A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells

Abstract

L-Isoleucine dioxygenase (IDO) specifically converts L-isoleucine(L-Ile) to 4-hydroxyisoleucine(4-HIL). To obtain IDO with improved activity, a strategy was developed that is dependent on the restoration of succinate-minus E. coli cell growth by the coupling of L-Ile hydroxylation and the oxidation of α-ketoglutarate(α-KGA) to succinate. Five mutants were obtained with this strategy, and the characteristics of IDOM3, which exhibited the highest activity, were studied. The catalytic efficiency, thermal stability and catalytic rate of IDOM3 were significantly improved compared with those of wild-type IDO. Moreover, an efficient method for the biotransformation of 4-HIL by resting cells expressing IDOM3 was developed, with which 151.9 mmol of 4-HIL/L (22.4 g/L) was synthesized in 12 h while the substrates seldom exhibited additional consumption.

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

“…Using this strain, 4-HIL yields were found to be 82%, employing 100 mmol L-isoleucine as a substrate [50]. Recently, Zhang et al enhanced the activity of IDO by introducing four mutations, which were identified by error-prone PCR [66]. Using this approach, the The enzymes typically accept free-standing amino acids as a substrate.…”

“…Using this strain, 4-HIL yields were found to be 82%, employing 100 mmol L-isoleucine as a substrate [50]. Recently, Zhang et al enhanced the activity of IDO by introducing four mutations, which were identified by error-prone PCR [66]. Using this approach, the k cat /K m value of IDO could be increased 1.5-fold to reach approximately 33 L/mmol/min, and the 4-HIL production doubled in a small-scale biocatalysis reaction [66].…”

“…Recently, Zhang et al enhanced the activity of IDO by introducing four mutations, which were identified by error-prone PCR [66]. Using this approach, the k cat /K m value of IDO could be increased 1.5-fold to reach approximately 33 L/mmol/min, and the 4-HIL production doubled in a small-scale biocatalysis reaction [66]. In a study to investigate IDO's substrate acceptance beyond L-isoleucine, recombinantly expressed IDO was successfully tested for the hydroxylation of the aliphatic amino acids L-leucin, L-norleucine, and L-norvaline and for the (S)-sulfoxidation of L-methionine and L-ethionine [67].…”

IGF-1/GLP-1 Signaling Activator 4-hydroxyisoleucine (4-HI) Prevent Neurobehavioral and Neurochemical Defects in Methylmercury-induced Experimental Model of ALS: Insights From CSF, Blood Plasma and Brain Homogenate Samples in Rats

“…4-HI is an amorphous, hydrophilic powder with a molecular weight of 147.17 grams per mole (Broca et al, 2004). It is produced from an amino acid, isoleucine, by the activity of bacterial isoleucine dioxygenase and has a high therapeutic potential in treating insulin dysregulation (Zhang et al, 2018). 4-HI has been reported to demonstrate peculiar therapeutic bene ts against insulin resistance, inadequate glucose absorption, lipotoxicity, obesity, and liver dysfunctions (Soriano et al, 2016).…”

Significantly enhancing production of trans -4-hydroxy- l -proline by integrated system engineering in Escherichia coli

“…Therefore, the -KG concentration has a significant effect on l-Pro hydroxylation. Zhang et al overexpressed -KG dehydrogenase (ODHC) gene and knocked out isocitrate lyase gene aceA in E. coli, where exogenous IDO shunted the flux of -KG to the pathway of 4-HIL production (10). The other approach to redirecting the flux of -KG to 4-HIL was ODHC inhibition.…”