Sustainable production of 4-hydroxyisoleucine with minimised carbon loss by simultaneously utilising glucose and xylose in engineered Escherichia coli

4-Hydroxyisoleucine is a promising drug for diabetes therapy; however, microbial production of 4-hydroxyisoleucine is not economically efficient because of the carbon loss in the form of CO2. This study aims to achieve de novo synthesis of 4-hydroxyisoleucine with minimised carbon loss in engineered Escherichia coli. Initially, an L-isoleucine-producing strain, ILE-5, was established, and the 4-hydroxyisoleucine synthesis pathway was introduced. The flux toward α-ketoglutarate was enhanced by reinforcing the anaplerotic pathway and disrupting competitive pathways. Subsequently, the metabolic flux for 4-hydroxyisoleucine synthesis was redistributed by dynamically modulating the α-ketoglutarate dehydrogenase complex activity, achieving a 4-hydroxyisoleucine production of 16.53 g/L. Finally, carbon loss was minimised by employing the Weimberg pathway, resulting in a 24.5% decrease in sugar consumption and a 31.6% yield increase. The 4-hydroxyisoleucine production by strain IEOH-11 reached 29.16 g/L in a 5-L fermenter. The 4-hydroxyisoleucine yield (0.29 mol/mol sugar) and productivity (0.91 g/(L⋅h)) were higher than those previously reported.