Toxicity of ricinoleic acid production in fission yeast Schizosaccharomyces pombe is suppressed by the overexpression of plg7, a phospholipase A2 of a platelet-activating factor (PAF) family homolog

Abstract

In an effort to produce ricinoleic acid (RA), an important natural raw material with great values as a petrochemical replacement, in Schizosaccharomyces pombe, we introduced Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12) to S. pombe, putting it under the control of an inducible nmt1 promoter. However, RA was toxic to S. pombe and the cells expressing CpFAH12 grew poorly at the normal growth temperature 30 °C. To address its toxic mechanism in S. pombe, we screened for a S. pombe cDNA library and identified plg7, which encodes a phospholipase A2, as a suppressor that restored the growth defect without affecting the RA production. A lacZ fusion experiment showed that the expression of plg7 was inducible by RA. Thin layer chromatographic analysis confirmed a reduction in RA moiety in phospholipids and a concomitant increase in free RA in the plg7 overexpressed strain. Since RA is synthesized at the sn-2 position of phosphatidylcholine by Fah12p, and phospholipase A2 hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and removal of RA moieties from phospholipids, major components of lipid bilayer membrane, by Plg7p would be its suppression mechanism.

Cellular factories for coenzyme Q10 production

“…More success has been attained in the production of ricinoleic acid, a fatty acid from castor oil in S. pombe [92], and it may be possible to hijack this system to co-produce both CoQ 10 and fatty acids, with CoQ 10 participating as a lipid-soluble antioxidant to protect polyunsaturated fatty acids (PUFA) against oxidative damage during storage. A similar approach has been explored in Yarrowia lipolytica , an oleaginous yeast, even though Y. lipolitica is a non-native producer of CoQ 10 , and this approach is currently undergoing approval for production [93].…”

The putative phospholipase Lip2 counteracts oxidative damage and influences the virulence of Ustilago maydis

“…We hypothesized that the virulence defect in Δlip2 mutants was caused by their increased susceptibility to oxidative stress, perhaps generated as part of the early plant defence response to infection. This would be consistent with the role of PAFAHs in the response to oxidative stress in fungi and other organisms (Foulks et al ., ; Rosenson and Stafforini, ; Yazawa et al ., ; Yu et al ., ). We therefore assessed the susceptibilities of the Δlip1 and Δlip2 mutants to oxidative stress caused by H 2 O 2 .…”

“…This PL was also identified as a suppressor of cell death in Sc. pombe strains engineered to produce high levels of ricinoleic acid (RA), a hydroxylated fatty acid capable of disrupting membrane function (Yazawa et al ., ,b, 2014). The PL encoded by a homologue of plg7 in Trichoderma harzianum is also a stress‐responsive protein.…”

Production of ricinoleic acid-containing monoestolide triacylglycerides in an oleaginous diatom, Chaetoceros gracilis

“…improved RA production and prevented its cellular toxicity, and Yazawa et al 5. reported that the co-expression of CpFAH and a phospholipase gene suppressed RA toxicity as well.…”

“…The RcFAH protein is localised in the endoplasmic reticulum of R. communis , where it preferentially hydroxylates oleic acid moieties linked at sn -2 positions in phosphatidylcholine34. Furthermore, the cellular localisation and substrate specificity of CpFAH are predicted to be similar to those of RcFAH5; however, CpFAH exhibits a higher sequence similarity to C. purpurea Δ 12 -desaturase than to RcFAH2.…”