枯草芽孢杆菌中高效响应N-乙酰神经氨酸生物传感器的构建

Development of biosensors highly responsive to N-acetylneuraminic acid in Bacillus subtilis

摘要:

枯草芽孢杆菌(Bacillus subtilis)是公认的食品安全菌株，目前已被用于多种高附加值产品的生物合成，包括被广泛用作营养化学品和药物中间体的N-乙酰神经氨酸(N-acetylneuraminic acid, NeuAc)。响应目标产物的生物传感器被广泛用于代谢工程中的动态调控和高通量筛选等方面，以提高生物合成效率。但是，枯草芽孢杆菌中缺乏可高效响应NeuAc的生物传感器。因此，本文首先测试和优化了能将胞外NeuAc转运进胞内的转运蛋白，获得了一系列具有不同转运能力的菌株，以用于后续响应NeuAc的生物传感器的验证；随后将响应NeuAc的转录因子Bbr_NanR的结合位点插入枯草芽孢杆菌组成型启动子的不同位置，筛选具有活性的杂合启动子；接下来，通过在具有NeuAc转运能力的枯草芽孢杆菌中表达Bbr_NanR，选择能响应NeuAc的杂合启动子，并进一步通过优化Bbr_NanR表达量获得了一系列动态范围广、激活倍数高的生物传感器，其中生物传感器P535-N2能灵敏地响应胞内NeuAc浓度的变化，具有最大的动态范围，为(180–20 245) AU/OD；P566-N2则具有最高的激活倍数，为122倍，是已报道的枯草芽孢杆菌中响应N-乙酰神经氨酸的生物传感器的2倍。本文构建的响应NeuAc的生物传感器可用于高产NeuAc的酶突变体和枯草芽孢杆菌菌株的筛选，为枯草芽孢杆菌生物合成NeuAc提供了高效、灵敏的分析和调控工具。

Abstract:

Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.