초록
<P><B>Abstract</B></P> <P>Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is used to edit eukaryotic genomes. Here, we show that CRISPRi can also be used for fine-tuning prokaryotic gene expression while simultaneously regulating multiple essential gene expression with less labor and time consumption. As a case study, CRISPRi was used to control polyhydroxyalkanoate (PHA) biosynthesis pathway flux and to adjust PHA composition. A pathway was constructed in <I>Escherichia coli</I> for the production of poly(3-hydroxybutyrate-<I>co</I>-4-hydroxybutyrate) [P(3HB-<I>co</I>-4HB)] from glucose. The native gene <I>sad</I> encoding <I>E. coli</I> succinate semi-aldehyde dehydrogenase was expressed under the control of CRISPRi using five specially designed single guide RNAs (sgRNAs) for regulating carbon flux to 4-hydroxybutyrate (4HB) biosynthesis. The system allowed formation of P(3HB-<I>co</I>-4HB) consisting of 1–9mol% 4HB. Additionally, succinate, generated by succinyl-coA synthetase and succinate dehydrogenase (respectively encoded by genes <I>sucC</I>, <I>sucD</I> and <I>sdhA</I>, <I>sdhB</I>) was channeled preferentially to the 4HB precursor by using selected sgRNAs such as sucC2, sucD2, sdhB2 and sdhA1 via CRISPRi. The resulting 4HB content in P(3HB-<I>co</I>-4HB) was found to range from 1.4 to 18.4mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in <I>E. coli</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> CRISPRi system is useful for prokaryotic metabolic engineering. </LI> <LI> CRISPRi system can simultaneously regulate multiple genes in prokaryotes. </LI> <LI> P(3HB-<I>co</I>-4HB) consisting of 1–18mol% 4HB was obtained using CRISPRi. </LI> <LI> CRISPRi system will become a powerful tool for synthetic biology. </LI> </UL> </P>