title: Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi.
abstract: BACKGROUND
  Clustered regularly interspaced short palindromic repeats interference (CRISPRi) has provided an efficient approach for targeted gene inhibition. A non-model microorganism Halomonas species TD01 has been developed as a promising industrial producer of polyhydroxyalkanoates (PHA), a family of biodegradable polyesters accumulated by bacteria as a carbon and energy reserve compound. A controllable gene repression system, such as CRISPRi, is needed for Halomonas sp. TD01 to regulate its gene expression levels.
  
  
  RESULTS
  For the first time CRISPRi was successfully used in Halomonas sp. TD01 to repress expression of ftsZ gene encoding bacterial fission ring formation protein, leading to an elongated cell morphology with typical filamentous shape similar to phenomenon observed with Escherichia coli. CRISPRi was employed to regulate expressions of prpC gene encoding 2-methylcitrate synthase for regulating 3-hydroxyvalerate monomer ratio in PHBV copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Percentages of HV in PHBV copolymers were controllable ranging from less than 1 to 13%. Furthermore, repressions on gltA gene encoding citrate synthase channeled more acetyl-CoA from the tricarboxylic acid (TCA) cycle to poly(3-hydroxybutyrate) (PHB) synthesis. The PHB accumulation by Halomonas sp. TD01 with its gltA gene repressed in various intensities via CRISPRi was increased by approximately 8% compared with the wild type control containing the CRISPRi vector without target.
  
  
  CONCLUSIONS
  It has now been confirmed that the CRISPRi system can be applied to Halomonas sp. TD01, a promising industrial strain for production of various PHA and chemicals under open and continuous fermentation process conditions. In details, the CRISPRi system was successfully designed in this study to target genes of ftsZ, prpC and gltA, achieving longer cell sizes, channeling more substrates to PHBV and PHB synthesis, respectively. CRISPRi can be expected to use for more metabolic engineering applications in non-model organisms.
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing, 100084, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tao
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing, 100084, China.
authors.forename: Li
authors.initials: L
authors.lastname: Lv
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing, 100084, China. chengq@mail.tsinghua.edu.cn.|Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China. chengq@mail.tsinghua.edu.cn.|Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China. chengq@mail.tsinghua.edu.cn.|Center for Nano- and Micro-Mechanics, Tsinghua University, Beijing, 100084, China. chengq@mail.tsinghua.edu.cn.|MOE Key Lab of Industrial Biocatalysis, Department Chemical Engineering, Tsinghua University, Beijing, 100084, China. chengq@mail.tsinghua.edu.cn.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D002950:Citrate (si)-Synthase / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000235:genetics; D020868:Gene Silencing; D020632:Halomonas / Q000235:genetics* / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D007652:Oxo-Acid-Lyases / Q000235:genetics; D011091:Polyesters / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis* / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods; D000094122:Polyhydroxybutyrates
chemical_list: D001426:Bacterial Proteins; D002950:Citrate (si)-Synthase; D003598:Cytoskeletal Proteins; D006885:Hydroxybutyrates; D007652:Oxo-Acid-Lyases; D011091:Polyesters; D054813:Polyhydroxyalkanoates; C071525:FtsZ protein, Bacteria; C052620:poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate); D000094122:Polyhydroxybutyrates; C003182:poly-beta-hydroxybutyrate; C112399:2-methylcitrate synthase
keywords: CRISPRi; PHB; PHBV; Synthetic biology; ftsZ; gltA; prpC