초록
<P><B>Background</B></P><P><I>Klebsiella pneumoniae</I> is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid.</P><P><B>Results</B></P><P>Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in <I>K</I>. <I>pneumoniae</I>. EGFP was recruited as a reporter of this CRISPRi system. Fluorescence assay of this CRISPRi system showed that enhanced green fluorescent protein (EGFP) expression level was repressed by 85–90%. To further test this CRISPRi system, guide RNAs were designed to individually or simultaneously target four lactate-producing enzyme genes. Results showed that all lactate-producing enzyme genes were significantly repressed. Notably, <SMALL>D</SMALL>-lactate dehydrogenase (ldhA) was shown to be the most influential enzyme for lactic acid formation in micro-aerobic conditions, as inhibiting <I>ldhA</I> alone led to lactic acid level similar to simultaneously repressing four genes. In shake flask cultivation, the strain coexpressing <I>puuC</I> (an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde to 3-HP) and dCas9-sgRNA inhibiting <I>ldhA</I> produced 1.37-fold 3-HP relative to the reference strain. Furthermore, in bioreactor cultivation, this CRISPRi strain inhibiting <I>ldhA</I> produced 36.7 g/L 3-HP, but only generated 1 g/L lactic acid. Clearly, this engineered CRISPRi system largely simplified downstream separation of 3-HP from its isomer lactic acid, an extreme challenge for 3-HP bioprocess.</P><P><B>Conclusions</B></P><P>This study offers a deep understanding of lactic acid metabolism in diverse species, and we believe that this CRISPRi system will facilitate biomanufacturing and functional genome studies of <I>K</I>. <I>pneumoniae</I> or beyond.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s12934-018-0903-1) contains supplementary material, which is available to authorized users.</P>