Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: Controllable P(3HB-co-4HB) biosynthesis.

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 Escherichia coli for the production of poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [P(3HB- co -4HB)] from glucose. The native gene sad encoding E. coli 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- co -4HB) consisting of 1–9 mol% 4HB. Additionally, succinate, generated by succinyl-coA synthetase and succinate dehydrogenase (respectively encoded by genes sucC , sucD and sdhA , sdhB ) 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- co -4HB) was found to range from 1.4 to 18.4 mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in E. coli . [ABSTRACT FROM AUTHOR]