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

Abstract

4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O2, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-P brnFE was researched. The native P brnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger P brnFE N promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9–74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong P tacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different P brnFE N-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O2 supply in ST04 by different combinations of P brnFE N-odhI and P brnFE N-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by P brnFE 7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.

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

“…This indicates that the deletion of ddh could promote the accumulation of 4-HIL, but could not attenuate the synthesis of Lys. Although the 4-HIL titer of SL09 in this study was lower than that of some improved strains, such as SZ05 [19], SF12 [20] and ST17 [21], reported so far (Table 3), SL09 accumulated almost no other by-products except Lys, indicating its potential for further reconstruction. The remained Lys accumulation in SL09 is contrary to the result reported previously [15], in which the deletion of ddh in recombinant C. glutamicum ATCC13869 weakened the Lys synthesis.…”

Synthetic Biology Toolkits and Metabolic Engineering Applied in Corynebacterium glutamicum for Biomanufacturing

“…For example, the transcriptional regulator Lrp can activate the expression of two-component permease BrnFE in response to the concentration of intracellular l -methionine and branched-chain amino acids l -valine, l -isoleucine, and l -leucine, maintaining the normal intracellular amino acid concentration . Tan et al (2020) constructed an engineered l -leucine biosensor with different combinations of natural or modified P brnFE promoters to regulate the expression of ido (encoding l -leucine dioxygenase), odhI (encoding ODHC inhibitor OdhI), and vgb ( Vitreoscilla hemoglobin VHb) . With this regulation, the 4-hydroxyisoleucine titer increased to 135.34 mM .…”

“…34 Tan et al ( 2020) constructed an engineered L-leucine biosensor with different combinations of natural or modified P brnFE promoters to regulate the expression of ido (encoding L-leucine dioxygenase), odhI (encoding ODHC inhibitor OdhI), and vgb (Vitreoscilla hemoglobin VHb). 35 With this regulation, the 4hydroxyisoleucine titer increased to 135.34 mM. 35 Nevertheless, the biosensor based on native TF had a poor dynamic regulation range and showed a certain delay in regulation.…”

“…35 With this regulation, the 4hydroxyisoleucine titer increased to 135.34 mM. 35 Nevertheless, the biosensor based on native TF had a poor dynamic regulation range and showed a certain delay in regulation.…”

Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

“…Subsequently, the promoter P brnFE in the lrp-brnEF regulation system was modified to the stronger promoter P brnFE N, which was used to regulate the transcription of l -isoleucine dioxygenase (IDO), that could convert l -isoleucine into 4-HIL in the presence of α-KG and oxygen. Meanwhile, the supply of α-KG and oxygen was coordinately modulated via this regulation system, which finally produced 19.9 g/L 4-HIL [ 88 ]. In general, regulating BCAAs transporter expression efficiently enhances BCAAs production.…”