Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
J Microbiol Biotechnol. 2023 Apr 28;33(4):552-558. doi: 10.4014/jmb.2301.01021. Epub 2023 Jan 27.
Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an strain and was found to be non-functional. When the host of the LA biosensor was switched from to KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.
乙酰丙酸（LA）是一种有价值的化学品，用于燃料添加剂、香料和聚合物。在这项研究中，我们提出了从木质素和聚对苯二甲酸乙二醇酯生产 LA 的可能生物合成途径。我们还创建了一种对 LA 有反应的遗传编码生物传感器，通过使用 KT2440 的 LvaR 转录调节剂来表达荧光报告基因，从而用于筛选和进化 LA 生物合成途径基因。LvaR 调节剂将 LA 识别为同源配体。首先在 菌株中检查了 LA 生物传感器，发现其无法正常工作。当 LA 生物传感器的宿主从 切换到 KT2440 时，LA 生物传感器在 0.156-10 mM LA 的 LA 浓度范围内显示出荧光强度与 LA 浓度之间的线性相关性。此外，我们确定在含有 LA 响应生物传感器的 KT2440 中，0.156 mM LA 是 LA 检测的下限。与不存在 LA 时相比，在存在 10 mM LA 时最大荧光增加了 12.3 倍。单个细胞对 LA 浓度的反应反映了群体平均反应，这使得能够对涉及 LA 生物合成的酶和代谢途径进行高通量筛选，并在工程微生物中可持续生产 LA。
