- Open Access
Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production
© The Author(s) 2018
- Received: 20 June 2018
- Accepted: 11 September 2018
- Published: 18 September 2018
Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural flavonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modification of ATP concentration in Escherichia coli was applied in efficient pinocembrin production.
In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efficiency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a final pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains.
Our results introduce a strategy to approach the efficient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condition have been optimized for pinocembrin production. The results and engineering strategies demonstrated here would hold promise for the ATP level improvement of other flavonoids by CRISPRi system, thereby facilitating other flavonoids production.
The biosynthetic pathways of natural products composed of enzymes with several protein subunits are active only in the presence of energy carriers (ATP) . For example, among the 971 reactions of natural product biosynthesis identified in Streptomyces. coelicolor, more than 21% rely on the participation of ATP . Also, ATP is required for reconstitution of heterocycle forming activity with the microcin B17 precursor . Previous research suggested that, ATP pathway modification might be an alternative way to enhance the final product biosynthesis. For instance, Zhao found that the increase of ATP supply would improve the production of terpenoids compounds .
ATP is synthesized through ATP synthase and electron transfer chain in E. coli . In order to increase ATP supply, different gene operons were modulated in E. coli with five regulatory parts, lead to 20%, 16%, 5% and 21% increase in β-carotene yield . In addition, the additive of sodium citrate acts as an auxiliary energy substrate for the accumulation of intracellular ATP and the increase of ATP/ADP ratio, lead to the overproduction of S-adenosylmethionine (SAM) and glutathione (GSH) . Although the overexpression of ATPase and optimization of fermentation process are effective ways to improve ATP supply, the repression of the consumption pathway to increase the ATP accumulation is an alternative option . It was reported that the disruption of the ATP consuming bypass pathway in Saccharomyces cerevisiae could achieve 3.1-fold higher ATP-generating activity and 1.7-fold higher glutathione productivity compared with the control strain. Furthermore, Chen  found that the sRNA-based downregulation strategy in E. coli could increase the ATP level and S-adenosylmethionine production. Recently, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system provides an efficient tool for targeted gene regulation in a sequence-specific manner in E. coli . Compared with sRNAs and antisense RNAs strategies [15, 16], this RNA-guided DNA recognition platform provides a simple approach for repression of multiple target genes simultaneously on a genome-wide scale [17–19]. Accordingly, the development of genetic engineering has offered an alternative approach utilizing a CRISPRi regulation strategy to control the intracellular ATP level. In the biosynthesis of flavonoid, ATP has been proved not only to provide adenosyl moiety in E. coli metabolism but also to play the role of an energy carrier in pinocembrin biosynthesis. However, there is no ATP engineering strategies for increasing pinocembrin biosynthesis has been proposed to date.
In this study, we demonstrated that the ATP additive was advantageous for pinocembrin production and 5 genes involved in ATP metabolic pathways were repressed by the CRISPRi system to identify individual target genes that could increase the ATP level and pinocembrin production. Furthermore, multiple genes repressing for ATP engineering was performed to achieve the most effective combination. Finally, the pinocembrin production was further improved by combination of ATP and malonyl-CoA engineering. The results and engineering strategies demonstrated here would provide an alternative platform to improve intracellular ATP level in the biosynthesis pathway of natural products.
Strains and media
Plasmids used in this study
P15A Replicon CmR
The dCas9 gene on plasmid 44249-dCas9 was inserted into the BglII and XhoI restriction sites of the pACYCDuet-1 plasmid
Trc promoter, CloE1 replicon, SmR
Plasmid is used for high intensity repression of gfp
Plasmid is used for medium intensity repression of gfp
Plasmid is used for low intensity repression of gfp
Plasmid is used for high intensity repression of aroK
Plasmid is used for high intensity repression of glnA
Plasmid is used for high intensity repression of argB
Plasmid is used for high intensity repression of metK
Plasmid is used for medium intensity repression of metK
Plasmid is used for low intensity repression of metK
Plasmid is used for high intensity repression of proB
Plasmid is used for medium intensity repression of proB
Plasmid is used for low intensity repression of proB
Plasmid is used for high intensity repression of metK and low intensity repression of proB
Plasmid is used for medium intensity repression of metK and low intensity repression of proB
Plasmid is used for low intensity repression of metK and low intensity repression of proB
Plasmid is used for high intensity repression of fabF
Plasmid is used for low intensity repression of fabB
Plasmid is used for medium intensity repression of fumC
Plasmid is used for medium intensity repression of sucC
Plasmid is used for low intensity repression of adhE
Plasmid is used for high intensity repression of fabF and low intensity repression of fabB
Plasmid is used for medium intensity repression of fumC and medium intensity repression of sucC
Plasmid is used for high intensity repression of fabF, low intensity repression of fabB, medium intensity repression of fumC and medium intensity repression of sucC
Plasmid is used for high intensity repression of metK, low intensity repression of proB, high intensity repression of fabF, low intensity repression of fabB, medium intensity repression of fumC, medium intensity repression of sucC and low intensity repression of adhE
Plasmid is used for high intensity repression of putA
Plasmid is used for high intensity repression of putA and low intensity repression of proB
Strains used in this study
BL21 (DE3) with pTrc-BOPAL-PA4CL, pRSF-CHS (Met) -CHI plasmid
Empty with pACYC-dCas9 plasmid
Control with pCDF303-gfp-H plasmid
Control with pCDF303-gfp-M plasmid
Control with pCDF303-gfp-L plasmid
Control with pCDF-aroK-H plasmid
Control with pCDF-glnA-H plasmid
Control with pCDF-argB-Hplasmid
Control with pCDF-metK-H plasmid
Control with pCDF-metK-M plasmid
Control with pCDF-metK-L plasmid
Control with pCDF-proB-H plasmid
Control with pCDF-proB-M plasmid
Control with pCDF-proB-L plasmid
Control with pCDF- metK-H-proB-L plasmid
Control with pCDF- metK-M-proB-L plasmid
Control with pCDF- metK-L-proB-L plasmid
Control with pCDF-fabF-H plasmid
Control with pCDF-fabB-L plasmid
Control with pCDF-fumC-M plasmid
Control with pCDF-sucC-M plasmid
Control with pCDF-adhE-L plasmid
Control with pCDF-fabF-H-fabB-L plasmid
Control with pCDF-fumC-M-sucC-M plasmid
Control with pCDF-metK-H-proB-L plasmid
Control with pCDF-fabF-H-fabB-L- fumC-M-sucC-M plasmid
Control with pCDF-metK-H-proB-L-fabF-H-fabB-L-fumC-M-sucC-M plasmid
Control with pCDF-putA-H plasmid
Control with pCDF-putA-H-proB-L plasmid
Construction of sgRNA-expressing plasmids
pACYC-dCas9 was constructed by amplifying dCas9 fragment from 44249-dCas9 plasmid (no. 44249) (Addgene, USA) into BglII/XhoI sites of pACYCDuet-1. The sgRNA chimera, which consists of five domains [a Trc-inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin, a 40-nt transcription terminator derived from Streptococcus pyogenes and a 46-nt rrnB transcription terminator] was synthesized by Genewiz (Suzhou, China) and inserted into EcoRI/BamHI sites of pCDF303. This resulted in the plasmid pCDF303-gfp-H, which was the template for PCR-based mutagenesis. Site-directed mutagenesis was performed using the overlap-extension PCR method with mutant-specific primers. Other plasmids for repression were constructed like pCDF303-gfp-H. But the primers are different. Oligo nucleotides used to generate sgRNA cassettes and the resultant sgRNA expression vectors are listed in Additional file 1: Tables S1 and S2. Inhibition of multiple genes is based on the ligation used isocaudomers BglII and BamHI. Plasmid pCDF-proB-L was cut with EcoRI and BglII and plasmid pCDF-metK-H was cut with EcoRI and BamHI. Plasmid pCDF-metK-H-proB-L was obtained after ligation.
To assess the levels of cinnamic acid and pinocembrin, the supernatant was extracted with an equivalent volume of ethyl acetate, vortexed, and centrifuged at 6000 rpm for 3 min at 4 °C. Then, the upper organic layer was removed and evaporated to dryness. The remaining residue was resolubilized with methanol (TEDIA, Fairfield, OH, USA). Samples were quantified by HPLC (Alltech, Deerfield, IL, USA) using an Alltech series 1500 instrument equipped with a prevail C18 reverse-phase column (5 μm, 250 × 4.6 mm; Grace, Deerfield, IL, USA) maintained at 25 °C. For detection, 0.1% acetic acid (solvent A) and acetonitrile supplemented with 0.1% acetic acid (solvent B) were applied as the mobile phases at a flow rate of 1.0 mL min−1. The elution was performed according to the following conditions: minute 0–1: 15% B; minutes 1–10: 15% to 40% B; minutes 10–15: 40% to 50% B; minutes 15–25: 50% to 85% B; minutes 25–30: 85% to 15% B; and minute 30–31: 15% B. Products were detected by monitoring the absorbance at 300 nm.
To quantify the malonyl-CoA and ATP concentration, the harvested cells were resuspended in 1 mL of 6% perchloric acid (ultrasonication in an ice-water bath) and neutralized with 0.3 mL of saturated potassium carbonate. The solution was centrifuged to pellet the cell debris. For detection of malonyl-CoA, 95% 0.1 M ammonium formate/5% MeOH (solvent A) and 50% 0.1 M ammonium formate/50% MeOH (solvent B) were applied as the mobile phases at a flow rate of 1.0 mL min−1. The elution was performed according to the following conditions: minutes 0–10: 100% A to 50% A; minutes 10–12: 50% to 100% A; minutes 12–13: 100% A. For detection of ATP and ADP, the mobile phase used was phosphate buffer consisting of 0.06 M K2HPO4 and 0.04 M KH2PO4 at pH 7.0 adjusted with 0.1 mol/L KOH and operated at a flow rate of 1 mL/min .
The effect of ATP on pinocembrin production
Design and construction of the CRISPRi systems for repression of the genes
According to previous research, the CRISPRi system can efficiently repress expression of targeted genes in E. coli, with no detectable off-target effects . In the present study, we constructed our adjustable CRISPRi system in E. coli BL21 (DE3) and confirmed its effect by repressing the green fluorescent protein (gfp) expression. Three sgRNAs with different repression efficiency were designed and constructed. Three corresponding protospacer adjacent motif (PAM) was located on the 29th, 293th and 683th of gfp gene . The closer the PAM region is to the initiation codon, the stronger the inhibition. As shown in the Additional file 1: Figure S1, compared with gfp-control, the fluorescence intensity of gfp-sgRNA-H, gfp-sgRNA-M and gfp-sgRNA-L could be decreased by 90.4%, 47.5% and 10.1% at 3 h, respectively. After 6 h, the fluorescence intensity of gfp-sgRNA-H, gfp-sgRNA-M and gfp-sgRNA-L could be decreased by 95.2%, 53.3% and 11.8% than that in gfp-control, respectively. These results indicated that CRISPRi system could be successfully repressing the expression of targeted protein in E. coli BL21(DE3).
Screening of different gens for increasing ATP levels and enhancing pinocembrin production
Optimization of repression efficiency of proB and metK
Optimization of pinocembrin production by combining ATP and malonyl-CoA engineering strategy
Optimization of cultivation and induction conditions
Biosynthesis of one molecule of pinocembrin requires one molecule of ATP in the pinocembrin metabolic pathway, thus, the intracellular ATP concentration might be significant for pinocembrin production. In this work, we identified a number of different genes-repression for increasing ATP and pinocembrin production in E. coli, and found that the repression of metK and proB based on CRISPRi system could enhance the pinocembrin production. The genes for the other enzyme tested, glnA, aroK and argB, had no effect. In addition, we optimized the gene repression level to increase the pinocembrin production and ATP level and further improved the pinocembrin production by multiple-gene repression of metK and proB. Combining optimization of culture, induction conditions and repression of the genes in the malonyl-CoA biosynthesis including fabF, fabB, fumC and sucC, a pinocembrin production of 11.2-fold higher than the Control were achieved.
In the central metabolism of E. coli, cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in metabolism . However, the low availability of cellular cofactor often impeded its utility for overproducing desired products. Although malonyl-CoA has been proved to be the limiting factor for flavonoids biosynthesis, the ATP availability in the synthetic pathway is also vital for pinocembrin production via ATP supplemented during the fermentation. In fact, the cell growth and maintenance, intracellular environment control, substrate transport and product export also require adequate supply of ATP . In many cases, the optimization of the fermentation process to enhance the ATP level is an option . ATP accumulation could also be elevated by gene knock-out strategies or small RNA regulation [13, 23, 24]. As CRISPRi system is advantageous over traditional gene-knockout strategies because of its ability to manipulate genes essential for growth , the development of CRISPRi system has offered an alternative approach to control the intracellular ATP level. The plasmid-based inducible CRISPRi system can regulate gene expression accurately at the transcriptional level in E. coli. Here, depending on the pET28-gfp plasmid locus, the non-template of gfp combined with dCas9-sgRNA complex, which had been successfully repressed in E. coli BL21(DE3) after induction by IPTG. It is found that CRISPRi system for ATP strategy did not generate growth repression in E. coli (data not show).
Compared with Cri-proB-H and Cri-proB-M, Cri-proB-L showed better effect on pinocembrin production. These finding indicate that the proline concentration might be important for E. coli metabolism and pinocembrin production. MetK was responsible for the final-product S-adenosylmethionine (SAM) in E. coli, while proB catalyze the reaction of the proline biosynthesis pathway and putA catalyze the reaction of the proline degradation pathway. Therefore, Cri-putA-H and Cri-putA-H-proB-L were constructed in order to detect whether the concentration of proline contribute to the improvement of pinocembrin production. As shown in Additional file 1: Figure S2, the strains Cri-putA-H decreased the pinocembrin production to 13.17 mg/L. The pinocembrin concentration in Cri-putA-H-proB-L was decreased by 30% when compared with that in Cri-proB-L. These results indicated that the further accumulation of proline concentration could not increase the ATP and pinocembrin production, and the repression of ATP consumption gene for proline or SAM biosynthesis was advantageous for pinocembrin production. In addition, we found that the combined strains Cri-A showed better effect than single gene interference. This demonstrated that coupling genetic modification to ATP strategy is benefit to identify the most suitable interventions. Further, the recombinant strains Cri-A and Cri-AM produced higher ATP, ATP/ADP ratio and pinocembrin production, compared with the control. Therefore, these results indicate that the ATP concentration and the ATP/ADP ratio are associated with the pinocembrin production, and high level of ATP was benefit for pinocembrin biosynthesis. Moreover, the metabolism of malonyl-CoA, as with ATP, is highly connected with the metabolic network in E. coli. Due to its direct association with cell growth and synthesis of phospholipids and fatty acids, the intracellular availability of malonyl-CoA is limited . Compared with ATP level, more malonyl-CoA was necessary for strains to achieve a high yield of pinocembrin. By coupling malonyl-CoA genetic modification to ATP strategy, it was found that, the combined effects obviously improved the malonyl-CoA level and pinocembrin production from 0 to 48 h. These results indicate that the malonyl-CoA concentration is associated with the pinocembrin production. The present work demonstrates a way to approach the efficient biosynthesis of pinocembrin via ATP level strengthen and induction conditions optimization in E. coli.
In this work, we found that the addition of ATP contributes to the synthesis of pinocembrin. Five ATP-related genes were screened using the CRISPRi system, and inhibition of proB and metK was found to contribute to the accumulation of ATP and pinocembrin. On this basis, the repression intensity of proB and metK were optimized, and the results showed that low intensity repression of proB or high intensity repression of metK could better increase the production of pinocembrin. The effects of low intensity repression of proB and high intensity repression of metK on the synthesis of pinocembrin were investigated. The results of the study showed that the recombinant strain Cri-A produced a higher yield of pinocembrin (40.59 mg/L). In addition, ATP strategy coupled with the malonyl-CoA engineering and optimization of culture and induction condition, the production of pinocembrin by the recombinant strain increased by more than 7 times (102.02 mg/L) compared to the control.
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The authors declare that they have no competing interests.
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