以异亮氨酰-tRNA合成酶为例构建一类新型氨基酸生物传感器

It is an efficient way to construct amino acid-producing strains by high-throughput screening with the aid of amino acid biosensors. However, most of the known biosensors have shortcomings of recognizing not only one amino acids, and/or responding to only a limited range of amino acid concentrations, and/or taking a long route to respond to the amino acids. The shortcomings can lead to high false positive rate during strain screenings, and therefore hinder the usage of the biosensors as a powerful tool to accelerate strain construction processes. In this project, by changing the characteristics of aminoacyl-tRNA synthetases (AARS), we are planning to construct a new kind of amino acid biosensors. The AARS based biosensors can couple the amino acid productivity with the cell growth rate, making it convenient to enrich high-producing strains from genetically modified or randomly mutated cells. Isoleucyl-tRNA synthetase (IleRS) will be taken as an example to construct an isoleucine biosensor. IleRS mutants will be obtained by in vivo selection and in vitro characterization. The Km(Ile) value of the mutants should be ranging from several to tens mM, and the other characteristics should not change too much. After constitutively expressed at different levels, the sensitivities of the IleRS mutant-based biosensors to the range of isoleucine concentrations will be determined by testing the cellular growth rates at different isoleucine levels. The sensitivity will be taken as an important parameter for introducing a suitable biosensor into a strain with known amino acid producing ability, and therefore to reduce the false positive rate of high throughput selection. Using the IleRS-based biosensor, the key enzymes of isoleucine metabolite pathway threonine dehydratase and acetolactate synthase, and an isoleucine producing strain will be improved. The successful construction of the isoleucine biosensor will prove the feasibility of using AARS to construct a new class of amino acid biosensors, which may provide a powerful tool for improving amino acid producing strains.

基于生物传感器（Biosensor）的高通量筛选技术逐渐成为氨基酸菌种构建的重要手段，目前已知的氨基酸Biosensor对氨基酸的专一性较差、或响应氨基酸的浓度范围较小、或信号响应线路长，导致假阳性率高，限制了其应用潜力。本项目将利用氨酰-tRNA合成酶（AARS）本身的优良特性，进一步对其改造构建氨基酸Biosensor，使菌株生长速率与氨基酸产量偶联。以大肠杆菌异亮氨酰-tRNA合成酶（IleRS）为例，通过体内筛选及体外表征，获得Km(Ile)值提高至几到几十mM的突变体，对不同突变体表达优化，使其适配不同异亮氨酸产量的菌株，降低假阳性率。利用IleRS突变体Biosensor对异亮氨酸代谢途径关键酶进行性能优化，对生产菌株连续进行诱变筛选，通过生长富集获得高产菌。本项目的研究将证实利用AARS构建一类较理想的新型氨基酸Biosensor的可行性，为提高氨基酸菌种的构建效率奠定基础。

氨基酸具有广泛用途，获取高性能微生物菌种是提高工业发酵生产水平的关键。基于生物传感器的高通量筛选可有效提高菌种水平。已知的氨基酸生物传感器种类少，特异性较差，应用受限。本研究提出了一种通过提高氨酰基-tRNA合成酶（AARS）对氨基酸的Km值，使得氨酰tRNA的合成水平受胞内氨基酸浓度控制，从而将胞内氨基酸浓度与菌株生长速度偶联，通过生长富集进行高通量筛选的策略。以大肠杆菌异亮氨酰-tRNA合成酶IleRS为例，利用Km(Ile)由3 μM变为33 mM的突变体IleRSG94R，结合异亮氨酸外排蛋白过表达，构建出专一性响应异亮氨酸、能够区分0-8g/L宽泛的异亮氨酸浓度范围的全细胞生物传感器菌株。从105级别的菌株随机突变库中传代培养富集到产量提高量2-10倍的突变株。对关键酶苏氨酸脱水酶IlvA突变库进行筛选，所获突变体活性提高20-30%。研究证明了基于AARS突变体的高通量筛选用于提高氨基酸菌种水平的可行性。项目执行过程中，我们还开发了多种氨基酸菌种的高效进化方法。包括敲除6-磷酸果糖激酶（PFK）基因，使菌株生长与外源引入的谷氨酸生产关键酶磷酸酮醇酶（PKT）活性偶联，建立了一种生长耦合进化PKT的策略,从104级别的突变库中传代富集了5个酶活提高的突变体，点突变组合PKTT2A/I6T /H260Y酶活提高了73%，使一株谷氨酸高产菌的产量和转化率分别提高了17％和18％。同时，开发了一种基于基因组复制工程的连续进化（GREACE）提高氨基酸生产菌株在发酵环境中耐受性的技术体系，用于赖氨酸高产大肠杆菌的适应性进化，所获突变体在发酵罐中产量和转化率分别提高15%和9%。解析发现speB、atpB、secY基因点突变促进菌株耐受和高产的新机制。该项目开发的氨基酸菌种高通量进化筛选技术对于提高工业菌种水平具有重要意义。