改造乙酰羟酸合成酶提高L-亮氨酸产量

Improvement of L--Leucine Production by Modifying Acetohydroxyacid Synthase

摘要:

乙酰羟酸合成酶（acetohydroxy acid synthase，AHAS，编码基因ilvBN）是L-亮氨酸合成途径的第一个限速酶。以谷氨酸棒杆菌XL-3（Corynebacterium glutamicum XL-3）为底盘细胞，通过分析并改造AHAS增加其对底物丙酮酸的偏好性，从而提高L--亮氨酸产量。首先利用AHAS的氨基酸序列进行同源建模，根据蛋白质结构进行丙氨酸扫描，找到突变的潜在位点，通过测定突变体酶活力和重组菌株的L--亮氨酸产量寻找最适突变体。测定结果发现将157位Gln突变成Arg能够有效提高AHAS催化丙酮酸的能力，最终重组菌株的L--亮氨酸产量达到（23.5±1.8）g/L，比出发菌株谷氨酸棒杆菌XL-3增加了51%，同时副产物L--异亮氨酸产量有所下降。因此，通过对AHAS的理性改造促进了L--亮氨酸的合成，该研究结果对后续利用蛋白质工程强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。

Abstract:

Acetohydroxy acid synthase (AHAS, encoded by the gene ilvBN) is the first rate-limiting enzyme in L--leucine synthesis pathway. Corynebacterium glutamicum XL-3 was used as the chassis cell to increase its preference for pyruvate and improve the yield of L--leucine by analyzing and modifying AHAS. Initially, the amino acid sequence of AHAS was used for homologous modeling. Then potential mutation sites were identified through alanine scanning of the protein structure. The optimal mutant was selected by measuring the catalytic activity of the mutant and theL--leucine yield of the recombinant strain. The results showed that substituting Gln157 with Arg effectively enhanced the ability of AHAS to catalyze pyruvate, resulting in a final L--leucine production of (23.5±1.8) g/L in the recombinant strain, which was a 51% increase compared to the parent strain Corynebacterium glutamicum XL-3. In addition, the yield of by-products L--isoleucine decreased. Therefore, the rational modification of AHAS could promote the synthesis of L--leucine. The research results have important implications for the subsequent use of protein engineering to strengthen the microbial synthesis of branched-chain amino acids such as L--leucine.