OBJECTIVE: To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. RESULTS: Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 μM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. CONCLUSION: H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.
Submitter: Shu-Ching Ou
Submission Date: March 8, 2019, 4:47 p.m.
|Number of data points||874|
|Assays/Quantities/Protocols||Experimental Assay: kcat ; Experimental Assay: Km ; Experimental Assay: kcat/Km ; Computational Protocol: Occurring Frequency at Histidine positions in ALAS family ; Computational Protocol: ΔΔG: FoldX|
|Libraries||Variants for RP-ALAS_Substrate ; Variants for RP-ALAS|