ObjectiveTo 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.ResultsEighteen 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 degrees C and 6.0 degrees C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1h incubation at 37 degrees C was obtained for H29R (2.0U/mg) and H15K (3.1U/mg) compared to wild-type (0.3U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10M 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.ConclusionH29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.