He Guimei, Jiang Meiru, Cui Zhenzhen, Sun Xi, Chen Tao, Wang Zhiwen
Key Laboratory of Systems Bioengineering (Ministry of Education), Frontier Science Center for Synthetic Biology (Ministry of Education), Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Key Laboratory of Systems Bioengineering (Ministry of Education), Frontier Science Center for Synthetic Biology (Ministry of Education), Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
J Biosci Bioeng. 2022 Nov;134(5):416-423. doi: 10.1016/j.jbiosc.2022.07.019. Epub 2022 Sep 9.
5-Aminolevulinic acid (5-ALA), a vital precursor for the biosynthesis of tetrapyrrole compounds, has been widely applied in agriculture and medicine, while extremely potential for the treatment of cancers, corona virus disease 2019 (COVID-19) and metabolic diseases in recent years. With the development of metabolic engineering and synthetic biology, the biosynthesis of 5-ALA has attracted increasing attention. 5-Aminolevulinic acid synthase (ALAS), the key enzyme for 5-ALA synthesis in the C4 pathway, is subject to stringent feedback inhibition by heme. In this work, cysteine-targeted mutation of ALAS was proposed to overcome this drawback. ALAS from Rhodopseudomonas palustris (RP-ALAS) and Rhodobacter capsulatus (RC-ALAS) were selected for mutation and eight variants were generated. Variants RP-C132A and RC-C201A increased enzyme activities and released hemin inhibition, respectively, maintaining 82.5% and 81.9% residual activities in the presence of 15 μM hemin. Moreover, the two variants exhibited higher stability than that of their corresponding wild-type enzymes. Corynebacterium glutamicum overexpressing RP-C132A and RC-C201A produced 14.0% and 21.6% higher titers of 5-ALA than the control, respectively. These results strongly suggested that variants RP-C132A and RC-C201A obtained by utilizing cysteine-targeted mutation strategy released hemin inhibition, broadening their applications in 5-ALA biosynthesis.
5-氨基乙酰丙酸（5-ALA）是四吡咯化合物生物合成的重要前体，已广泛应用于农业和医学领域，近年来在癌症、2019冠状病毒病（COVID-19）和代谢性疾病的治疗方面极具潜力。随着代谢工程和合成生物学的发展，5-ALA的生物合成受到了越来越多的关注。5-氨基乙酰丙酸合酶（ALAS）是C4途径中5-ALA合成的关键酶，受到血红素的严格反馈抑制。在这项工作中，提出了对ALAS进行半胱氨酸靶向突变以克服这一缺点。选择来自沼泽红假单胞菌（RP-ALAS）和荚膜红细菌（RC-ALAS）的ALAS进行突变，产生了8个变体。变体RP-C132A和RC-C201A分别提高了酶活性并解除了血红素抑制，在存在15μM血红素的情况下保持了82.5%和81.9%的残余活性。此外，这两个变体表现出比其相应野生型酶更高的稳定性。过表达RP-C132A和RC-C201A的谷氨酸棒杆菌产生的5-ALA滴度分别比对照高14.0%和21.6%。这些结果有力地表明，通过利用半胱氨酸靶向突变策略获得的变体RP-C132A和RC-C201A解除了血红素抑制，拓宽了它们在5-ALA生物合成中的应用。
