Programming adaptive laboratory evolution of 4-hydroxyisoleucine production driven by a lysine biosensor in Corynebacterium glutamicum

Abstract

Abstract4-Hydroxyisoleucine (4-HIL) is a promising drug for treating diabetes. In our previous study, 4-HIL was synthesized from self-produced L-isoleucine (Ile) in Corynebacterium glutamicum by expressing an Ile dioxygenase gene. Although the 4-HIL production of recombinant strain SZ06 increased significantly, a by-product, L-lysine (Lys) was accumulated because of the share of the first several enzymes in Ile and Lys biosynthetic pathways. In this study, programming adaptive laboratory evolution (ALE) was designed and conducted in SZ06 to promote 4-HIL biosynthesis. At first, a programming evolutionary system pMK was constructed, which contains a Lys biosensor LysG-PlysE and an evolutionary actuator composed of a mutagenesis gene and a fluorescent protein gene. The evolutionary strain SZ06/pMK was then let to be evolved programmatically and spontaneously by sensing Lys concentration. After successive rounds of evolution, nine mutant strains K1 − K9 with significantly increased 4-HIL production and growth performance were obtained. The maximum 4-HIL titer was 152.19 ± 14.60 mM, 28.4% higher than that in SZ06. This titer was higher than those of all the metabolic engineered C. glutamicum strains ever constructed. The whole genome sequencing of the nine evolved strains revealed approximately 30 genetic mutations in each strain. Only one mutation was directly related to the Lys biosynthetic pathway. Therefore, programming ALE driven by Lys biosensor can be used as an effective strategy to increase 4-HIL production in C. glutamicum.

Metabolic Engineering of Corynebacterium glutamicum for Sustainable Production of the Aromatic Dicarboxylic Acid Dipicolinic Acid

“…The slight inhibitory effect of DPA on growth of C. glutamicum WT is not yet understood, however, the structure of its 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase also contains a divalent cation, namely Mn 2+ [132]. In recent years, adaptive laboratory evolution has emerged as an excellent approach to select for improved growth, tolerance and production [133][134][135][136]. Tolerance of C. glutamicum to methanol [137,138], anthranilate [139] and indole [140] was improved by this strategy that may be applicable to increasing tolerance to DPA.…”

Directed Evolution of 4-Hydroxyphenylpyruvate Biosensors Based on a Dual Selection System

“…Their detectable fluorescence is often used as the output signal in quantitative metabolite determination and high-throughput screening [12,13]. Thus, aTF-based biosensors with innately high ligand specificity are widely used in molecule detection, enzyme-directed evolution, dynamic control of metabolic pathways and adaptive laboratory evolution [14][15][16][17]. Despite their promising applications in synthetic biology, only a handful of TFs have been developed as biosensors due to the limited number of reported ligand compounds and their effector aTFs [18].…”

Improving Furfural Tolerance of Escherichia coli by Integrating Adaptive Laboratory Evolution with CRISPR-Enabled Trackable Genome Engineering (CREATE)

“…1,2 ALE was successfully applied to improve bacterial growth, 3,4 substrate consumption, 5−7 strain robustness, 8−10 as well as the titer, yield, and productivity (TRY) of products. 11,12 It is also often combined with metabolic engineering and synthetic biology. 13,14 Adaptive evolution functions by subjecting a microbial population to selection pressure, and it traditionally relies on spontaneously arising genetic diversity to drive.…”

“…Adaptive laboratory evolution (ALE) is a widespread used strain-improvement method in which bacteria are continually cultured in stressful conditions for several generations to improve fitness by accumulating beneficial mutations. , ALE was successfully applied to improve bacterial growth, , substrate consumption, − strain robustness, − as well as the titer, yield, and productivity (TRY) of products. , It is also often combined with metabolic engineering and synthetic biology. , …”