High yield production of L-isoleucine through readjusting the ratio of two direct precursors in Escherichia coli

• Published in: Bioresource technology Vol. 418; p. 131889
• Main Authors: Shi, Congrong, Huo, Xiaojing, You, Ran, Tao, Yong, Lin, Baixue
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.02.2025
• Subjects: biochemical pathways; biosynthesis; Biosynthetic Pathways; byproducts; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; essential amino acids; glucose; Isoleucine - biosynthesis; Keto Acids - metabolism; Metabolic Engineering - methods; technology; threonine; Threonine - metabolism; valine; Valine - metabolism; L-Val; L-Ile; OD600; L-Thr; PEP; C. glutamicum; E. coli; HPLC; PTS
• ISSN: 0960-8524; 1873-2976; 1873-2976
• DOI: 10.1016/j.biortech.2024.131889

[Display omitted] •E. coli strains were metabolically engineered for efficient production of L-Ile.•Readjusting the ratio of two precursors enhanced L-Ile yield and reduced L-Val.•Enzymes with high substrate specificity were screened for L-Ile synthesis.•High yield and productivity of L-Ile was achieved. L-isoleucine (L-Ile) is an essential amino acid, which biosynthesis has been studied extensively. L-valine (L-Val) is a major by-product that limits L-Ile yield. Here, multiple strategies were employed to enhance L-Ile production and reduce L-Val in Escherichia coli. The biosynthetic pathway of L-Ile was divided into L-threonine (L-Thr) and L-Ile modules. In the L-Ile module, AHAS and IlvC were screened for high affinity toward 2-oxobutanoate and α-aceto-α-hydroxybutyrate, respectively, enhancing L-Ile yield. The L-Thr module was optimized via the overexpression of key enzymes, increasing 2-oxobutanoate supply. Furthermore, the deletion ofptsGandpykFand overexpression of citramalate synthase were employed to balance the precursors ratio. The engineered strain achieved a high yield of 0.40 mol L-Ile/mol glucose, a high productivity of 0.83 g/L/h, and significantly reduced L-Val accumulation, which would facilitate the subsequent separation and purification of L-Ile. This work provides a sustainable platform for the production of L-Ile derivatives.