title: Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: Controllable P(3HB-co-4HB) biosynthesis.
abstract: 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 1-9mol% 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 18.4mol% depending on the expression levels of down-regulated genes. The results show that CRISPRi is a feasible method to simultaneously manipulate multiple genes in E. coli.
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Li
authors.initials: L
authors.lastname: Lv
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Yi-Lin
authors.initials: YL
authors.lastname: Ren
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Jin-Chun
authors.initials: JC
authors.lastname: Chen
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China; Center for Nano and Micro-Mechanics, Tsinghua University, Beijing 100084, China. Electronic address: chengq@mail.tsinghua.edu.cn.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D064113:CRISPR-Cas Systems; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D006885:Hydroxybutyrates / Q000378:metabolism*; D060847:Metabolic Engineering; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D029968:Escherichia coli Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; C068056:poly(3-hydroxybutyrate-co-4-hydroxybutyrate); D000094122:Polyhydroxybutyrates
keywords: 4-hydroxybutyrate; CRISPRi; Metabolic engineering; P3HB4HB; PHB; Synthetic biology