ABSTRACT
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.