Tan Zijian, Zhao Jing, Chen Jiuzhou, Rao Deming, Zhou Wenjuan, Chen Ning, Zheng Ping, Sun Jibin, Ma Yanhe
College of Chemical Engineering and Materials Science, Tianjin University of Science & Technology, Tianjin, 300457, China.
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Biotechnol Lett. 2019 Jan;41(1):181-191. doi: 10.1007/s10529-018-2627-z. Epub 2018 Nov 29.
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.
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.
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.
通过计算机辅助的合理设计策略，提高沼泽红假单胞菌5-氨基乙酰丙酸合酶（RP-ALAS）的热稳定性并解除血红素对其的抑制作用。
通过测量18种RP-ALAS单变体加热后的残余活性，对其进行了合理设计和筛选。其中，H29R和H15K的解链温度分别比野生型高2.3℃和6.0℃。与野生型（0.3 U/mg）相比，H29R（2.0 U/mg）和H15K（3.1 U/mg）在37℃孵育1小时后的比活性分别提高了6.7倍和10.3倍。此外，H29R和H15K在血红素存在下具有更高的残余活性（例如，在10μM血红素时分别为64%和76%，而野生型为27%）。在使用表达H29R和H15K的谷氨酸棒杆菌ATCC 13032进行发酵时，ALA产量分别提高了6%和22%。
H29R和H15K表现出高热稳定性、降低的血红素抑制作用和略高的活性，表明这两个变体是生物生产ALA的良好候选者。
