Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.
Appl Microbiol Biotechnol. 2012 Jul;95(1):179-87. doi: 10.1007/s00253-012-3959-6. Epub 2012 Feb 29.
In an effort to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: C18:1-OH) as a petrochemical replacement in a variety of industrial processes, we introduced Claviceps purpurea oleate ∆12-hydroxylase gene (CpFAH12) to Schizosaccharomyces pombe, putting it under the control of inducible nmt1 promoter. Since Fah12p is able to convert oleic acid to ricinoleic acid, we thought that S. pombe, in which around 75% of total fatty acid (FA) is oleic acid, would accordingly be an ideal microorganism for high production of ricinoleic acid. Unfortunately, at the normal growth temperature of 30 °C, S. pombe cells harboring CpFAH12 grew poorly when the CpFAH12 gene expression was induced, perhaps implicating ricinoleic acid as toxic in S. pombe. However, in line with a likely thermoinstability of Fah12p, there was almost no growth inhibition at 37 °C or, by contrast with 30 °C and lower temperatures, ricinoleic acid accumulation. Accordingly, various optimization steps led to a regime with preliminary growth at 37 °C followed by a 5-day incubation at 20 °C, and the level of ricinoleic acid reached 137.4 μg/ml of culture that corresponded to 52.6% of total FA.
为了在各种工业过程中用蓖麻油酸（12-羟基-十八碳-顺-9-烯酸：C18:1-OH）替代石化产品，我们将 Claviceps purpurea 油醇 ∆12-羟化酶基因（CpFAH12）引入酿酒酵母（Schizosaccharomyces pombe），并使其受诱导型 nmt1 启动子的控制。由于 Fah12p 能够将油酸转化为蓖麻油酸，我们认为大约 75%的总脂肪酸（FA）为油酸的 S. pombe 将是生产蓖麻油酸的理想微生物。不幸的是，在 30°C 的正常生长温度下，当诱导 CpFAH12 基因表达时，携带 CpFAH12 的 S. pombe 细胞生长不良，这可能表明蓖麻油酸对 S. pombe 有毒。然而，与 Fah12p 可能的热不稳定性一致，在 37°C 时几乎没有生长抑制，与 30°C 和更低的温度相比，蓖麻油酸积累。因此，各种优化步骤导致了在 37°C 下初步生长，然后在 20°C 下孵育 5 天的方案，蓖麻油酸的水平达到了 137.4μg/ml 的培养物，相当于总 FA 的 52.6%。
