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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

The tandem condensation reactions by decaprenyl diphosphate synthase result in the polymerization of isopentenyl diphosphate (IPP) molecules into decaprenyl diphosphate. The indicated enzymes are as follow: FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; DPPS, decaprenyl diphosphate synthase and UbiA, 4HB-polyprenyl transferase (Zahiri et al, 2009).
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Figure 1: The tandem condensation reactions by decaprenyl diphosphate synthase result in the polymerization of isopentenyl diphosphate (IPP) molecules into decaprenyl diphosphate. The indicated enzymes are as follow: FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; DPPS, decaprenyl diphosphate synthase and UbiA, 4HB-polyprenyl transferase (Zahiri et al, 2009).

Mentions: Ubiquinone has been used as a medicine, but is now widely used as a nutritional supplement 9,10. The effect of ubiquinone on the lifespan of humans is currently being studied extensively while studies on other roles of ubiquinone are also underway 11. Ubiquinone is generally composed of a benzene ring and an isoprenoid side chain, which is a homopolymer of Isopentenyl diphosphate (IPP), also known as the isoprene unit. The length of the side chain determines the type of CoQ in various organisms. For example, the CoQ side chain in human cells is comprised of ten isoprene units and forms CoQ10, while Escherichia coli (E. coli) contains CoQ8which possesses eight isoprene units in its side chain 12–16. The synthesis of the CoQ10side chain is catalyzed by Dds, whereas Octaprenyl diphosphate synthase (Ods) is the enzyme responsible for biosynthesis of the corresponding side chain in E. coli. Therefore, the expression of the heterologous gene encoding for Dds can lead to the production of CoQ10in transformed E. coli cells along with CoQ8. Accordingly, several CoQ10-producing E. coli strains have been devolved by introducing Dds encoding gene from various sources either via plasmid-expression system or chromosomal integration 4,15–18. The initial step in biosynthesis of isoprenoid side chain involves a condensation reaction between an IPP and its allelic isomer, Dime-thylallyl diphosphate (DMADP) by Geranyl diphosphate synthase (GPPS) which results in the formation of Geranyl diphosphate (GPP) with ten carbons. FPPS adds another IPP to GPP and produces Farnesyl diphosphate (FPP) with 15 carbons. Likewise, Geranylgeranyl diphosphate synthase (GGPPS) uses FPP as a substrate and adds another IPP to form Geranylgeranyl diphosphate (GGPP) with 20 carbons. Finally, the polyprenyl diphosphate synthases such as Dds can use the above mentioned molecules (IPP, GPP, FPP and GGPP) as substrates for addition of more IPP molecules to make corresponding isoprenoid side chain. Finally, attachment of the synthesized side chain to benzene ring and subsequent modifications of the ring result in CoQ production 3,16,17,19,20. Figure 1 illustrates the biosynthetic steps for CoQ10production.


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)

The tandem condensation reactions by decaprenyl diphosphate synthase result in the polymerization of isopentenyl diphosphate (IPP) molecules into decaprenyl diphosphate. The indicated enzymes are as follow: FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; DPPS, decaprenyl diphosphate synthase and UbiA, 4HB-polyprenyl transferase (Zahiri et al, 2009).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The tandem condensation reactions by decaprenyl diphosphate synthase result in the polymerization of isopentenyl diphosphate (IPP) molecules into decaprenyl diphosphate. The indicated enzymes are as follow: FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; DPPS, decaprenyl diphosphate synthase and UbiA, 4HB-polyprenyl transferase (Zahiri et al, 2009).
Mentions: Ubiquinone has been used as a medicine, but is now widely used as a nutritional supplement 9,10. The effect of ubiquinone on the lifespan of humans is currently being studied extensively while studies on other roles of ubiquinone are also underway 11. Ubiquinone is generally composed of a benzene ring and an isoprenoid side chain, which is a homopolymer of Isopentenyl diphosphate (IPP), also known as the isoprene unit. The length of the side chain determines the type of CoQ in various organisms. For example, the CoQ side chain in human cells is comprised of ten isoprene units and forms CoQ10, while Escherichia coli (E. coli) contains CoQ8which possesses eight isoprene units in its side chain 12–16. The synthesis of the CoQ10side chain is catalyzed by Dds, whereas Octaprenyl diphosphate synthase (Ods) is the enzyme responsible for biosynthesis of the corresponding side chain in E. coli. Therefore, the expression of the heterologous gene encoding for Dds can lead to the production of CoQ10in transformed E. coli cells along with CoQ8. Accordingly, several CoQ10-producing E. coli strains have been devolved by introducing Dds encoding gene from various sources either via plasmid-expression system or chromosomal integration 4,15–18. The initial step in biosynthesis of isoprenoid side chain involves a condensation reaction between an IPP and its allelic isomer, Dime-thylallyl diphosphate (DMADP) by Geranyl diphosphate synthase (GPPS) which results in the formation of Geranyl diphosphate (GPP) with ten carbons. FPPS adds another IPP to GPP and produces Farnesyl diphosphate (FPP) with 15 carbons. Likewise, Geranylgeranyl diphosphate synthase (GGPPS) uses FPP as a substrate and adds another IPP to form Geranylgeranyl diphosphate (GGPP) with 20 carbons. Finally, the polyprenyl diphosphate synthases such as Dds can use the above mentioned molecules (IPP, GPP, FPP and GGPP) as substrates for addition of more IPP molecules to make corresponding isoprenoid side chain. Finally, attachment of the synthesized side chain to benzene ring and subsequent modifications of the ring result in CoQ production 3,16,17,19,20. Figure 1 illustrates the biosynthetic steps for CoQ10production.

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