초록
<P><B>Abstract</B></P> <P>Cofactor engineering is a common strategy to improve amino acid production. 4-hydroxyisoleucine (4-HIL), a nonproteinogenic amino acid, exhibits unique insulinotropic and insulin-sensitizing activities, therefore has potential medical value in treating diabetes. In our previous study, <SMALL>L</SMALL>-isoleucine (Ile) dioxygenase gene <I>ido</I> was overexpressed in an Ile-producing <I>Corynebacterium glutamicum</I> strain, and 4-HIL was <I>de novo</I> synthesized from glucose. In this study, to increase the NADPH supply, the endogenous NAD<SUP>+</SUP> kinase gene <I>ppnK</I> and glucose-6-phosphate dehydrogenase gene <I>zwf</I> were co-expressed with <I>ido</I>. The resulting strain SL01 produced 81.12 ± 5.96 mM 4-HIL, 62% higher than the <I>ido</I>-mere expressing strain SN02. However, the strain SL02 co-expressing exogenous NADH kinase gene <I>POS5</I> with <I>ido</I> grew slowly and its 4-HIL production decreased by 12%, perhaps due to the lower 2-oxoglutarate (OG) level and slightly weaker membrane permeability. To increase OG availability for 4-HIL conversion, the serine/threonine protein kinase G gene <I>pknG</I> was deleted and replaced by <I>ido</I> gene in SL02. The growth of the resulting strain SL04 was restored and 4-HIL production was improved to 84.14 ± 6.38 mM; meanwhile, the conversion ratio of Ile to 4-HIL reached up to 0.98 ± 0.01 mol/mol. Therefore, sufficient NADPH supply and OG availability may be benefit to 4-HIL <I>de novo</I> biosynthesis in recombinant <I>C. glutamicum</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>De novo</I> biosynthesis of an antidiabete, 4-HIL, was improved in <I>C. glutamicum</I>. </LI> <LI> Co-expression of <I>zwf</I>-<I>ppnK</I> with <I>ido</I> was benefit to NADPH supply and 4-HIL synthesis. </LI> <LI> Co-expression of <I>POS5</I> with <I>ido</I> was benefit to Ile synthesis but not Ile conversion. </LI> <LI> Δ<I>pknG</I>::<I>ido</I> substitution improved growth and 4-HIL synthesis as <I>POS5</I> overexpressed. </LI> </UL> </P>