] Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating oxygenase) pathway. Here, the intermediates of fatty acid synthesis pathway are used as substrate by the AAR enzyme to make fatty aldehyde, which is then deformylated by ADO to make linear chain alkane. However, the variation in substrate availability to the first enzyme of the pathway, i.e., AAR, via fatty acid synthesis pathway and low turnover of the ADO enzyme make calculation of yields and titers under in vivo conditions extremely ...
[摘要] [摘要] 由于烷烃的高能量密度和与现有石油燃料的相似性，使用合成生物学工具生产烷烃的微生物受到了广泛关注。最常研究的途径之一是通过AAR（酰基-ACP （酰基）载体蛋白）还原酶）-ADO（醛Deformylating 加氧酶）途径。在这里，中间体脂肪酸合成途径被用作基材由AAR Enzym E要使脂肪醛，然后是Deformylated 通过ADO，使线性链烷烃。但是，即该途径的第一种酶的底物利用率的变化，即，AAR，通过脂肪酸合成途径和ADO酶的低周转率，使得在体内条件下的产量和效价的计算极为困难。在体内测定中，将确定的ADO酶底物外加到培养基中有助于监测菌体的流入。因此，该底物提供了更准确的产物收率测量方法。在此方案中，我们包括用于实施体内测定法以监测大肠杆菌中烷烃生产的详细指南。
[背景] 利用工程微生物生产烷烃的研究已广受欢迎，因为它提供了一种有吸引力的替代方案，可减少对化石燃料的依赖，同时减轻气候变化的影响（Lee 等，2008；Knothe ，2010； Lu，2010； Schirmer 等。人，2010;谭等人。，2011）各种途径已被发现或人工Assemb ...
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