Ma Zeng-Xin, Zhang Min, Zhang Chang-Tai, Zhang Hui, Mo Xu-Hua, Xing Xin-Hui, Yang Song
School of Life Sciences, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao Agricultural University, Qingdao, Shandong Province, People's Republic of China.
Shandong Longkete Enzyme Co., Ltd., Linyi, Shandong, People's Republic of China.
Biotechnol J. 2021 Jun;16(6):e2000413. doi: 10.1002/biot.202000413. Epub 2021 Mar 23.
Methylorubrum extorquens AM1 can be engineered to convert methanol to value-added chemicals. Most of these chemicals derive from acetyl-CoA involved in the serine cycle. However, recent studies on methylotrophic metabolism have suggested that C3 pyruvate is a good potential precursor for broadening the types of synthesized products.
In the present study, we found that isobutanol was a model chemical that could be generated from pyruvate through a 2-keto acid pathway. Initially, the engineered M. extorquens AM1 could only produce a trace amount of isobutanol at 0.62 mgL after introducing the heterologous 2-ketoisovalerate decarboxylase and alcohol dehydrogenase. Furthermore, the metabolomic analysis revealed that insufficient carbon fluxes through 2-ketoisovalerate and pyruvate were the key limitation steps for efficient biosynthesis of isobutanol. Based on this analysis, the titer of isobutanol was improved by over 20-fold after overexpressing alsS gene encoding acetolactate synthase and deleting ldhA gene for lactate dehydrogenase. Moreover, substituting the cell chassis with the isobutanol-tolerant strain isolated from adaptive evolution of M. extorquens AM1 further increased the production of isobutanol by 1.7-fold, resulting in the final titer of 19 mgL in flask cultivation.
Our current findings provided promising insights into engineering methylotrophic cell factories capable of converting methanol to isobutanol or value-added chemicals using pyruvate as the precursor.
甲基营养型外排甲基杆菌AM1可被改造用于将甲醇转化为高附加值化学品。这些化学品大多来源于丝氨酸循环中涉及的乙酰辅酶A。然而，最近关于甲基营养代谢的研究表明，C3丙酮酸是拓宽合成产品类型的良好潜在前体。
在本研究中，我们发现异丁醇是一种可通过2-酮酸途径由丙酮酸生成的模型化学品。最初，经工程改造的甲基营养型外排甲基杆菌AM1在引入异源2-酮异戊酸脱羧酶和乙醇脱氢酶后，仅能产生痕量的异丁醇，浓度为0.62 mg/L。此外，代谢组学分析表明，通过2-酮异戊酸和丙酮酸的碳通量不足是异丁醇高效生物合成的关键限制步骤。基于此分析，在过表达编码乙酰乳酸合酶的alsS基因并删除乳酸脱氢酶的ldhA基因后，异丁醇的产量提高了20多倍。此外，用从甲基营养型外排甲基杆菌AM1的适应性进化中分离出的耐异丁醇菌株替代细胞底盘，进一步使异丁醇产量提高了1.7倍，在摇瓶培养中最终产量达到19 mg/L。
我们目前的研究结果为工程化甲基营养型细胞工厂提供了有前景的见解，该工厂能够以丙酮酸为前体将甲醇转化为异丁醇或高附加值化学品。
