Functional Conservation of Coenzyme Q Biosynthetic Genes among Yeasts, Plants, and Humans

Abstract

Coenzyme Q (CoQ) is an essential factor for aerobic growth and oxidative phosphorylation in the electron transport system. The biosynthetic pathway for CoQ has been proposed mainly from biochemical and genetic analyses of Escherichia coli and Saccharomyces cerevisiae; however, the biosynthetic pathway in higher eukaryotes has been explored in only a limited number of studies. We previously reported the roles of several genes involved in CoQ synthesis in the fission yeast Schizosaccharomyces pombe. Here, we expand these findings by identifying ten genes (dps1, dlp1, ppt1, and coq3–9) that are required for CoQ synthesis. CoQ10-deficient S. pombe coq deletion strains were generated and characterized. All mutant fission yeast strains were sensitive to oxidative stress, produced a large amount of sulfide, required an antioxidant to grow on minimal medium, and did not survive at the stationary phase. To compare the biosynthetic pathway of CoQ in fission yeast with that in higher eukaryotes, the ability of CoQ biosynthetic genes from humans and plants (Arabidopsis thaliana) to functionally complement the S. pombe coq deletion strains was determined. With the exception of COQ9, expression of all other human and plant COQ genes recovered CoQ10 production by the fission yeast coq deletion strains, although the addition of a mitochondrial targeting sequence was required for human COQ3 and COQ7, as well as A. thaliana COQ6. In summary, this study describes the functional conservation of CoQ biosynthetic genes between yeasts, humans, and plants.

Biosynthesis of coenzyme Q in eukaryotes

“…The decarboxylation stage of the ring modification procedure precedes the hydroxylation and methylation stages in E. coli, but the order of these reactions is thought to be different in S. cerevisiae. The species-specific phenotypes of all coq gene-knockout S. pombe 3,15) and C. elegans 16) strains have been reported. It is generally considered that the CoQ biosynthesis process is common in eukaryotes (Table 1); however, some differences have been reported: (i) polyprenyl diphosphate synthase forms a homodimeric or homotetrameric structure in S. cerevisiae, 17) but the homologous enzyme forms a heterotetramer structure composed of two subunits in S. pombe, mouse, and human 18,19) ; (ii) orthologs of Coq7 are missing in plant genomic databases; and (iii) Coq9 is missing in C. elegans.…”

Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis

“…Eco_ppsA, phosphoenolpyruvate synthase; Eco_aroF FBR , feedback-inhibitionresistant (FBR) 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase; Eco_ubiC, chorismate lyase; Spo_aro2, chorismate synthase; Spo_aro7, chorismate mutase; Sce_thmgr1, truncated 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase 1; Sce_mvk FBR , FBR mevalonate kinase; Spo_erg8, phosphomevalonate kinase; Spo_mvd1, diphosphomevalonate decarboxylase; dps1-dlp1, decaprenyl diphosphate synthase; ppt1, PHB-decaprenyl diphosphate transferase. CoQ 10 biosynthesis 2,19,24,26) ; thus, it was a promising organism to use with this approach.…”

“…pREP1-dps1-dlp1 was constructed by inserting the dlp1 expression cassette (nmt1 promoter-dlp1 gene-nmt1 terminator), which had been amplified using the nmt1-pro/ PstI and nmt1-term/PstI primers from pREP1-dlp1, 19) into the PstI site of pREP1-dps1. 19) pREP1-HIS7-ppt1-coq5 was constructed by inserting the coq5 expression cassette (nmt1 promoter-coq5 gene-nmt1 terminator), which had been amplified using the nmt1-pro/PstI and nmt1-term/PstI primers from pREP1-coq5, 19) into the PstI site of pREP1-HIS7-ppt1. pREP1-HIS7-ppt1 was constructed by inserting the ppt1 gene into the SalI and SmaI sites of pREP1-HIS7, which was constructed by removing LEU2 by HindIII digestion of pREP1 and inserting his7 into the SacI site.…”

“…pREP1-AUR1-coq4 was constructed by inserting the coq4 gene into the BamHI and SmaI sites of pREP1-AUR1, which was constructed by removing LEU2 by HindIII digestion of pREP1 and inserting aur1 20) into the SacI site. pREP1-KAN-coq7-coq9 was constructed by inserting the coq9 expression cassette (nmt1 promoter-coq9 gene-nmt1 terminator), which had been amplified from pREP1-coq9 using the nmt1pro-n-SphI and nmt1term-c-SphI, 19) into the SphI site of pREP1-KAN-coq7. pREP1-KAN-coq7 was constructed by inserting the coq7 gene 21) into the SalI and SmaI sites of pREP1-KAN, which was constructed by removing LEU2 by HindIII digestion of pREP1 and inserting kan r into the SacI site.…”

“…19) First, CoQ biosynthetic genes were individually overexpressed to determine their impact on CoQ production. Individual genes (dps1, dlp1, ppt1, and coq3-coq9) were cloned into the pREP1 vector for expression under a strong promoter (nmt1).…”