Alkaline pH enhances farnesol production by Saccharomyces cerevisiae Masayoshi Muramatsu, 1 Chikara Ohto, 1 Shusei Obata, 1 Eiji Sakuradani, 2 and Sakayu Shimizu 2, ⁎ Bio Research Lab., Toyota Motor Corporation,1 Toyota-cho, Toyota 471-8572, Japan 1 and Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan 2 Received 6 November 2008; accepted 13 February 2009 External environments affect prenyl alcohol production by squalene synthetase-deficient mutant Saccharomyces cerevisiae ATCC 64031. Cultivation of the yeast in medium with an initial pH ranging from 7.0 to 8.0 increased the amount of secreted farnesol (FOH). In contrast, acidic medium with a pH below 4.0 increased the intracellular FOH and its isomer nerolidol. These effects of alkaline pH were also observed on constant pH cultivation in a jar fermenter. On cultivation for 133 h, the FOH production reached 102.8 mg/l. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Farnesol; Farnesyl diphosphate; Nerolidol; Prenyl alcohol; Terpenoid] (E,E)-Farnesol (FOH) is considered to be produced in organisms through the hydrolysis of (E,E)-farnesyl diphosphate (FPP) by a peculiar phosphatase (1). It has been utilized as not only a perfume for cosmetics but also as a starting material for the chemical synthesis for various supplements: carotenoids, tocopherol, and co-enzyme Q10. Commercially available FOH has so far been produced through chemical synthesis not a fermentation process because of the too low productivity for application to an industrial process. The first microorganism for which FOH was detected in the culture broth was squalene synthase-deficient Saccharomyces cerevisiae ATCC 64031 (1). Candida albicans also excretes FOH, which suppresses mycelial development by the cells, acting as a quorum-sensing compound (2). Although the FOH production by these microorgan- isms is quite low, medium containing a squalene synthase inhibitor, detergents, and oils increases the extracellular production of FOH. Cultivation of C. albicans with zaragozic acid B, a potent squalene synthase inhibitor, leads to an 8-fold increase in the amount of FOH (3). The combined addition of this inhibitor and soybean oil to the culture medium increases approximately 100 times the extracellular production of FOH by various microorganisms (4, 5). These findings suggest the possibility of enhancing the production by changing the extracellular environment around the cells. We examined many cultivation conditions to improve prenyl alcohol production by S. cerevisiae ATCC 64031, and found that neutral and mild alkaline pHs accelerate the conversion of FPP to FOH significantly. In this report, we describe the detailed conditions causing this unique conversion by the strain. pH control of the culture medium is considered to be an extremely valuable technique for large- scale production of prenyl alcohol with microorganisms. MATERIALS AND METHODS Materials YM broth and Yeast Nitrogen Base (YNB) were purchased from Becton, Dickinson and Company (NJ, USA). Acid phosphatase and FPP were from Sigma- Aldrich (St. Louis, MO, USA) and Cosmo Bio Co., Ltd. (Tokyo, Japan), respectively. Other chemicals were purchased from Nacalai Tesuque Inc. (Kyoto, Japan). Strain and culture conditions S. cerevisiae ATCC 64031, a squalene synthetase- deficient mutant, was used. All media used here contained 0.002% ergosterol supplemented with a 1000× solution. The stock solution was prepared by dissolving 20 mg ergosterol powder in 1 ml of ethanol containing 50% tergitol. The details of cultivations are given in the legend to each figure. Analysis of FOH and nerolidol (NOH) Culture broth (2 ml) was centrifuged to separate supernatants (extracellular fractions) and cells at 2000 ×g for 10 min at room temperature. The cells, after washing twice with physiological saline, were suspended in 0.75 ml of the saline, and then treated with 0.5 mm glass beads using a multi-bead shocker (Yasui Kikai, Osaka, Japan) for 20 min. The prenyl alcohols were extracted separately from the extracellular and intracellular fractions with a 1.6 volume of extraction solvent (methanol: pentane, 3:5; v/v). After vigorous mixing and following centrifugation (2000 ×g), the upper organic layer was transferred for gas chromato- graphy/mass spectroscopy (GC/MS) in a vial in which 10 μl of an internal standard solution (0.1% v/v undecanol in ethanol) had been placed in advance. FOH and NOH were analyzed with an Agilent 5973 GC/MS system (Agilent Technologies, Santa Clara, CA, USA; column, HP-5MS, 0.25 mm id×30 m; column temperature, 115 °C, hold 1.5 min, raise at 70 °C/min to 250 °C, hold 0 min, raise at 70 °C/min to 300 °C, and hold 7 min). The amount of a prenyl alcohol was calculated from the ratio of the selected ion peak area (69 mass fragment) to that of undecanol. All data presented in the present paper are the means of three independent determinations (SD, less than 7%). Analysis of glucose, ethanol, and acetic acid The concentrations of glucose, and ethanol and acetic acid in the broth were measured with an F-Kit (J. K. International Inc., Tokyo, Japan) based on the hexose kinase method, and enzyme assays involving the combination of alcohol dehydrogenase and acetaldehyde dehydrogenase, respectively. Determination of degraded FPP The culture broth (3.5 ml, O.D.660 = 8.5) at 3rd day on the bench-scale cultivation was centrifuged to separate supernatants (extracellular fractions) and cells at 2000 ×g for 10 min at room temperature. The cells were suspended in 3.5 ml of deionized water, and then treated with the same volume of glass beads (0.5 mm) using a multi-bead shocker (Yasui Kikai) for 20 min. The intracellular fraction was prepared from the disrupted cell suspension by the centrifugation. The fractions were autoclaved at 121 °C for 20 min, if necessary. The extracellular or intracellular fraction (250 μl) was added to the FPP solution (0.15 mg/ 750 μl), followed by incubation at 30 °C overnight without shaking. The pH value of the Journal of Bioscience and Bioengineering VOL. 108 No. 1, 52 – 55, 2009 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 75 753 6113; fax: +81 75 753 6128. E-mail address: sim@kais.kyoto-u.ac.jp (S. Shimizu). 1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.02.012