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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

A CRISPRi-based strategy for targeting gene identification.All target genes were silenced with high efficacy. It was found that sgRNAs targeting ppsA, eno, glyA, adhE, mdh, fumC, sdhABCD, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA concentration (increased by over 180%). Meanwhile, sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA and malonyl-CoA concentrations (increased by over 223%). 1 mL of cell culture was harvested at the mid-log phase of growth to quantify the intracellular concentrations of malonyl-CoA and acetyl-CoA.
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f3: A CRISPRi-based strategy for targeting gene identification.All target genes were silenced with high efficacy. It was found that sgRNAs targeting ppsA, eno, glyA, adhE, mdh, fumC, sdhABCD, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA concentration (increased by over 180%). Meanwhile, sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA and malonyl-CoA concentrations (increased by over 223%). 1 mL of cell culture was harvested at the mid-log phase of growth to quantify the intracellular concentrations of malonyl-CoA and acetyl-CoA.

Mentions: The results showed that sgRNAs targeting ppsA, eno, glyA, adhE, mdh, fumC, sdhABCD, sucC and citE produced a dramatic increase in acetyl-CoA concentration (over 180%), while sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC and citE produced a dramatic increase in malonyl-CoA concentration (over 223%). It was also found that sgRNAs targeting fabH, fabB, fabF and fabI produced a simultaneous increase in acetyl-CoA and malonyl-CoA concentration (over 244%) (Fig. 3). Hence, ppsA, eno, adhE, mdh, fumC, sdhA, sucC, citE, fabH, fabB, fabF and fabI were chosen as target genes.


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)

A CRISPRi-based strategy for targeting gene identification.All target genes were silenced with high efficacy. It was found that sgRNAs targeting ppsA, eno, glyA, adhE, mdh, fumC, sdhABCD, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA concentration (increased by over 180%). Meanwhile, sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA and malonyl-CoA concentrations (increased by over 223%). 1 mL of cell culture was harvested at the mid-log phase of growth to quantify the intracellular concentrations of malonyl-CoA and acetyl-CoA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: A CRISPRi-based strategy for targeting gene identification.All target genes were silenced with high efficacy. It was found that sgRNAs targeting ppsA, eno, glyA, adhE, mdh, fumC, sdhABCD, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA concentration (increased by over 180%). Meanwhile, sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC, cite, fabH, fabB, fabF and fabI showed dramatic increases in acetyl-CoA and malonyl-CoA concentrations (increased by over 223%). 1 mL of cell culture was harvested at the mid-log phase of growth to quantify the intracellular concentrations of malonyl-CoA and acetyl-CoA.
Mentions: The results showed that sgRNAs targeting ppsA, eno, glyA, adhE, mdh, fumC, sdhABCD, sucC and citE produced a dramatic increase in acetyl-CoA concentration (over 180%), while sgRNAs targeting ppsA, eno, adhE, mdh, fumC, sdhA, sucC and citE produced a dramatic increase in malonyl-CoA concentration (over 223%). It was also found that sgRNAs targeting fabH, fabB, fabF and fabI produced a simultaneous increase in acetyl-CoA and malonyl-CoA concentration (over 244%) (Fig. 3). Hence, ppsA, eno, adhE, mdh, fumC, sdhA, sucC, citE, fabH, fabB, fabF and fabI were chosen as target genes.

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