Photocontrol of Itaconic Acid Synthesis in Escherichia coli

Abstract

Metabolic engineering aims to control cellular metabolic flow and maximize the production of a product of interest. Photocontrol of the activities of proteins is an effective method for accurately regulating metabolic pathways. In this study, we inserted the photosensor light-oxygen-voltage-sensing domain 2 of Avena sativa (AsLOV2) into selected sites of isocitrate dehydrogenase (IDH), the key enzyme in the competitive pathway of itaconic acid (ITA) synthesis, to construct photoswitchable IDH-AsLOV2 (ILOVs). These engineered light-sensitive proteins were used to regulate the metabolic flux of the tricarboxylic acid (TCA) cycle in Escherichia coli to improve ITA production. The engineered fusion proteins ILOV2, ILOV3, ILOV6, and ILOV7 exhibited effective reversibility under the oscillation of darkness and blue light illumination in vitro. The efficacies of the intracellular photoswitches were evaluated, and an optimal photocontrol strategy was established in vivo. The ITA titer was significantly enhanced to 3.30 g/L for strain ITAΔ43, which displayed superior photoswitchable potency for ITA production compared with the strains that completely deleted the icd gene. The photocontrol strategy developed here can be extended for process optimization and titer improvement of other high-value bioengineering chemicals.

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production

“…By applying nitrogen-limiting conditions, C. glutamicum ITA1 produced 0.32 ± 0.09 g IA L −1 after 72 h, corresponding to a Y P/S of 8 ± 2 mmol IA per mol acetate (Table 1 ). Due to the rather low affinity of CAD towards its substrate cis -aconitate (K M of 2.45 mM) [ 35 ], reducing carbon flux via IDH is a common strategy for IA production in bacterial hosts [ 33 , 36 , 37 ]. Therefore, we exchanged the native icd gene in C. glutamicum ITA1 by three versions that code for IDH A94D , IDH G407S and IDH R453C , which possess 10, 55 and 29% of the wild type IDH activity, respectively [ 38 ].…”

Recent Progress in Metabolic Engineering of Escherichia coli for the Production of Various C4 and C5-Dicarboxylic Acids

“…This light-mediated regulation enables precise control over metabolic flux, leading to enhanced itaconate production. 99 In comparison to the dark phase of the culture, isocitrate dehydrogenase activity was suppressed during the light phase, resulting in an enhancement of itaconate titer by up to 1.2-fold due to prolonged irradiation time (from 2 to 5 min). This photoregulation technique led to a substantial increase in itaconate production, reaching up to 3.30 g/L, which represents a 1.4-fold improvement over the results obtained from dark treatment alone (2.36 g/L).…”

“…Any imbalance can result in a metabolic burden that can impede effective production . Addressing these challenges required dynamic regulation that replicates the metabolic control systems of various organisms, allowing cells to alter metabolic flux by detecting external stress or intracellular conditions, thereby mitigating the metabolic burden and facilitating efficient chemical production. ,− …”

“…This photoregulation technique led to a substantial increase in itaconate production, reaching up to 3.30 g/L, which represents a 1.4-fold improvement over the results obtained from dark treatment alone (2.36 g/L). 99 The CRISPRi tool was used to regulate the expression of various target genes, dynamically controlling the metabolic flux between TCA cycle and itaconate synthesis pathway. A selfinducing system (CiMS) was developed using an itaconatesensing module comprised of an itaconate-responsive transcription factor, ItcR, from Yersinia pseudotuberculosis, and a flux interference module using CRISPRi.…”

“…Engineered proteins, known as ILOVs, demonstrate reversible dehydrogenase activity in vitro by alternating between darkness and blue light in the range of 420–480 nm. This light-mediated regulation enables precise control over metabolic flux, leading to enhanced itaconate production . In comparison to the dark phase of the culture, isocitrate dehydrogenase activity was suppressed during the light phase, resulting in an enhancement of itaconate titer by up to 1.2-fold due to prolonged irradiation time (from 2 to 5 min).…”