title: Accumulation of squalene in a microalga Chlamydomonas reinhardtii by genetic modification of squalene synthase and squalene epoxidase genes.
abstract: Several microalgae accumulate high levels of squalene, and as such provide a potentially valuable source of this useful compound. However, the molecular mechanism of squalene biosynthesis in microalgae is still largely unknown. We obtained the sequences of two enzymes involved in squalene synthesis and metabolism, squalene synthase (CrSQS) and squalene epoxidase (CrSQE), from the model green alga Chlamydomonas reinhardtii. CrSQS was functionally characterized by expression in Escherichia coli and CrSQE by complementation of a budding yeast erg1 mutant. Transient expression of CrSQS and CrSQE fused with fluorescent proteins in onion epidermal tissue suggested that both proteins were co-localized in the endoplasmic reticulum. CrSQS-overexpression increased the rate of conversion of 14C-labeled farnesylpyrophosphate into squalene but did not lead to over-accumulation of squalene. Addition of terbinafine caused the accumulation of squalene and suppression of cell survival. On the other hand, in CrSQE-knockdown lines, the expression level of CrSQE was reduced by 59-76% of that in wild-type cells, and significant levels of squalene (0.9-1.1 μg mg-1 cell dry weight) accumulated without any growth inhibition. In co-transformation lines with CrSQS-overexpression and CrSQE-knockdown, the level of squalene was not increased significantly compared with that in solitary CrSQE-knockdown lines. These results indicated that partial knockdown of CrSQE is an effective strategy to increase squalene production in C. reinhardtii cells.
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kajikawa
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
authors.forename: Seiko
authors.initials: S
authors.lastname: Kinohira
authors.affiliation: Graduate School of Humanities and Science, Ochanomizu University, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ando
authors.affiliation: Graduate School of Humanities and Science, Ochanomizu University, Tokyo, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Shimoyama
authors.affiliation: Graduate School of Humanities and Science, Ochanomizu University, Tokyo, Japan.
authors.forename: Misako
authors.initials: M
authors.lastname: Kato
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
authors.forename: Hideya
authors.initials: H
authors.lastname: Fukuzawa
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics* / Q000378:metabolism; D055785:Gene Knockdown Techniques; D009154:Mutation; D009281:Naphthalenes / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013185:Squalene / Q000378:metabolism*; D050603:Squalene Monooxygenase / Q000235:genetics* / Q000378:metabolism; D013261:Sterols / Q000096:biosynthesis; D000077291:Terbinafine; D015003:Yeasts / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011994:Recombinant Proteins; D013261:Sterols; D013185:Squalene; D050603:Squalene Monooxygenase; D013186:Farnesyl-Diphosphate Farnesyltransferase; D000077291:Terbinafine