초록
<P>Biosynthesis of 1,4-butanediol (1,4-BDO) in <I>E.？coli</I> requires an artificial pathway that involves six genes and time-consuming, iterative genome engineering. CRISPR is an effective gene editing tool, while CRISPR interference (CRISPRi) is repurposed for programmable gene suppression. This study aimed to combine both CRISPR and CRISPRi for metabolic engineering of <I>E.？coli</I> and 1,4-BDO production. We first exploited CRISPR to perform point mutation of <I>glt</I>A, replacement of native <I>lpdA</I> with heterologous <I>lpdA</I>, knockout of <I>sad</I> and knock-in of two large (6.0 and 6.3 kb in length) gene cassettes encoding the six genes (<I>cat</I>1, <I>suc</I>D, 4<I>hbd</I>, <I>cat</I>2, <I>bld</I>, <I>bdh</I>) in the 1,4-BDO biosynthesis pathway. The successive <I>E.？coli</I> engineering enabled production of 1,4-BDO to a titer of 0.9 g/L in 48 h. By combining the CRISPRi system to simultaneously suppress competing genes that divert the flux from the 1,4-BDO biosynthesis pathway (<I>gabD</I>, <I>ybgC</I> and <I>tesB</I>) for >85%, we further enhanced the 1,4-BDO titer for 100% to 1.8 g/L while reducing the titers of byproducts gamma-butyrolactone and succinate for 55% and 83%, respectively. These data demonstrate the potential of combining CRISPR and CRISPRi for genome engineering and metabolic flux regulation in microorganisms such as <I>E.？coli</I> and production of chemicals (<I>e.g.</I>, 1,4-BDO).</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2017/asbcd6.2017.6.issue-12/acssynbio.7b00251/production/images/medium/sb-2017-00251d_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/sb7b00251'>ACS Electronic Supporting Info</A></P>