Limits...
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

Effects of single or multiple genetic perturbations on (2S)-naringenin production.Control strains contained the (2S)-naringenin heterologous pathway without an RNA-guided dCas9:sgRNA system. The sgRNA-expressing plasmids repressing single or multiple genes were further transformed into the control strain to investigate the effects of these systems on (2S)-naringenin production. Final OD600 values, average specific growth rates and concentrations of p-coumaric acid and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4555050&req=5

f5: Effects of single or multiple genetic perturbations on (2S)-naringenin production.Control strains contained the (2S)-naringenin heterologous pathway without an RNA-guided dCas9:sgRNA system. The sgRNA-expressing plasmids repressing single or multiple genes were further transformed into the control strain to investigate the effects of these systems on (2S)-naringenin production. Final OD600 values, average specific growth rates and concentrations of p-coumaric acid and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h.

Mentions: Based on a previous study24, the control strain overproducing (2S)-naringenin from L-tyrosine was constructed. The plasmids pCDF-Trc-TAL-Trc-4CL and pET-CHS-CHI24 were transformed into E. coli BL21 (DE3), which yielded a production titer of 50.5 mg/L. Appropriate repressing efficacy toward a target gene that could increase the malonyl-CoA concentration without significantly altering the final biomass was ranked as a beneficial genetic perturbation. The impacts of these independent genetic interventions, namely low repressing efficacy toward eno, adhE, mdh and fabB, medium repressing efficacy toward sucC and fumC and high repressing efficacy toward fabF, on (2S)-naringenin production were analyzed. Single perturbations of eno, adhE, mdh, fabB, fabF, sucC and fumC increased production by up to 38.6%, 74.3%, 86.1%, 54.5%, 135.6%, 78.2% and 96.1%, respectively, and perturbation of fabF resulted in the highest (2S)-naringenin production (119.6 mg/L) (Fig. 5).


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)

Effects of single or multiple genetic perturbations on (2S)-naringenin production.Control strains contained the (2S)-naringenin heterologous pathway without an RNA-guided dCas9:sgRNA system. The sgRNA-expressing plasmids repressing single or multiple genes were further transformed into the control strain to investigate the effects of these systems on (2S)-naringenin production. Final OD600 values, average specific growth rates and concentrations of p-coumaric acid and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Effects of single or multiple genetic perturbations on (2S)-naringenin production.Control strains contained the (2S)-naringenin heterologous pathway without an RNA-guided dCas9:sgRNA system. The sgRNA-expressing plasmids repressing single or multiple genes were further transformed into the control strain to investigate the effects of these systems on (2S)-naringenin production. Final OD600 values, average specific growth rates and concentrations of p-coumaric acid and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h.
Mentions: Based on a previous study24, the control strain overproducing (2S)-naringenin from L-tyrosine was constructed. The plasmids pCDF-Trc-TAL-Trc-4CL and pET-CHS-CHI24 were transformed into E. coli BL21 (DE3), which yielded a production titer of 50.5 mg/L. Appropriate repressing efficacy toward a target gene that could increase the malonyl-CoA concentration without significantly altering the final biomass was ranked as a beneficial genetic perturbation. The impacts of these independent genetic interventions, namely low repressing efficacy toward eno, adhE, mdh and fabB, medium repressing efficacy toward sucC and fumC and high repressing efficacy toward fabF, on (2S)-naringenin production were analyzed. Single perturbations of eno, adhE, mdh, fabB, fabF, sucC and fumC increased production by up to 38.6%, 74.3%, 86.1%, 54.5%, 135.6%, 78.2% and 96.1%, respectively, and perturbation of fabF resulted in the highest (2S)-naringenin production (119.6 mg/L) (Fig. 5).

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