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Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12934-017-0655-3) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Controllable repression of prpC gene transcription in recombinant Halomonas sp. TD01. The relative binding positions of sgRNAs targeting prpC gene (a) and RT-PCR study of prpC transcription levels (b). All data were the average of three independent studies with standard deviations. Mean ± SE (n = 3). *p < 0.05 and **p < 0.01. TD01, Halomonas sp. TD wild type; TD-sgRNA, TD01 strain harboring the pli-dCas9-sgRNA plasmid without any target site; TD-prpC1, TD-prpC2, TD-prpC3, TD-prpC4, TD-prpC5, TD-prpC6, TD-prpC7, TD-prpC6prpC7, TD01 strains harboring the pli-dCas9-sgRNA plasmid with different DNA targets on gene prpC
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Fig4: Controllable repression of prpC gene transcription in recombinant Halomonas sp. TD01. The relative binding positions of sgRNAs targeting prpC gene (a) and RT-PCR study of prpC transcription levels (b). All data were the average of three independent studies with standard deviations. Mean ± SE (n = 3). *p < 0.05 and **p < 0.01. TD01, Halomonas sp. TD wild type; TD-sgRNA, TD01 strain harboring the pli-dCas9-sgRNA plasmid without any target site; TD-prpC1, TD-prpC2, TD-prpC3, TD-prpC4, TD-prpC5, TD-prpC6, TD-prpC7, TD-prpC6prpC7, TD01 strains harboring the pli-dCas9-sgRNA plasmid with different DNA targets on gene prpC

Mentions: As shown in Fig. 4a, seven sgRNAs were designed along the prpC gene, mostly near the start site of the coding sequence or within 260 bp from the ATG sequence. CRISPRi plasmids are constructed and named as pli-dCas9-prpC1, pli-dCas9-prpC2, pli-dCas9-prpC3, pli-dCas9-prpC4, pli-dCas9-prpC5, pli-dCas9-prpC6 and pli-dCas9-prpC7, respectively (Additional file 1: Table S1; Fig. 4a). Various sgRNA binding sites led to different repressive effects. Two effective inhibition sites, namely prpC6 and prpC7, were combined together to form pli-dCas9-prpC6prpC7 to verify possible enhanced combinatory inhibition effect. All the plasmids were transferred via E. coli conjugation into Halomonas sp. TD01, forming strains TD-prpC1, TD-prpC2, TD-prpC3, TD-prpC4, TD-prpC5, TD-prpC6, TD-prpC7 and TD-prpC6prpC7, respectively.


Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi
Controllable repression of prpC gene transcription in recombinant Halomonas sp. TD01. The relative binding positions of sgRNAs targeting prpC gene (a) and RT-PCR study of prpC transcription levels (b). All data were the average of three independent studies with standard deviations. Mean ± SE (n = 3). *p < 0.05 and **p < 0.01. TD01, Halomonas sp. TD wild type; TD-sgRNA, TD01 strain harboring the pli-dCas9-sgRNA plasmid without any target site; TD-prpC1, TD-prpC2, TD-prpC3, TD-prpC4, TD-prpC5, TD-prpC6, TD-prpC7, TD-prpC6prpC7, TD01 strains harboring the pli-dCas9-sgRNA plasmid with different DNA targets on gene prpC
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Controllable repression of prpC gene transcription in recombinant Halomonas sp. TD01. The relative binding positions of sgRNAs targeting prpC gene (a) and RT-PCR study of prpC transcription levels (b). All data were the average of three independent studies with standard deviations. Mean ± SE (n = 3). *p < 0.05 and **p < 0.01. TD01, Halomonas sp. TD wild type; TD-sgRNA, TD01 strain harboring the pli-dCas9-sgRNA plasmid without any target site; TD-prpC1, TD-prpC2, TD-prpC3, TD-prpC4, TD-prpC5, TD-prpC6, TD-prpC7, TD-prpC6prpC7, TD01 strains harboring the pli-dCas9-sgRNA plasmid with different DNA targets on gene prpC
Mentions: As shown in Fig. 4a, seven sgRNAs were designed along the prpC gene, mostly near the start site of the coding sequence or within 260 bp from the ATG sequence. CRISPRi plasmids are constructed and named as pli-dCas9-prpC1, pli-dCas9-prpC2, pli-dCas9-prpC3, pli-dCas9-prpC4, pli-dCas9-prpC5, pli-dCas9-prpC6 and pli-dCas9-prpC7, respectively (Additional file 1: Table S1; Fig. 4a). Various sgRNA binding sites led to different repressive effects. Two effective inhibition sites, namely prpC6 and prpC7, were combined together to form pli-dCas9-prpC6prpC7 to verify possible enhanced combinatory inhibition effect. All the plasmids were transferred via E. coli conjugation into Halomonas sp. TD01, forming strains TD-prpC1, TD-prpC2, TD-prpC3, TD-prpC4, TD-prpC5, TD-prpC6, TD-prpC7 and TD-prpC6prpC7, respectively.

View Article: PubMed Central - PubMed

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.

Electronic supplementary material: The online version of this article (doi:10.1186/s12934-017-0655-3) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus