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Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate.

Samoudi M, Omid Yeganeh N, Shahbani Zahiri H, Shariati P, Hajhosseini R - Avicenna J Med Biotechnol (2015 Jul-Sep)

Bottom Line: Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively.The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other.The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration.

View Article: PubMed Central - PubMed

Affiliation: Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

ABSTRACT

Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli).

Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters.

Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other.

Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .

No MeSH data available.


Related in: MedlinePlus

Plasmid pTispA was introduced into E. coli Br, and the coenzyme Qs content was quantified in the resulting strain, as referred to E. coli BrTi. Error bars indicate the standard error of the mean of three independent experiments.
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Figure 7: Plasmid pTispA was introduced into E. coli Br, and the coenzyme Qs content was quantified in the resulting strain, as referred to E. coli BrTi. Error bars indicate the standard error of the mean of three independent experiments.

Mentions: In order to confirm that the reduction in CoQs production is due to the inhibitory effect of high FPP concentration, ispA was expressed under the control of trc promoter which is a stronger promoter than PBADand regulated alternatively by adding IPTG to the culture media. So, the ispA gene was ligated into the pTrc99A plasmid, leading to the formation of pTispA (Figure 3B). This recombinant plasmid was used for transformation of E. coli Ba and E. coli Br. The resulting recombinant cells, designated as E. coli BaTi and E. coli BrTi, were cultured in 2YTG medium under the same conditions, as described previously. Then, their CoQs were extracted and quantified by HPLC. The results of this study demonstrated that the over-expression of ispA was highly inhibiting for CoQ10biosynthesis in both E. coli BaTi (Figure 6) and E. coli BrTi (Figure 7) and reduced CoQ10production levels in the two strains from 629±40 to 289±16 μg/g DCW and from 564±28 to 110±20 μg/g DCW, respectively, even in the absence of an inducer. The presence of the inducer, IPTG (0.05 mM), led to a further decrease in CoQ10production to 30±13 μg/g DCW in E. coli BaTi and 80±14 μg/g DCW in E. coli BrTi. These results show that the inhibition of CoQ10production was more severe when compared to the E. coli BrDi and E. coli BaDi where the ispA gene is controlled by PBADpromoter.


Inhibition of Coenzyme Qs Accumulation in Engineered Escherichia coli by High Concentration of Farnesyl Diphosphate.

Samoudi M, Omid Yeganeh N, Shahbani Zahiri H, Shariati P, Hajhosseini R - Avicenna J Med Biotechnol (2015 Jul-Sep)

Plasmid pTispA was introduced into E. coli Br, and the coenzyme Qs content was quantified in the resulting strain, as referred to E. coli BrTi. Error bars indicate the standard error of the mean of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4508334&req=5

Figure 7: Plasmid pTispA was introduced into E. coli Br, and the coenzyme Qs content was quantified in the resulting strain, as referred to E. coli BrTi. Error bars indicate the standard error of the mean of three independent experiments.
Mentions: In order to confirm that the reduction in CoQs production is due to the inhibitory effect of high FPP concentration, ispA was expressed under the control of trc promoter which is a stronger promoter than PBADand regulated alternatively by adding IPTG to the culture media. So, the ispA gene was ligated into the pTrc99A plasmid, leading to the formation of pTispA (Figure 3B). This recombinant plasmid was used for transformation of E. coli Ba and E. coli Br. The resulting recombinant cells, designated as E. coli BaTi and E. coli BrTi, were cultured in 2YTG medium under the same conditions, as described previously. Then, their CoQs were extracted and quantified by HPLC. The results of this study demonstrated that the over-expression of ispA was highly inhibiting for CoQ10biosynthesis in both E. coli BaTi (Figure 6) and E. coli BrTi (Figure 7) and reduced CoQ10production levels in the two strains from 629±40 to 289±16 μg/g DCW and from 564±28 to 110±20 μg/g DCW, respectively, even in the absence of an inducer. The presence of the inducer, IPTG (0.05 mM), led to a further decrease in CoQ10production to 30±13 μg/g DCW in E. coli BaTi and 80±14 μg/g DCW in E. coli BrTi. These results show that the inhibition of CoQ10production was more severe when compared to the E. coli BrDi and E. coli BaDi where the ispA gene is controlled by PBADpromoter.

Bottom Line: Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively.The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other.The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration.

View Article: PubMed Central - PubMed

Affiliation: Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

ABSTRACT

Background: Coenzyme Q 10 (CoQ 10 ) is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase (FPPS) is a responsible enzyme for biosynthesis of farnesyl diphosphate (FPP), a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ 10 -producing Escherichia coli (E. coli).

Methods: Two CoQ 10 -producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS (ispA) under the control of either the trc or P BAD promoters.

Results: Over-expression of ispA under the control of P BAD promoter led to a relative increase in CoQ 10 production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ 10 production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629±40 to 30±13 and 564±28 to 80±14 μg/g Dried Cell Weight (DCW), respectively. The results showed high level of FPP reduces endogenous CoQ 8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other.

Conclusion: The reduction in CoQ 10 production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ 10 .

No MeSH data available.


Related in: MedlinePlus