4-Hydroxyisoleucine production of recombinant Corynebacterium glutamicum ssp. lactofermentum under optimal corn steep liquor limitation

Abstract

4-Hydroxyisoleucine (4-HIL) is a nonproteinogenic amino acid that exhibits insulinotropic biological activity. Here, L-isoleucine dioxygenase gene (ido) derived from Bacillus thuringiensis YBT-1520 was cloned and expressed in an L-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, in order to directly convert its endogenous L-isoleucine (Ile) into 4-HIL through single-step fermentation. The effects of corn steep liquor limitation as well as ido and truncated idoΔ6 overexpression on 4-HIL production were researched. 4-HIL production by ido-overexpressing strain was improved to 65.44 ± 2.27 mM after fermented for 144 h under corn steep liquor-subsufficient condition, obviously higher than that under corn steep liquor-rich and insufficient conditions. The conversion ratio of Ile to 4-HIL increased to 0.85 mol/mol. In addition, 4-HIL production by ido-overexpressing strain was higher than that by idoΔ6-overexpressing strain, in accord with the relatively higher affinity of Ido as compared to IdoΔ6. This research generated a novel system for 4-HIL de novo biosynthesis and demonstrated corn steep liquor limitation as a useful strategy for improving 4-HIL production in recombinant C. glutamicum ssp. lactofermentum.

Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified l-Isoleucine Biosensor in Recombinant Corynebacterium glutamicum

“…The conversion ratio of Ile to 4-HIL was calculated as the mole of 4-HIL divided by the total moles of Ile and 4-HIL. The intracellular amino acid concentration was measured as described previously . 4-HIL fermentation in a fermenter was conducted in a 2.4-L bioreactor (T&J-Minbox 2.4L*4; T&J Bioengineering Co. LTD, Shanghai, China) containing 1.0 L of optimized fermentation medium with 100 g/L glucose.…”

“…The intracellular amino acid concentration was measured as described previously. 40 4-HIL fermentation in a fermenter was conducted in a 2.4-L bioreactor (T&J-Minbox 2.4L*4; T&J Bioengineering Co. LTD, Shanghai, China) containing 1.0 L of optimized fermentation medium with 100 g/L glucose. The temperature and pH were kept at 30 °C and 6.8, respectively.…”

“…glutamicum ssp. lactofermentum strain SN01, 4-HIL was de novo synthesized from glucose, and neither Ile nor α-KG was added . To improve 4-HIL production, static metabolic engineering was then applied, such as enhancing substrates supply and/or IDO activity by gene expression and deletion. − However, low titer and yield still restricted the engineering of this strain, likely due to the burden caused by constitutive gene expression and the imbalance of metabolic flux in such static engineering.…”

2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis

“…Hence, it is feasible to scale up the foregoing 4-HIL production system. In contrast, an in vivo 2-KG supply for 4-HIL synthesis has been successfully implemented by redirecting the TCA cycle [ 21 , 38 ]. However, metabolic engineering strategies of shifting the carbon flux from l -Ile to 2-KG by boosting the TCA cycle and further increasing the pool of oxaloacetate were adopted to obtain 34.21 g/L (232.45 mM) of 4-HIL at 64 h, inevitably hindering normal cell growth [ 21 ].…”

Comparative transcriptome analysis of global effect of ddh and lysE deletion on 4-hydroxyisoleucine production in Corynebacterium glutamicum

“…In our previous study, the ido gene of B. thuringiensis YBT-1520 was expressed in the Ile-producing C. glutamicum strain SN01 to synthesize 4-HIL. The yield of 4-HIL is 65.44 ± 2.27 mM after a 144-h fermentation [12]. In another research, the codon-optimized ido gene was expressed in C. glutamicum YI, another Ile-producing strain, and 2.31 g/L of 4-HIL was accumulated after a 48-h fermentation [13].…”