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Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

Wu J, Du G, Chen J, Zhou J - Sci Rep (2015)

Bottom Line: The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%).Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin.By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

ABSTRACT
The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain. The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for producing other important products that require malonyl-CoA as a precursor.

No MeSH data available.


Related in: MedlinePlus

Construction of the CRISPRi system for controlling gene expression.(A) Sequence of the designed sgRNA template. sgRNA targets the non-template DNA strand of the gene-coding region. Base-pairing nucleotides (20 bp) are shown in orange. The dCas9-binding hairpin is in blue. The PAM sequence is shown in red. The Trc promoter is shown in grey. (B) This CRISPRi system consists of an inducible dCas9 protein and a designed sgRNA chimera. The dCas9 mutant gene contains two silencing mutations of the RuvC1 and HNH nuclease domains. The sgRNA chimera contains four functional domains: a Trc-inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes15.
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f1: Construction of the CRISPRi system for controlling gene expression.(A) Sequence of the designed sgRNA template. sgRNA targets the non-template DNA strand of the gene-coding region. Base-pairing nucleotides (20 bp) are shown in orange. The dCas9-binding hairpin is in blue. The PAM sequence is shown in red. The Trc promoter is shown in grey. (B) This CRISPRi system consists of an inducible dCas9 protein and a designed sgRNA chimera. The dCas9 mutant gene contains two silencing mutations of the RuvC1 and HNH nuclease domains. The sgRNA chimera contains four functional domains: a Trc-inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes15.

Mentions: To implement the CRISPRi platform in E. coli, a catalytically dead Cas9 mutant (dCas9) derived from Streptococcus pyogenes cas9 gene15, which acts as an RNA-guided DNA-binding complex, was expressed under T7 promoter. The sgRNA molecule, which consists of four domains (a Trc promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes), was coexpressed with dCas915 (Fig. 1).


Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

Wu J, Du G, Chen J, Zhou J - Sci Rep (2015)

Construction of the CRISPRi system for controlling gene expression.(A) Sequence of the designed sgRNA template. sgRNA targets the non-template DNA strand of the gene-coding region. Base-pairing nucleotides (20 bp) are shown in orange. The dCas9-binding hairpin is in blue. The PAM sequence is shown in red. The Trc promoter is shown in grey. (B) This CRISPRi system consists of an inducible dCas9 protein and a designed sgRNA chimera. The dCas9 mutant gene contains two silencing mutations of the RuvC1 and HNH nuclease domains. The sgRNA chimera contains four functional domains: a Trc-inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes15.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555050&req=5

f1: Construction of the CRISPRi system for controlling gene expression.(A) Sequence of the designed sgRNA template. sgRNA targets the non-template DNA strand of the gene-coding region. Base-pairing nucleotides (20 bp) are shown in orange. The dCas9-binding hairpin is in blue. The PAM sequence is shown in red. The Trc promoter is shown in grey. (B) This CRISPRi system consists of an inducible dCas9 protein and a designed sgRNA chimera. The dCas9 mutant gene contains two silencing mutations of the RuvC1 and HNH nuclease domains. The sgRNA chimera contains four functional domains: a Trc-inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes15.
Mentions: To implement the CRISPRi platform in E. coli, a catalytically dead Cas9 mutant (dCas9) derived from Streptococcus pyogenes cas9 gene15, which acts as an RNA-guided DNA-binding complex, was expressed under T7 promoter. The sgRNA molecule, which consists of four domains (a Trc promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes), was coexpressed with dCas915 (Fig. 1).

Bottom Line: The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%).Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin.By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

ABSTRACT
The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain. The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for producing other important products that require malonyl-CoA as a precursor.

No MeSH data available.


Related in: MedlinePlus