Engineering Bacillus subtilis Isoleucine Dioxygenase for Efficient Synthesis of (2S,3R,4S)-4-Hydroxyisoleucine

Abstract

Isoleucine dioxygenase (IDO)-catalyzed hydroxylation of isoleucine is a promising method for the synthesis of the diabetic drug (2S,3R,4S)-4-hydroxyisoleucine [(2S,3R,4S)-4-HIL]. However, the low activity of IDO significantly limits its practical application. In this work, a high-throughput screening method was developed and directed evolution was performed on the IDO from Bacillus subtilis, resulting in a double mutant with improvements in specific activity, protein expression level, and fermentation titer of 3.2-, 2.8-, and 9.4-fold, respectively. l-Isoleucine (228 mM) was completely converted to (2S,3R,4S)-4-HIL by the best variant with a space-time yield of up to 80.8 g L–1 d–1, which is the highest record reported so far. With a further increase of the substrate loading to 1 M, a high conversion of 91% could also be achieved. At last, enzymatic synthesis of (2S,3R,4S)-4-HIL was successfully carried out on a 3 L scale, indicating tremendous potential of the IDO variant I162T/T182N for green and efficient production of (2S,3R,4S)-4-HIL.

Exploring l-isoleucine riboswitches for enhancing 4-hydroxyisoleucine production in Corynebacterium glutamicum

“…7B). Compared with previously constructed strain D-In order to achieve the overproduction of 4-hydroxyisoleucine (4-HIL), variety of approaches were conducted in bacteria (Du et al 2020;Wei et al 2022;Zhang et al 2018). Kivero et al (2012) rstly introduced ido (coding isoleucine dioxygenase, IDO) into E. coli to produce 4-HIL.…”

Molecular Insights into the Regioselectivity of the Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Catalyzed C–H Hydroxylation of Amino Acids

“…Various l -proline hydroxylases have been explored to facilitate hydroxylation of substrates at different positions. − l -Lysine dioxygenase (KDO) has also been well studied for catalyzing hydroxylation. − As an efficient biocatalyst, GlbB can hydroxylate l -lysine with high regioselectivity in the preparation of key dipeptide fragments of griseofulvin and has received more attention regarding catalytic performance and thermostability in recent years. − In addition, PolL enables the sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid . Besides, there are Fe/2-KG DOs that act on amino acids bound to peptidyl carrier proteins. , According to the established phylogenetic tree, Fe/2-KG DOs that hydroxylate amino acids can be generally divided into three clusters based on the properties of their substrates: (i) those that act on hydroxylated heterocyclic amino acid substrates, (ii) those involved in peptide antibiotic synthesis, and (iii) those involved in the oxidation of aliphatic amino acids.…”

“…The enzymatic synthesis of hydroxy amino acids with high regioselectivity offers remarkable advantages over chemical syntheses involving complex steps. , IDO from Bacillus thuringiensis can directly catalyze the C–H hydroxylation of various aliphatic amino acids. , However, IDO engineering is mainly focused on enhancing the catalytic activity and thermostability of IDO using an error-prone polymerase chain reaction or site-directed mutagenesis ,, without consideration for its regioselectivity. Understanding enzymatic regioselectivity is necessary to engineer enzymes that can synthesize high-purity hydroxy amino acids, specifically by regulating the C4 positional preference of IDO toward l -Nle for the highly regioselective synthesis of 4-HNL.…”