Pang Cuiping, Zhang Guoqiang, Liu Song, Zhou Jingwen, Li Jianghua, Du Guocheng
National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.
Biotechnol Biofuels Bioprod. 2022 Oct 10;15(1):105. doi: 10.1186/s13068-022-02206-x.
Lipoxygenase (EC. 1.13.11.12, LOX) can catalyze the addition of oxygen into polyunsaturated fatty acids to produce hydroperoxides, which are widely used in the food, chemical, and pharmaceutical industries. In recent years, the heterologous production of LOX by Escherichia coli has attracted extensive attention. However, overexpressed recombinant LOX in E. coli aggregates and forms insoluble inclusion bodies owing to protein misfolding.
In this study, a split green fluorescent protein-based screening method was developed to screen sigma (σ) factors and molecular chaperones for soluble LOX expression. Three mutant libraries of Skp, GroES, and RpoH was analyzed using the high-throughput screening method developed herein, and a series of mutants with significantly higher yield of soluble heterologous LOX were obtained. The soluble expression level of LOX in the isolated mutants increased by 4.2- to 5.3-fold. Further, the highest LOX activity (up to 6240 ± 269 U·g-DCW) was observed in E. coli REopt, with the regulatory factor mutants, RpoH and GroES. Based on RNA-Seq analysis of the selected strains, E. coli Eopt, E. coli Sopt, E. coli Ropt, and wild type, amino acid substitutions in σ factors and molecular chaperones regulated the expression level of genes related to gene replication, recombination, and repair. Furthermore, the regulatory factor mutants were identified to be beneficial to the soluble expression of two other heterologous proteins, amylase and bone morphological protein 12.
In this study, a high-throughput screening method was developed for improved soluble LOX expression. The obtained positive mutants of the regulatory factor were analyzed and employed for the expression of other heterologous proteins, thus providing a potential solution for the inclusion-body protein.
脂氧合酶（EC. 1.13.11.12，LOX）可催化将氧添加到多不饱和脂肪酸中以产生氢过氧化物，其在食品、化工和制药行业中广泛应用。近年来，大肠杆菌异源生产LOX引起了广泛关注。然而，在大肠杆菌中过表达的重组LOX由于蛋白质错误折叠而聚集并形成不溶性包涵体。
在本研究中，开发了一种基于分裂绿色荧光蛋白的筛选方法，以筛选用于可溶性LOX表达的σ因子和分子伴侣。使用本文开发的高通量筛选方法分析了Skp、GroES和RpoH的三个突变体文库，并获得了一系列可溶性异源LOX产量显著更高的突变体。分离出的突变体中LOX的可溶性表达水平提高了4.2至5.3倍。此外，在含有调节因子突变体RpoH和GroES的大肠杆菌REopt中观察到最高的LOX活性（高达6240±269 U·g-DCW）。基于对所选菌株大肠杆菌Eopt、大肠杆菌Sopt、大肠杆菌Ropt和野生型的RNA测序分析，σ因子和分子伴侣中的氨基酸取代调节了与基因复制、重组和修复相关基因的表达水平。此外，已确定调节因子突变体有利于另外两种异源蛋白淀粉酶和骨形态发生蛋白12的可溶性表达。
在本研究中，开发了一种高通量筛选方法以提高可溶性LOX的表达。对获得的调节因子阳性突变体进行了分析并用于其他异源蛋白的表达，从而为包涵体蛋白提供了一种潜在的解决方案。
