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Noda Institute for Scientific Research GRANT2013 Research Grant

Development of efficient secretory production of ricinoleicacid, a petrochemical replacement of bioplastics, in fissionyeast Schizosaccharomyces pombeHiroshi UemuraNational Institute of Advanced Industrial Science and Technology

Research aimsRicinoleic acid (RA, 12-hydroxy-octadeca-cis-9-enoicacid: C18:1-OH), an important natural raw material, isvaluable as a petrochemical replacement in a variety of industrial processes. RA biosynthesis is catalyzed by the oleate Δ12-hydroxylase (FAH12), which adds a hydroxylgroup (-OH) to the 12th carbon of oleic acid. Seeds of thecastor bean plant (Ricinus communis) are a major source ofRA, and RA constitutes approximately 90% of the totalfatty acid (FA) content of the seed oil. However, castorbeans are not an ideal source crop, because the productionof RA from R. communis involves technical problems, suchas the presence of toxic and allergic substances that causehealth complications in workers involved in planting, harvesting, and processing; and that its cultivation is limited totropical and sub-tropical regions. Although extensive efforts were undertaken for the heterologous production ofRA in other plants or microorganisms, including yeast, ahigh level of RA production has never been achieved because RA is an unusual fatty acid containing a hydroxylgroup (-OH) and causes toxicity in the host cells. Recently,we successfully produced RA (more than 50% of the totalFA) by introducing the Claviceps purpurea D12-hydroxylase gene (CpFAH12) into Schizosaccharomyces pombe,despite its toxicity to S. pombe cells.1, 2) Subsequent studieshave demonstrated that overexpression of plg7, encodingphospholipase A2, suppresses the toxic effects of RA3), andsuggested the possibility of RA secretion into the culturemedia. The main goal of this project is to study the mechanisms that allow RA tolerance and its secretion in S. pombe.
MethodsThe S. pombe strain ARC010-1 (h− leu1-32 ura4-D18)was used in this study. An integration plasmid containingCpFAH12 under the nmt1 promoter of S. pombe, which isrepressed by thiamine, was integrated at the ura4 locus onchromosome III, as previously described.3) The S. pombestrains were grown in EMMS or EMMS dropout media, depending on the selection pressure required to maintain theplasmids. EMMS was used for normal yeast growth, but anitrogen-limited minimal medium (EMM-C/N3) was usedfor the production of RA. In EMM-C/N3, the concentrationof ammonium chloride and glucose was reduced and increased to 0.1% and 10%, respectively. For the repressionof gene expression under the nmt1 promoter, 15 µM thiamine (5 µg/mL) was used. Total FA content was determinedby gas chromatographic (GC) analysis.1) Lipid compositionwas analyzed by thin layer chromatography (TLC: silicagel 60 plate, Merck).3)ResultsSecretory production of RA by plg7 overexpression in S.
pombe4)To produce RA in S. pombe, we introduced CpFAH12into S. pombe. However, RA proved toxic to S. pombe andthe cells expressing CpFAH12 showed poor growth at thenormal growth temperature of 30°C.1) To explore thismechanism of toxicity in S. pombe, we screened an S.
pombe cDNA library and identified plg7, which encodesphospholipase A2, as a suppressor that restored the growthdefect without affecting the RA production. TLC analysisconfirmed the reduction in RA moiety in phospholipids anda concomitant increase in free RA in the plg7-overexpressed strain. Since RA is synthesized at the sn-2 positionof phosphatidylcholine by Fah12p, and phospholipase A2hydrolyzes the sn-2 acyl bond of phospholipids, we speculate that plg7 is a stress-responsive gene, and that Plg7pacts as a suppressor by removing the RA moieties fromphospholipids, the major components of lipid bilayer membranes.3)During the course of additional studies on plg7, we noticed that a large amount of free RA was secreted in the culture media when the FAH12 integrant harboring the plg7multicopy plasmid was grown at 20°C, a temperature thatis more favorable for RA production. We speculated thatthe turbidity of the culture was caused by cell lysis. However, the constant increase in turbidity of the supernatantsduring cultivation in 10% glucose and the highly selectivecomposition of RA in the media indicated that the turbidityof the supernatant is due to the secretion of RA and not dueto cell lysis. Besides, viability tests clearly demonstratedthat there is no correlation between the viability and turbidity. In addition, TLC analysis confirmed that the majorcomponent of the turbid supernatant is free RA. Taken together, our results show that the FAH12 integrant harboring

48Fig. 1. Growth recovery of CpFAH12 integrant by the overexpression ofplg7 and ptl2 genes.
The Pnmt1-CpFAH12 integrants harboring various genomic clone plasmids were grown in EMM-C/N3 in the presence (dotted line) or absence(solid line) of thiamine at 30°C. Vector (diamonds): empty vector pALKS. plg7 (triangles): genomic plg7 plasmid. ptl1 (squares): genomic ptl1plasmid. ptl12 (circles): genomic ptl2 plasmid. ptl3 (asterisks): genomicptl3 plasmid. Ratio indicates doubling time ratio to that of the controlstrain under the same growth condition.

the plg7 multicopy plasmid secretes free RA into the media.
The strain secretes more than 50% of the produced RA intothe culture media.
Suppression of RA toxicity by ptl2 overexpression in S.
pombe5)To further increase RA tolerance and enhance RA secretion, we extended our analysis and examined the effect oftriglyceride (TG) lipase overexpression as TG metabolismis closely related to phospholipid metabolism and fatty acidremodeling. S. pombe has three TG lipase genes, ptl1, ptl2,and ptl3, which have high protein sequence similarity toeach other and to the S. cerevisiae counterparts TGL3,TGL4, and TGL5, respectively.2) However, only ptl2 overexpression suppressed the growth defect of RA production,and the culture grown at 20°C secreted free RA into themedia. The suppression by ptl2 was independent of plg7and a large amount of free RA was accumulated in thecells, along with a concomitant decrease in the RA moietiesin phospholipids. Furthermore, the suppression by ptl2 wasattenuated by bromoenol lactone (BEL), a phospholipaseA2-specific inhibitor, suggesting that Ptl2p has phospholipase activity. These results suggest that ptl2 suppresses thetoxicity of RA with its phospholipase activity.

Fig. 2. Overexpression of plg7 and ptl2 in the CpFAH12 integrant facilitates the secretion of RA.
The Pnmt1-CpFAH12 integrants with various plasmids were grown inEMM-C/N3 in the absence of thiamine at 20°C for 5 d starting withOD600=2. Vector: empty vector pREP1. plg7: plg7 expressed in pREP41.
ptl1: ptl1 expressed in pREP41. ptl2: ptl2 expressed in pREP41. ptl3: ptl3expressed in pREP3. The upper and lower parts of the graph represent intracellular FA composition and culture supernatant, respectively. Fattyacids are categorized into three groups: (1) C18:1-OH (RA), (2) C18-2n-6(linoleic acid, by-product of FAH12), and (3) others (all other original fattyacids). Values in the graph indicate the percentage of RA (out of total FA)inside the cells and culture supernatants.

pressor by removing RA moieties from phospholipids. Wealso discovered the secretion of RA into the growth medium when a large amount of RA was produced at low temperature. Furthermore, we found that one of the TG lipasegenes, ptl2, is also involved in the suppression of RA toxicity. Our identification of plg7 and ptl2 as suppressors of RAtoxicity and their analyses have opened up new avenues forthe efficient production of RA without disturbing the cellgrowth. Since the long-term goal of our study is to produceRA in sustainable quantities using transgenic S. pombe, thediscovery of the secretion of free RA into the media may beadvantageous for the industrial application of this system.
References1)

2)

3)ConclusionsThe lipid RA has a wide range of industrial applications.
Since RA is an unusual fatty acid containing a hydroxylgroup (-OH), it is toxic to the host cells, and only a fewplants (R. communis and Lesquerella fendleri) and fungi(C. purpurea) are natural producers of RA. Although RAproduction in S. pombe also shows toxicity, we circumvented this problem by isolating plg7 as a multicopy suppressor. Our research suggests that plg7 functions as a sup-4)

5)

Holic R., Yazawa H., Kumagai H., and Uemura H. (2012) Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe. Appl. Microbiol. Biotechnol. 95: 179–187.
Yazawa H., Kumagai H., and Uemura H. (2012) Characterization oftriglyceride lipase genes of fission yeast Schizosaccharomycespombe. Appl. Microbiol. Biotechnol. 96: 981–991.
Yazawa H., Holic R., Kumagai H., and Uemura H. (2013) Toxicityof ricinoleic acid production in fission yeast Schizosaccharomycespombe is suppressed by the overexpression of plg7, a phospholipaseA2 of a platelet-activating factor (PAF) family homolog. Appl. Microbiol. Biotechnol. 97: 8193–8203.
Yazawa H., Kumagai H., and Uemura H. (2013) Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe.
Appl. Microbiol. Biotechnol. 97: 8663–8671.
Yazawa H., Ogiso M., Kumagai H., and Uemura H. (2014) Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeastSchizosaccharomyces pombe. Appl. Microbiol. Biotechnol. In press(DOI: 10.1007/s00253-014-6006-y).