The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi, People's Republic of China.
State Key Laboratory of Food Science and Technology, School of Biotechnology, Jiangnan University, WuXi, People's Republic of China.
Biotechnol Bioeng. 2018 Jul;115(7):1764-1777. doi: 10.1002/bit.26591. Epub 2018 Apr 16.
l-lysine is an important amino acid in animals and humans and NADPH is a vital cofactor for maximizing the efficiency of l-lysine fermentation. Dihydrodipicolinate reductase (DHDPR), an NAD(P)H-dependent enzyme, shows a variance in nucleotide-cofactor affinity in bacteria. In this study, we rationally engineered Corynebacterium glutamicum DHDPR (CgDHDPR) to switch its nucleotide-cofactor specificity resulting in an increase in final titer (from 82.6 to 117.3 g L ), carbon yield (from 0.35 to 0.44 g [g glucose] ) and productivity (from 2.07 to 2.93 g L hr ) of l-lysine in JL-6 ΔdapB::Ec-dapB in fed-batch fermentation. To do this, we comparatively analyzed the characteristics of CgDHDPR and Escherichia coli DHDPR (EcDHDPR), indicating that hetero-expression of NADH-dependent EcDHDPR increased l-lysine production. Subsequently, we rationally modified the conserved structure of cofactor-binding motif, and results indicated that introducing the mutation K11A or R13A in CgDHDPR and introducing the mutation R16A or R39A in EcDHDPR modifies the nucleotide-cofactor affinity of DHDPR. Lastly, the effects of these mutated DHDPRs on l-lysine production were investigated. The highest increase (26.2%) in l-lysine production was observed for JL-6 ΔdapB::Ec-dapB , followed by JL-6 Cg-dapB (21.4%) and JL-6 ΔdapB::Ec-dapB (15.2%). This is the first report of a rational modification of DHDPR that enhances the l-lysine production and yield through the modulation of nucleotide-cofactor specificity.
L-赖氨酸是动物和人类的一种重要氨基酸，NADPH 是最大限度提高 L-赖氨酸发酵效率的重要辅因子。二氢二吡啶羧酸还原酶（DHDPR）是一种依赖 NAD(P)H 的酶，在细菌中显示出核苷酸辅因子亲和力的差异。在本研究中，我们通过理性工程改造谷氨酸棒杆菌 DHDPR（CgDHDPR），使其核苷酸辅因子特异性发生转变，从而提高终浓度（从 82.6 克/升到 117.3 克/升）、碳产率（从 0.35 克/[g 葡萄糖]到 0.44 克/[g 葡萄糖]）和生产效率（从 2.07 克/升/小时到 2.93 克/升/小时）。为了实现这一点，我们比较分析了 CgDHDPR 和大肠杆菌 DHDPR（EcDHDPR）的特性，表明异源表达 NADH 依赖性 EcDHDPR 增加了 L-赖氨酸的产量。随后，我们对辅因子结合基序的保守结构进行了理性修饰，结果表明，在 CgDHDPR 中引入突变 K11A 或 R13A，以及在 EcDHDPR 中引入突变 R16A 或 R39A，改变了 DHDPR 的核苷酸辅因子亲和力。最后，研究了这些突变 DHDPR 对 L-赖氨酸生产的影响。在 JL-6 ΔdapB::Ec-dapB 中，L-赖氨酸产量的最高增加（26.2%），其次是 JL-6 Cg-dapB（21.4%）和 JL-6 ΔdapB::Ec-dapB（15.2%）。这是首次报道通过调节核苷酸辅因子特异性，理性修饰 DHDPR 可提高 L-赖氨酸的产量和产率。
