32

Noda Institute for Scientific Research GRANT
2012 Research Grant

Development of yeast strains that produce high levels of CoQ10
Makoto KawamukaiLife and Environmental Science, Shimane University

Research aims
Coenzyme Q (CoQ, ubiquinone) is a component of theelectron transport system in the mitochondria of eukaryotes
and is essential for energy production. CoQ consists of a quinone moiety and an isoprenoid chain. Almost all living cells
produce CoQ with different isoprenoid chain lengths. For
example, CoQ10, which is a popular food supplement, possesses 10 isoprene units. CoQ10 is commercially producedby microorganisms, and as of yet, genetic engineering approaches to commercially synthesize this molecule have notbeen reported. Despite the importance of CoQ in living cells,
the biosynthetic pathway of CoQ remains to be elucidated.1)
There were two major aims in the present study. The firstwas to unravel the biosynthetic pathway for Coenzyme Q
in the fission yeast Schizosaccharomyces pombe. The second was to use the knowledge gained to produce CoQ10 in
S. pombe. In contrast to Saccharomyces cerevisiae, which
produces CoQ6, S. pombe naturally produces CoQ10, thesame molecule found in humans, so it is beneficial to utilize
S. pombe to produce CoQ10. As a first step, the genes for
CoQ10 biosynthesis were investigated. The genes responsible for the synthesis of CoQ10 were then overexpressed
and CoQ10 production was evaluated. Finally, CoQ10 wasmeasured in protein kinase deletion strains.
Methods
Standard yeast culture media and genetic manipulations
were used. S. pombe strains were grown in complete YES

medium (0.5% yeast extract, 3% glucose, 225 mg/L each ofadenine, leucine, uracil, histidine, and lysine hydrochloride)or in EMM medium (0.3% potassium hydrogen phthalate,
0.56% sodium phosphate, 0.5% ammonium chloride, 2%
glucose, vitamins, minerals, and salts). Standard procedures
were used for cloning genes in Escherichia coli and construction of gene deletion strains of S. pombe. PCR was done
according to commonly used methods. Nucleotide sequenceswere determined by the dideoxynucleotide chain-termination
method using an Applied Biosystems 3500 Genetic Analyzer. Crude lipid extracts were analyzed by normal phasethin layer chromatography (TLC) with authentic coenzyme
Q10 as the standard. Normal-phase TLC was carried out on
a Kieselgel 60 F254 plate with benzene/acetone (97 : 3, v/v).
The band containing coenzyme CoQ10 was collected fromthe TLC plate following UV visualization and extracted withchloroform/methanol (1 : 1, v/v). The samples were dried andresolved in ethanol. Purified CoQ was further analyzed by
HPLC with ethanol as the solvent. The production of H2S
was measured by the methylene blue method.2)
Results
The biosynthetic pathway that converts para-hydroxybenzoic acid (PHB) into CoQ10 consists of at least eightsteps in S. pombe. Starting from the synthesis of PHB,
seven enzymes are thought to be involved in CoQ10 synthesis. These steps include the condensation and transfer ofthe isoprenoid side chain to PHB, followed by methylations, decarboxylation and hydroxylations (Fig. 1). PHB-Fig. 1. Biosynthetic pathway for Coenzyme Q in fission yeast Schizosaccharomyces pombe. Name of gene products involved in CoQ synthesis are indicated. There are two undefined reactions in the pathway. Three genes (coq4, coq8, and coq9) are known to be essential for CoQ biosynthesis, but their rolesin this pathway were not determined.

		33Fig. 2. cAMP-PKA signaling and the production of CoQ10. When glucose levels are decreased, the synthesis of cAMP becomes lower due tolack of activation of adenylyl cyclase. Under low levels of cAMP, PKA isinactive due to formation of a complex between the catalytic subunit(Pka1) and regulatory subunit (Cgs1). Under low glucose conditions,
CoQ10 is highly produced.

polyprenyl diphosphate transferase (Ppt1/Coq2) catalyzes
the condensation reaction between PHB and the isoprenoidside chain. Coq3 (O-methyltransferase) catalyzes the twoO-methylation steps in the CoQ biosynthetic pathway.
Coq4 is absolutely required for biosynthesis, but its enzymatic function remains to be elucidated. Coq5 (C-methyltransferase) catalyzes the single C-methylation step in theCoQ10 biosynthetic pathway. Coq6 has been identified as
C5 oxygenase. Coq7 (mono-oxygenase) is involved in thepenultimate step of CoQ biosynthesis. Coq8 functions as aprotein kinase that phosphorylates some of the Coq proteinsand stabilizes the protein complex for CoQ synthesis. Coq9
is required for CoQ biosynthesis, but its enzymatic function
is not yet known. Recently it has been shown that para-aminobenzoic acid also serves as a precursor for CoQ biosynthesis in budding yeast, but it is not clear if this is true inother organisms.
Ten deletion strains for CoQ10 synthesis were constructed in S. pombe. These included three genes that have
not yet been assigned to the CoQ biosynthetic pathway. We
then investigated the phenotypes of these deletion strains
and observed that they did not grow well on the EMM minimum plate. Because of the lack of CoQ10 synthesis, thesestrains were unable to survive in stationary phase, produced
a high amount of H2S3) and lack of de novo uridine monophosphate synthesis.4) We then determined the product ofthe precursor(s) of CoQ10 and identified DMQ10 in an S.
pombe coq7 deletion mutant. We also tried to characterize
CoQ10 precursors in the coq5 deletion strain but were unable to determine the structures of these products.
The effect of CoQ10 encapsulated with cyclodextrin wasalso investigated. Cyclodextrin allowed solubilization ofCoQ10 so that it could be dissolved in water. We found that
CoQ10 encapsulated with cyclodextrin restored the growthof S. pombe coq deletion strains by supporting the antioxidant property of CoQ10. 5)

Each gene that was involved in CoQ biosynthesis wasthen expressed in S. pombe. Ten genes were individually
expressed and CoQ10 production was measured. As a result, coq2, which encodes PHB-prenyltransferase, increased
the production of CoQ10 effectively, although only by30%. We attempted to express all 10 coq genes in the samecells, but this resulted in growth inhibition in many cases.
We then tested the deletion strains of protein kinases tomeasure the amount of CoQ. We found that loss of Pka1 increased the production of CoQ10, and loss of its regulatory
subunit Cgs1 lowered the production of CoQ10 (Fig. 2).
Pka1 is the catalytic subunit of cAMP-dependent protein
kinase A (PKA), which is activated by glucose in yeast toregulate cellular proliferation. Consistently, CoQ10 was
highly produced under low glucose conditions and was decreased in the presence of high glucose. Therefore, wefound that PKA contributes to the production of CoQ10 infission yeast.
Conclusion
There are at least ten genes involved in CoQ10 synthesis.
Individual coq gene deletion strains displayed phenotypes
related to respiration and oxidation-reduction of enzymes.
Some strains accumulated putative precursors. DMQ10 was
found in S. pombe coq7 deletion mutant. Expression ofCoQ biosynthetic genes in S. pombe led to the increase ofCoQ10 synthesis by up to 30%. By deletion of pka1, a significant increase in CoQ10 was observed. The PKA pathway regulates the synthesis of CoQ10 in fission yeast S.
pombe.
References
1)Kawamukai, M. (2009) Biosynthesis and bioproduction of coenzyme Q10 by yeasts and other organisms. Biotechnol. Appl. Biochem. 53: 217–226
2) Miki, R., Saiki, R., Ozoe, Y., and Kawamukai, M. (2008) Comparison of a coq7 deletion mutant with other respiration-defectivemutants in fission yeast. FEBS J. 275: 5309–5324
3) Zhang, M., Wakitani, S., Hayashi, K., Miki, M., and Kawamukai, M.
(2008) High production of sulfide in coenzyme Q deficient fission
yeast. Biofactors 32, 91–98
4) Matsuo, Y., Nishino, K., Mizuno, K., Akihiro, T., Toda, T., Matsuo,Y., Kaino, T., and Kawamukai, M. (2013) Polypeptone induces dramatic cell lysis in ura4 deletion mutants in fission yeast PLoS ONE
8(3): e59887
5) Nishida, T., Kaino, T., Ikarashi, R., Nakata, D., Terao, K., Ando, M.,
Hamaguchi, H., Kawamukai, M., and Yamamoto, T. (2013) The effect of coenzyme Q10 included by gamma-cyclodextrin on thegrowth of fission yeast studied by microscope Raman spectroscopy.
J. Molecular Structure 375–381