초록
<P><B>Abstract</B></P> <P>The present study describes the use of metabolic engineering to achieve the production of <I>R,R</I>-2,3-butanediol (<I>R,R</I>-2,3-BD) of ultra-high optical purity (>99.99%). To this end, the diacetyl reductase (DAR) gene (<I>dud A</I>) of <I>Paenibacillus polymyxa</I> ZJ-9 was knocked out via homologous recombination between the genome and the previously constructed targeting vector pRN5101-L′C in a process based on homologous single-crossover. PCR verification confirmed the successful isolation of the <I>dud</I> A gene disruption mutant <I>P. polymyxa</I> ZJ-9-△<I>dud</I> A. Moreover, fermentation results indicated that the optical purity of <I>R,R</I>-2,3-BD increased from about 98% to over 99.99%, with a titer of 21.62 g/L in Erlenmeyer flasks. The latter was further increased to 25.88 g/L by fed-batch fermentation in a 5-L bioreactor.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A ultra-high optical purity (>99.99%) of <I>R,R</I>-2,3-butanediol was obtained. </LI> <LI> The diacetyl reductase gene of <I>P. polymyxa</I> ZJ-9 was knocked out via HR. </LI> <LI> A new designed gene-knockout processing by HR technology has performed. </LI> <LI> Homologous single-crossover technology is easy to operate and be applied. </LI> <LI> A new version of the strategy for the pure targeted compound production was provided. </LI> </UL> </P>