Overcoming glutamate auxotrophy in Escherichia coli itaconate overproducer by the Weimberg pathway

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Ken W. Lu, Chris T. Wang, Hengray Chang, Ryan S. Wang, Claire R. Shen
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引用次数: 4

Abstract

Biosynthesis of itaconic acid occurs through decarboxylation of the TCA cycle intermediate cis-aconitate. Engineering of efficient itaconate producers often requires elimination of the highly active isocitrate dehydrogenase to conserve cis-aconitate, leading to 2-ketoglutarate auxotrophy in the producing strains. Supplementation of glutamate or complex protein hydrolysate then becomes necessary, often in large quantities, to support the high cell density desired during itaconate fermentation and adds to the production cost. Here, we present an alternative approach to overcome the glutamate auxotrophy in itaconate producers by synthetically introducing the Weimberg pathway from Burkholderia xenovorans for 2-ketoglutarate biosynthesis. Because of its independence from natural carbohydrate assimilation pathways in Escherichia coli, the Weimberg pathway is able to provide 2-ketoglutarate using xylose without compromising the carbon flux toward itaconate. With xylose concentration carefully tuned to minimize excess 2-ketoglutarate flux in the stationary phase, the final strain accumulated 20 g/L of itaconate in minimal medium from 18 g/L of xylose and 45 g/L of glycerol. Necessity of the recombinant Weimberg pathway for growth also allowed us to maintain multi-copy plasmids carrying in operon the itaconate-producing genes without addition of antibiotics. Use of the Weimberg pathway for growth restoration is applicable to other production systems with disrupted 2-ketoglutarate synthesis.

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温伯格途径克服衣康酸过量大肠杆菌谷氨酸缺失症
衣康酸的生物合成是通过三羧酸循环中间体顺-aconitate的脱羧发生的。高效的衣康酸生产者工程通常需要消除高活性的异柠檬酸脱氢酶来保存顺式乌头酸,导致生产菌株中2-酮戊二酸萎缩。为了支持衣康酸发酵过程中所需的高细胞密度,通常需要大量补充谷氨酸或复合蛋白水解物,这增加了生产成本。在这里,我们提出了一种替代方法,通过综合引入来自异种伯克霍尔德菌的Weimberg途径,用于2-酮戊二酸的生物合成,来克服衣康酸生产者的谷氨酸缺失。由于其独立于大肠杆菌的天然碳水化合物同化途径,Weimberg途径能够利用木糖提供2-酮戊二酸,而不会影响衣康酸的碳通量。仔细调整木糖浓度以减少固定相中过量的2-酮戊二酸通量,最终菌株在最小培养基中从18 g/L木糖和45 g/L甘油积累了20 g/L衣康酸。重组Weimberg途径对生长的必要性也使我们能够在不添加抗生素的情况下维持在操纵子中携带产itaconate基因的多拷贝质粒。使用Weimberg途径恢复生长适用于其他2-酮戊二酸合成中断的生产系统。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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