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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.


CRISPRi system used for Halomonas sp. TD01 (a) and relative binding positions of sgRNAs targeting ftsZ gene (b). Pli-dCas9-sgRNA, plasmid carrying the CRISPRi system; Ptrc, trc promoter; CmR, chloramphenicol resistance gene; oriT, origin of transfer; ftsZ, filamenting temperature-sensitive mutant Z. The length of ftsZ gene is 1179 bp, while the length of sgRNAs is around 20 bp. The promoter of ftsZ gene is 35 bp to 10 bp upstream of ftsZ gene. To inhibit ftsZ gene expression, ftsZ1 is designed 4 bp upstream from ATG sequence, from position −26 to −5, after the PAM sequence CGG (from position −29 to −27). FtsZ2 is designed 89 bp downstream of ATG sequence, from position 90 to 112, after the PAM sequence TGG (from position 87 to 89)
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Fig2: CRISPRi system used for Halomonas sp. TD01 (a) and relative binding positions of sgRNAs targeting ftsZ gene (b). Pli-dCas9-sgRNA, plasmid carrying the CRISPRi system; Ptrc, trc promoter; CmR, chloramphenicol resistance gene; oriT, origin of transfer; ftsZ, filamenting temperature-sensitive mutant Z. The length of ftsZ gene is 1179 bp, while the length of sgRNAs is around 20 bp. The promoter of ftsZ gene is 35 bp to 10 bp upstream of ftsZ gene. To inhibit ftsZ gene expression, ftsZ1 is designed 4 bp upstream from ATG sequence, from position −26 to −5, after the PAM sequence CGG (from position −29 to −27). FtsZ2 is designed 89 bp downstream of ATG sequence, from position 90 to 112, after the PAM sequence TGG (from position 87 to 89)

Mentions: The sgRNAs were designed in the promoter region or near the ATG sequence in the targeted gene, and were right after a NGG sequence, namely, PAM sequence (protospacer adjacent motif sequence) [4]. All the sgRNAs could bind to the non-template DNA strand with sequence specificity. Thus, two sgRNAs were designed near the ATG sequence in ftsZ gene (Fig. 2b). CRISPRi inhibition systems pli-dCas9-ftsZ1 and pli-dCas9-ftsZ2 were constructed. The plasmids were then transferred via E. coli conjugation into Halomonas sp. TD01, forming the recombinants Halomonas sp. TD-ftsZ1 and TD-ftsZ2 strains. Halomonas sp. TD01 containing the non-target plasmid pli-dCa9-sgRNA, was named Halomonas sp. TD-sgRNA strain. Wild type Halomonas sp. TD01 and Halomonas sp. TD-sgRNA were used as control groups.


Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi
CRISPRi system used for Halomonas sp. TD01 (a) and relative binding positions of sgRNAs targeting ftsZ gene (b). Pli-dCas9-sgRNA, plasmid carrying the CRISPRi system; Ptrc, trc promoter; CmR, chloramphenicol resistance gene; oriT, origin of transfer; ftsZ, filamenting temperature-sensitive mutant Z. The length of ftsZ gene is 1179 bp, while the length of sgRNAs is around 20 bp. The promoter of ftsZ gene is 35 bp to 10 bp upstream of ftsZ gene. To inhibit ftsZ gene expression, ftsZ1 is designed 4 bp upstream from ATG sequence, from position −26 to −5, after the PAM sequence CGG (from position −29 to −27). FtsZ2 is designed 89 bp downstream of ATG sequence, from position 90 to 112, after the PAM sequence TGG (from position 87 to 89)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: CRISPRi system used for Halomonas sp. TD01 (a) and relative binding positions of sgRNAs targeting ftsZ gene (b). Pli-dCas9-sgRNA, plasmid carrying the CRISPRi system; Ptrc, trc promoter; CmR, chloramphenicol resistance gene; oriT, origin of transfer; ftsZ, filamenting temperature-sensitive mutant Z. The length of ftsZ gene is 1179 bp, while the length of sgRNAs is around 20 bp. The promoter of ftsZ gene is 35 bp to 10 bp upstream of ftsZ gene. To inhibit ftsZ gene expression, ftsZ1 is designed 4 bp upstream from ATG sequence, from position −26 to −5, after the PAM sequence CGG (from position −29 to −27). FtsZ2 is designed 89 bp downstream of ATG sequence, from position 90 to 112, after the PAM sequence TGG (from position 87 to 89)
Mentions: The sgRNAs were designed in the promoter region or near the ATG sequence in the targeted gene, and were right after a NGG sequence, namely, PAM sequence (protospacer adjacent motif sequence) [4]. All the sgRNAs could bind to the non-template DNA strand with sequence specificity. Thus, two sgRNAs were designed near the ATG sequence in ftsZ gene (Fig. 2b). CRISPRi inhibition systems pli-dCas9-ftsZ1 and pli-dCas9-ftsZ2 were constructed. The plasmids were then transferred via E. coli conjugation into Halomonas sp. TD01, forming the recombinants Halomonas sp. TD-ftsZ1 and TD-ftsZ2 strains. Halomonas sp. TD01 containing the non-target plasmid pli-dCa9-sgRNA, was named Halomonas sp. TD-sgRNA strain. Wild type Halomonas sp. TD01 and Halomonas sp. TD-sgRNA were used as control groups.

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.