文献类型：期刊文献

英文题名：Inside-Out Rational Design of Ornithine Cyclodeaminase RlOCD from Rhizobium leguminosarum by a Multiregion Synergy Strategy for Efficient Synthesis of l-Pipecolic Acid

作者：Gao, Weijie[1];Jing, Zijian[1];Meng, Yifang[1];Liu, Qinghai[1];Wang, Hualei[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2024

卷号：72

期号：46

起止页码：25782

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20244317248288);WOS:【SCI-EXPANDED(收录号:WOS:001336908300001)】；

基金：This study was supported by the National Key Research and Development Program of China (grant number: 2021YFC2102100) and the Fundamental Research Funds for the Central Universities.

语种：英文

外文关键词：l-pipecolic acid; ornithine cyclodeaminase; rational design

摘要：Lysine cyclodeaminase (LCD)-mediated synthesis of l-pipecolic acid (l-PA) from l-lysine (l-Lys) is a promising approach. However, only one LCD has been reported, and its inadequate activity limits industrial applications. To address this problem, a substrate analogue-guided enzyme mining strategy was employed. A novel ornithine cyclodeaminase (OCD) from Rhizobium leguminosarum (RlOCD) was identified in combination with directed macrogenomic approaches. RlOCD displayed a conversion rate of 28% at a substrate loading as high as 1000 mM. A multiregion synergy strategy consisting of pocket reshaping, dynamical cross-correlation matrix-guided coevolutionary design, and surface modification was used to design RlOCD from the inside-out. A quadruple mutant (V93C/L119C/I170T/R90L) designated Mu4 with significantly increased activity was obtained, which showed a 28.46-fold increase in the catalytic efficiency. The conversion of Mu4 was 91% within 10 h at 1000 mM (146.19 g L-1) loading. The space-time yield of 282.1 g L-1 d(-1) is the highest level ever reported. Molecular dynamics simulations and interaction analyses revealed that efficient pocket expansion and unique conformational rearrangements increased the affinity for the substrate, resulting in a more catalytically active conformation. This study expands the toolbox for the production of l-PA and demonstrates the effectiveness and potential of Mu4 for its production.