A Transcription Factor-Based Biosensor for Detection of Itaconic Acid.
A Transcription Factor-Based Biosensor for Detection of Itaconic Acid.
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DOI：
10.1021/acssynbio.8b00057
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发表时间：
2018-05-18
影响因子：
4.7
通讯作者：
Malys N
中科院分区：
文献类型：
--
作者：
Hanko EKR;Minton NP;Malys N
衣康酸是一种重要的平台化学品，可以很容易地结合到聚合物中，并有可能取代石油化工基丙烯酸或甲基丙烯酸。许多微生物已被开发用于衣壳酸酯的生物合成，包括土曲霉、大肠杆菌和酿酒酵母。然而，由于缺乏高通量筛选方法，目前可以检测衣康酸滴度增加的菌株数量和条件有限。在这里，我们从假结核耶尔森菌和铜绿假单胞菌中鉴定出了itaconate诱导启动子及其相应的lysr型转录调控子。我们发现，衣康酸在模型大肠杆菌和铜杆菌中高度诱导了YpItcR/Pccl诱导系统（分别为215倍和105倍）。我们研究了YpItcR/Pccl诱导系统的动力学和动力学，并证明，除了衣康酸外，遗传编码的生物传感器还能够以剂量依赖的方式检测间萨酸、顺式和反式衣康酸。此外，利用荧光生物传感器在大肠杆菌中鉴定cadA的最佳表达水平，其产物催化顺式aconate转化为衣康酸。荧光输出显示与高效液相色谱和紫外光谱定量的衣康酸浓度有很好的相关性。这项工作强调了YpItcR/Pccl诱导系统作为高通量微生物菌株开发的生物传感器的潜力，以促进衣康酸的生物合成。
Itaconic acid is an important platform chemical that can easily be incorporated into polymers and has the potential to replace petrochemical-based acrylic or methacrylic acid. A number of microorganisms have been developed for the biosynthesis of itaconate including Aspergillus terreus, Escherichia coli, and Saccharomyces cerevisiae. However, the number of strains and conditions that can be tested for increased itaconate titers are currently limited because of the lack of high-throughput screening methods. Here we identified itaconate-inducible promoters and their corresponding LysR-type transcriptional regulators from Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that the YpItcR/Pccl inducible system is highly inducible by itaconic acid in the model gammaproteobacterium E. coli and the betaproteobacterium Cupriavidus necator (215- and 105-fold, respectively). The kinetics and dynamics of the YpItcR/Pccl inducible system are investigated, and we demonstrate, that in addition to itaconate, the genetically encoded biosensor is capable of detecting mesaconate, cis-, and trans-aconitate in a dose-dependent manner. Moreover, the fluorescence-based biosensor is applied in E. coli to identify the optimum expression level of cadA, the product of which catalyzes the conversion of cis-aconitate into itaconate. The fluorescence output is shown to correlate well with itaconate concentrations quantified using high-performance liquid chromatography coupled with ultraviolet spectroscopy. This work highlights the potential of the YpItcR/Pccl inducible system to be applied as a biosensor for high-throughput microbial strain development to facilitate improved itaconate biosynthesis.
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影响因子：
4.7
作者：
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