Jia Yuan-Yuan, Xie Yu-Li, Yang Lu-Lu, Shi Hong-Ling, Lu Yun-Feng, Zhang Si-Pu, Tang Cun-Duo, Yao Lun-Guang, Kan Yun-Chao
Henan Provincial Engineering Laboratory of Insect Bio-reactor, Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Nanyang Normal University, Nanyang, China.
School of Life Sciences and Agricultural Engineering, Nanyang Normal University, Nanyang, China.
Front Bioeng Biotechnol. 2021 Mar 30;9:655522. doi: 10.3389/fbioe.2021.655522. eCollection 2021.
Leucine dehydrogenase (LDH) is a NAD-dependent oxidoreductase, which can selectively catalyze α-keto acids to obtain α-amino acids and their derivatives. It plays a key role in the biosynthesis of -tert-leucine (-Tle). As a non-naturally chiral amino acid, -Tle can be used as an animal feed additive, nutrition fortifier, which is a perspective and important building block in the pharmaceutical, cosmetic, and food additive industry. In this study, four hypothetical leucine dehydrogenases were discovered by using genome mining technology, using the highly active leucine dehydrogenase LeuDH as a probe. These four leucine dehydrogenases were expressed in BL21(DE3), respectively, and purified to homogeneity and characterized. Compared with the other enzymes, the specific activity of LeuDH also shows stronger advantage. In addition, the highly selective biosynthesis of -Tle from trimethylpyruvic acid (TMP) was successfully carried out by whole-cell catalysis using engineered cells as biocatalyst, which can efficiently coexpress leucine dehydrogenase and formate dehydrogenase. One hundred-millimolar TMP was catalyzed for 25 h, and the yield and space-time yield of -Tle reached 87.38% ( >99.99%) and 10.90 g L day. In short, this research has initially achieved the biosynthesis of -Tle, laying a solid foundation for the realization of low-cost and large-scale biosynthesis of -Tle.
亮氨酸脱氢酶（LDH）是一种依赖烟酰胺腺嘌呤二核苷酸（NAD）的氧化还原酶，它能够选择性地催化α-酮酸生成α-氨基酸及其衍生物。它在叔亮氨酸（-Tle）的生物合成中起着关键作用。作为一种非天然手性氨基酸，-Tle可用作动物饲料添加剂、营养强化剂，是制药、化妆品和食品添加剂行业中一种具有前景且重要的结构单元。在本研究中，以高活性亮氨酸脱氢酶LeuDH为探针，利用基因组挖掘技术发现了四种假定的亮氨酸脱氢酶。这四种亮氨酸脱氢酶分别在大肠杆菌BL21(DE3)中表达，并纯化至均一性且进行了表征。与其他酶相比，LeuDH的比活性也显示出更强的优势。此外，以工程化细胞作为生物催化剂，通过全细胞催化成功地从三甲基丙酮酸（TMP）高效选择性地生物合成了-Tle，该工程化细胞能够高效共表达亮氨酸脱氢酶和甲酸脱氢酶。催化100 mM的TMP反应25 h，-Tle的产率和时空产率分别达到87.38%（>99.99%）和10.90 g L-1 day-1。简而言之，本研究初步实现了-Tle的生物合成，为低成本、大规模生物合成-Tle奠定了坚实基础。
