초록
<P><B>Abstract</B></P> <P> <I>t</I>-Butyl 6-cyano-(3<I>R</I>,5<I>R</I>)-dihydroxyhexanoate ((3<I>R</I>,5<I>R</I>)-<B>2</B>) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant <I>Km</I>AKR-W297H (M1) of <I>Kluyveromyces marxianus</I> aldo-keto reductase (<I>Km</I>AKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards <I>t</I>-butyl 6-cyano-(5<I>R</I>)-hydroxy-3-oxohexanoate ((5<I>R</I>)-<B>1</B>). In this work, <I>Km</I>AKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5<I>R</I>)-<B>1</B>. The <I>K</I> <SUB>m</SUB> values of M3 for NADPH and (5<I>R</I>)-<B>1</B> were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate <I>v</I> <SUB>max</SUB> was 55.56 μmol/min/mg. Compared with M1, the catalytic efficiency <I>k</I> <SUB>cat</SUB>/<I>K</I> <SUB>m</SUB> of M3 was increased 2.64-fold. Coupled with <I>Exiguobacterium sibiricum</I> glucose dehydrogenase (<I>Es</I>GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5<I>R</I>)-<B>1</B> at up to 100.0 g/L, producing 237.4 mmol/L (3<I>R</I>,5<I>R</I>)-<B>2</B> in <I>d.e.</I> <SUB>P</SUB> value above 99.5%. The space-time yield (STY) of M3-catalyzed (3<I>R</I>,5<I>R</I>)-<B>2</B> synthesis was 372.8 g/L/d.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Robust <I>Km</I>AKR<I>-</I>W297H/Y296W/K29H (M3) was developed after two rounds of SSM library screening. </LI> <LI> M3 took only 3.5 h to completely reduce 100.0 g/L (5<I>R</I>)-<B>1</B>. </LI> <LI> The space-time yield (STY) of M3-catalyzed (3<I>R</I>,5<I>R</I>)-<B>2</B> synthesis reached 372.8 g/L/d. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>Semi-rational engineering of M1 and its variants catalyzed <I>t</I>-butyl 6-cyano- (3<I>R</I>,5<I>R</I>)-dihydroxyhexanoate synthesis.</P> <P>[DISPLAY OMISSION]</P>