Jha Ramesh K, Narayanan Niju, Pandey Naresh, Bingen Jeremy M, Kern Theresa L, Johnson Christopher W, Strauss Charlie E M, Beckham Gregg T, Hennelly Scott P, Dale Taraka
Bioscience Division, MS M888 , Los Alamos National Laboratory , Los Alamos , New Mexico 87545 , United States.
Theoretical Biology and Biophysics, Theoretical Division , Los Alamos National Laboratory , Los Alamos , New Mexico 87545 , United States.
ACS Synth Biol. 2019 Apr 19;8(4):775-786. doi: 10.1021/acssynbio.8b00465. Epub 2019 Mar 25.
Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.
产物抑制是工业酶中常见的瓶颈，通过传统方法测试缓解产物抑制的突变仍然具有挑战性，因为需要针对多种底物和产物浓度测试许多变体。此外，传统的筛选方法是在体外进行的，所得的酶变体在全细胞代谢和调节的背景下在体内可能表现不同。在本研究中，我们通过建立一种高通量筛选方法来解决这两个问题，以缓解工业相关酶分支酸丙酮酸裂解酶（UbiC）中的产物抑制。首先，我们在工业相关宿主恶臭假单胞菌KT2440中设计了一种针对4-羟基苯甲酸（4HB）的高度特异性的基因编码生物传感器。随后，我们应用该生物传感器检测将分支酸转化为4HB和丙酮酸的异源表达UbiC的活性。通过使用苯甲酸作为抑制UbiC而不激活生物传感器的产物替代物，我们能够有效地创建和筛选一个多样化的文库，以寻找产物抑制降低的UbiC变体。将改良的UbiC酶变体引入工业前体顺，顺-粘康酸（粘康酸）的实验生产菌株中，当新的UbiC变体从质粒表达时，葡萄糖到粘康酸的转化率提高了2倍以上，当相同的UbiC变体基因组整合到该菌株中时，产量提高了60%。总体而言，这项工作表明，通过将酶变体文库与全细胞催化和生物传感相结合，可以鉴定出产物抑制降低的变体，并且这种改良的酶可以提高下游目标分子的滴度。
