Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a “push” (synthesis) and “pull” (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses. Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a “push” (synthesis) and “pull” (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses. Hydroxy fatty acids are important raw materials with a wide range of industrial uses. In particular, ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid), a long-chain hydroxy fatty acid produced by castor bean (Ricinus communis), has many specialized uses in the manufacturing of a variety of industrial products such as nylons, lubricants, ink, and paints, as well as pharmaceuticals and cosmetics (1McKeon T.A. Lin J.T. Stafford A.E. Biosynthesis of ricinoleate in castor oil.Adv. Exp. Med. Biol. 1999; 464: 37-47Crossref PubMed Scopus (25) Google Scholar, 2Jaworski J. Cahoon E.B. Industrial oils from transgenic plants.Curr. Opin. Plant Biol. 2003; 6: 178-184Crossref PubMed Scopus (144) Google Scholar). However, due to the presence of a highly potent toxin (ricin), this native oilseed plant is not considered an ideal source for hydroxy fatty acid production. Therefore, tremendous effort has recently been made in identifying genes involved in the biosynthesis of this fatty acid and using the genes to engineer oilseed crops for producing ricinoleic acid (3van de Loo F.J. Broun P. Turner S. Somerville C. An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.Proc. Natl. Acad. Sci. USA. 1995; 92: 6743-6747Crossref PubMed Scopus (310) Google Scholar, 4Burgal J. Shockey J. Lu C. Dyer J. Larson T. Graham I. Browse J. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil.Plant Biotechnol. J. 2008; 6: 819-831Crossref PubMed Scopus (258) Google Scholar, 5Lu C. Fulda M. Wallis J.G. Browse J. A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis.Plant J. 2006; 45: 847-856Crossref PubMed Scopus (117) Google Scholar, 6van Erp H. Bates P.D. Burgal J. Shockey J. Browse J. Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis.Plant Physiol. 2011; 155: 683-693Crossref PubMed Scopus (143) Google Scholar, 7Snapp A.R. Kang J. Qi X. Lu C. A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa.Planta. 2014; 240: 599-610Crossref PubMed Scopus (43) Google Scholar). However, to date these attempts have met with only partial success. When a hydroxylase gene from a native plant species was introduced into oilseed crops, the hydroxy fatty acid content in transgenic seeds rarely exceeded 20% of the total fatty acids (8Broun P. Somerville C. Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean.Plant Physiol. 1997; 113: 933-942Crossref PubMed Scopus (177) Google Scholar, 9Broun P. Boddupalli S. Somerville C. A bifunctional oleate 12-hydroxylase: desaturase from Lesquerella fendleri.Plant J. 1998; 13: 201-210Crossref PubMed Scopus (140) Google Scholar, 10Smith M.A. Moon H. Chowrira G. Kunst L. Heter­ologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana.Planta. 2003; 217: 507-516Crossref PubMed Scopus (120) Google Scholar). In 2008, a new hydroxylase for the biosynthesis of ricinoleic acid was identified from a nonplant origin, and expression of this gene in Arabidopsis resulted in accumulation of a slightly higher level of hydroxy fatty acids in transgenic seeds (11Meesapyodsuk D. Qiu X. An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.Plant Physiol. 2008; 147: 1325-1333Crossref PubMed Scopus (37) Google Scholar). Very recently, coexpression of genes encoding factors involved in the networks of the hydroxylation or acyl trafficking process along with a hydroxylase has also been attempted (4Burgal J. Shockey J. Lu C. Dyer J. Larson T. Graham I. Browse J. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil.Plant Biotechnol. J. 2008; 6: 819-831Crossref PubMed Scopus (258) Google Scholar, 5Lu C. Fulda M. Wallis J.G. Browse J. A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis.Plant J. 2006; 45: 847-856Crossref PubMed Scopus (117) Google Scholar, 6van Erp H. Bates P.D. Burgal J. Shockey J. Browse J. Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis.Plant Physiol. 2011; 155: 683-693Crossref PubMed Scopus (143) Google Scholar, 7Snapp A.R. Kang J. Qi X. Lu C. A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa.Planta. 2014; 240: 599-610Crossref PubMed Scopus (43) Google Scholar). Although the improved production of hydroxy fatty acids is observed, the amount in transgenic seeds still rarely exceeds 25% of the total fatty acids. It appears that selection of genes from diverse sources and addition of cofactors can make a difference for transgenic production of hydroxyl fatty acids, but production of a single hydroxy fatty acid at the commercially viable level in plants still remains as a challenging task. Microorganisms have recently emerged as promising systems for producing biofuel for transportation and platform chemicals for biopolymers because some microbes offer high output of biomass with good oil content in a short period of time and are easily used for genetic manipulation to implement a metabolic engineering strategy. Exploitation of metabolic engineering of lipid pathways in microorganisms based on fatty acid biosynthesis and assembly frameworks provides a promising solution to bioproducts from microorganisms to replace petrochemicals, as fatty acids and derivatives share many similar chemical properties with petroleum fuels and petrochemicals such as reduced state of hydrocarbons and high density of energy (12Lennen R.M. Pfleger B.F. Microbial production of fatty acid-derived fuels and chemicals.Curr. Opin. Biotechnol. 2013; 24: 1044-1053Crossref PubMed Scopus (138) Google Scholar, 13Steen E.J. Kang Y. Bokinsky G. Hu Z. Schirmer A. McClure A. Del Cardayre S.B. Keasling J.D. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass.Nature. 2010; 463: 559-562Crossref PubMed Scopus (1074) Google Scholar, 14Runguphan W. Keasling J.D. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.Metab. Eng. 2014; 21: 103-113Crossref PubMed Scopus (288) Google Scholar, 15Xu P. Gu Q. Wang W. Wong L. Bower A.G. Collins C.H. Koffas M.A. Modular optimization of multi-gene pathways for fatty acids production in E. coli.Nat. Commun. 2013; 4: 1409Crossref PubMed Scopus (379) Google Scholar, 16Xu P. Li L. Zhang F. Stephanopoulos G. Koffas M. Improving fatty acids production by engineering dynamic pathway regulation and metabolic control.Proc. Natl. Acad. Sci. USA. 2014; 111: 11299-11304Crossref PubMed Scopus (368) Google Scholar). Therefore, metabolic engineering of microbial systems has potential in providing an alternative to plant sources for hydroxy fatty acid for industrial uses (17Holic R. Yazawa H. Kumagai H. Uemura H. Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe.Appl. Microbiol. Biotechnol. 2012; 95: 179-187Crossref PubMed Scopus (42) Google Scholar, 18Beopoulos A. Verbeke J. Bordes F. Guicherd M. Bressy M. Marty A. Nicaud J.M. Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica.Appl. Microbiol. Biotechnol. 2014; 98: 251-262Crossref PubMed Scopus (98) Google Scholar). This study describes metabolic engineering of ricinoleic acid in Pichia pastoris, a yeast with a good yield of biomass and oil. Two genes encoding CpFAH, a fatty acid hydroxylase we cloned previously from the fungus Claviceps (11Meesapyodsuk D. Qiu X. An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.Plant Physiol. 2008; 147: 1325-1333Crossref PubMed Scopus (37) Google and CpDGAT1, a diacylglycerol acyltransferase for the triacylglycerol biosynthesis cloned from the same species, in a diploid strain and in a haploid mutant resulted in the production of a high amount of ricinoleic acid mainly in the free fatty acid by the with little in the polar lipids. This work the effectiveness of the metabolic engineering strategy and excellent capacity of the yeast for hydroxy fatty acid production. also a new strategy to produce a higher level of fatty acids in transgenic plants free fatty acids as C. was and on the at Saccharomyces cerevisiae mutant L. A. E. A. M. H. S. lipid synthesis is in Biol. PubMed Scopus Google used as a to study the of CpDGAT1 was on yeast Pichia and used to study the expression of CpFAH and haploid P. was used as a strain for of Δ12 desaturase gene was made by of and of of Δ12 desaturase from P. into the and of in Δ12 strain was made by the by mutant strain was by and the to produce acid. from C. was using to the was by with was from the total by of was used as a to for and using the cDNA and used to the and was used for the cDNA of CpDGAT1 was by using and and and to used in this study are in the products and into of the yeast expression of to was by and of CpFAH and CpDGAT1 from (11Meesapyodsuk D. Qiu X. An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.Plant Physiol. 2008; 147: 1325-1333Crossref PubMed Scopus (37) Google and by and into with the same enzyme to and CpFAH and CpDGAT1, a of CpFAH and was with and cloned by into with was into S. cerevisiae using the expression yeast with CpDGAT1 or the at for in yeast and to an density of in a and to at the same for lipid Pichia of gene or of genes was into Pichia by using an on or or expression the at with in yeast acids, and the and to an of in yeast acids, and to at with for lipid and total fatty acids analysis, and was to the to from yeast with Dyer A of total lipid and J. Physiol. PubMed Scopus Google Scholar). in on with a system acid by with in and used to fatty acid In the to total fatty acids free fatty acid and in the and used as previously using (11Meesapyodsuk D. Qiu X. An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.Plant Physiol. 2008; 147: 1325-1333Crossref PubMed Scopus (37) Google Scholar). at for In the fatty acids and fatty by using at for X. R.M. E. of a of from developing seed the production of Biol. 2013; PubMed Scopus Google Scholar). the was used as to with fatty acids fatty in the presence of a little the to D. Qiu X. A pathway involved in the biosynthesis of fatty acids in Physiol. 2011; PubMed Scopus Google Scholar). by of and at for (11Meesapyodsuk D. Qiu X. An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis.Plant Physiol. 2008; 147: 1325-1333Crossref PubMed Scopus (37) Google Scholar). Two of total on an with a with and was at for and to at a of analysis, the was an range of at oil content was by using as was to the free fatty acid was used as for free fatty acids. acyltransferase the of the biosynthesis has been to a in accumulation of fatty acids (4Burgal J. Shockey J. Lu C. Dyer J. Larson T. Graham I. Browse J. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil.Plant Biotechnol. J. 2008; 6: 819-831Crossref PubMed Scopus (258) Google Scholar). this gene from C. purpurea, an from the was using Arabidopsis as a resulted in of a partial cDNA of and cDNA using a of cDNA with a of and of CpDGAT1 encoding a of acids with a of CpDGAT1 to the a of the transferase is distributed in such as plants and as well as H. diacylglycerol to oil biosynthesis and in Yarrowia 2012; PubMed Scopus Google Scholar). acid of CpDGAT1 to a previously identified from the same species I. D. P. M. Qiu X. diacylglycerol acyltransferase from Claviceps with ricinoleic acid, a hydroxyl fatty acid of industrial as Microbiol. 2010; PubMed Scopus (25) Google was at acid of CpDGAT1 with from R. communis Arabidopsis C. S. cerevisiae and that CpDGAT1 from C. was with a of from and but not with from the same species that the from C. and have activity of CpDGAT1, the cDNA was cloned and in a S. cerevisiae mutant defective in the synthesis of and L. A. E. A. M. H. S. lipid synthesis is in Biol. PubMed Scopus Google as a similar mutant of P. was not for the mutant yeast with the and the with was used as a and a neutral from the gene and by in CpDGAT1 produced while the not produce of the from C. that CpDGAT1 produce an amount of higher than that the CpDGAT1 gene a functional involved in the biosynthesis of and that the activity was higher than that of in the strategy to produce ricinoleic acid in Pichia was to genes encoding CpFAH and CpDGAT1 involved in biosynthesis and assembly of the fatty acid. This strategy has to produce fatty acids (4Burgal J. Shockey J. Lu C. Dyer J. Larson T. Graham I. Browse J. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil.Plant Biotechnol. J. 2008; 6: 819-831Crossref PubMed Scopus (258) Google Scholar, 6van Erp H. Bates P.D. Burgal J. Shockey J. Browse J. Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis.Plant Physiol. 2011; 155: 683-693Crossref PubMed Scopus (143) Google Scholar, M. Stephanopoulos G. the and of lipid biosynthesis in oleaginous yeast Yarrowia for biofuel Eng. 2013; PubMed Scopus Google Scholar). with or gene of CpFAH and CpDGAT1 the of an of the yeast expression and introduced into a diploid yeast P. strain the gene into the yeast of the the in a time for production of ricinoleic acid. the the diploid the CpFAH, and similar and produced a new fatty acid with the was identified as ricinoleic acid a time the amount of ricinoleic acid produced in the from the of the at the time and However, with CpFAH the coexpression and produced a higher amount of ricinoleic acid at time in the time at CpFAH produced the level of ricinoleic acid was slightly higher than the the amount of ricinoleic acid in CpFAH and at was at the of the time the amount of ricinoleic acid only at of the total fatty acids. In the amount of ricinoleic acid produced in the coexpression the level at at of the total fatty acids. the amount of ricinoleic acid but and at of the was at the amount of ricinoleic acid at of the total fatty acids, was higher than that of CpFAH at the same in the diploid CpFAH and CpDGAT1 produced a amount of ricinoleic acid, acid was still of the fatty acids in the due to a highly Δ12 desaturase in P. Pichia Δ12 desaturase is a enzyme the synthesis of acid using acid as acid is also the for CpFAH to produce ricinoleic acid, for the same by the of the hydroxy fatty acid production in the the and boost the level for the the Pichia Δ12 desaturase gene was by in a haploid P. strain was by using of as well as fatty acid in the strain acid and acid This mutant strain was used as a to express CpFAH or CpFAH and the the haploid similar in and of the fatty acids in the the haploid strain produced fatty acids, acid and acid, while and acids the the Δ12 strain produced only a single fatty acid, acid and acid and acid with the strain. In the strain CpFAH or CpFAH and CpDGAT1 ricinoleic acid was produced as a fatty acid with acid, acid, and acid at a high production in the time It was that acid and acid also produced in the of acid was due to the Δ12 desaturase activity of CpFAH, while a amount of acid resulted from a of the Δ12 desaturase activity of CpFAH and an desaturase activity in to the production of ricinoleic acid in the diploid the hydroxyl fatty acid production in the haploid the of a and in the time amount of ricinoleic acid produced in CpFAH was at the time of the time with the of ricinoleic acid in CpFAH was at at and at a level of at However, in the coexpression of CpFAH and CpDGAT1, the hydroxy fatty acid level was the at at of the total fatty acids. only and at the level of at was than that in CpFAH at the same the production time the total lipid content of the coexpression was at of ricinoleic acid, while the lipid content of CpFAH was and the was lipid content in the coexpression strain by to CpFAH and with the strain. the hydroxy fatty acid at the level in the the lipid content in the coexpression was by and with CpFAH and the acid of Pichia in the Δ12 and at of ricinoleic acid. from the of are of with in a new contents in the haploid mutant strain CpFAH or CpFAH and CpDGAT1 at and from the of are of with in a new ricinoleic acid. from the of are of with from the of are of with into of ricinoleic acid in lipid of the haploid mutant strain CpFAH and CpDGAT1, the total of the coexpression strain at by and fatty acid of lipid in with the mutant the mutant CpFAH or CpFAH and CpDGAT1 produce or new of neutral with ricinoleic acid, hydroxy fatty acid and hydroxy fatty acid with in the the of ricinoleic acid in this lipid not with of castor oil of ricinoleic acid, was with the of with ricinoleic acids. free hydroxyl fatty acid was of total fatty acids as ricinoleic acid, with a single fatty acid in the It was that the polar in the of the of ricinoleic acid. In was produced in the CpFAH but a amount of in the CpFAH and CpDGAT1, the effectiveness of CpDGAT1 to ricinoleic acid to the that a amount of in the haploid expression and the of CpDGAT1 in the of ricinoleic acid into the total from the of CpFAH and at and in a time and by the total and the the while the the free fatty acids D. Qiu X. A pathway involved in the biosynthesis of fatty acids in Physiol. 2011; PubMed Scopus Google Scholar). in at time the coexpression a higher of ricinoleic acid in the than CpFAH alone. a of ricinoleic acid was in the in CpFAH and in the coexpression for of the total lipids. the of in the CpFAH at of the total while the amount in the coexpression was of the total lipids. of the amount of ricinoleic acid in the coexpression in CpFAH at was while the as the time and at the of the amount of ricinoleic acid in the was to This that the of CpDGAT1 for of ricinoleic acid to the as the time acid contents in lipid of the haploid mutant strain CpFAH or CpDGAT1 at and lipid from the of are of with in a new from the of are of with Although tremendous have been made in metabolic engineering of ricinoleic acid in oilseed crops as a alternative for industrial a commercially viable level of this fatty acid has not been in transgenic In this we producing ricinoleic acid by CpFAH and CpDGAT1 in a diploid strain and a haploid mutant strain of P. In particular, the level of ricinoleic acid can of the total fatty acids at expression in the haploid mutant strain defective in the Δ12 desaturase This demonstrates the effectiveness of the metabolic strategy used for metabolic engineering of the hydroxy fatty acid in of a highly CpDGAT1 increases not only the total level of ricinoleic acid, but also the lipid content and the amount of ricinoleic acid in the lipids. a similar level of hydroxyl fatty acid was also recently in yeast species (17Holic R. Yazawa H. Kumagai H. Uemura H. Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe.Appl. Microbiol. Biotechnol. 2012; 95: 179-187Crossref PubMed Scopus (42) Google Scholar, 18Beopoulos A. Verbeke J. Bordes F. Guicherd M. Bressy M. Marty A. Nicaud J.M. Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica.Appl. Microbiol. Biotechnol. 2014; 98: 251-262Crossref PubMed Scopus (98) Google Scholar). these the capacity and potential of microbial systems for the production of hydroxyl fatty acid. Pichia for producing and fatty acids for It is to genetic and genetic are for metabolic It is with a and has been used for producing and In is well that the fatty acid biosynthesis by long-chain and fatty acid for the metabolism (12Lennen R.M. Pfleger B.F. Microbial production of fatty acid-derived fuels and chemicals.Curr. Opin. Biotechnol. 2013; 24: 1044-1053Crossref PubMed Scopus (138) Google Scholar, 13Steen E.J. Kang Y. Bokinsky G. Hu Z. Schirmer A. McClure A. Del Cardayre S.B. Keasling J.D. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass.Nature. 2010; 463: 559-562Crossref PubMed Scopus (1074) Google Scholar, 14Runguphan W. Keasling J.D. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.Metab. Eng. 2014; 21: 103-113Crossref PubMed Scopus (288) Google Scholar). In this ricinoleic acid in Pichia is mainly distributed in free fatty acid form, a the of a strategy to the in the metabolic engineering of biofuels W. Keasling J.D. Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals.Metab. Eng. 2014; 21: 103-113Crossref PubMed Scopus (288) Google Scholar). these make Pichia a promising system for producing hydroxyl fatty acid as an alternative to plant sources for industrial uses. When systems and used for metabolic engineering of ricinoleic acid are appears that the is than transgenic plants in producing hydroxyl fatty acid. It is that ricinoleic acid by oleate hydroxylase on acid to the of the by a hydroxyl to the at the of the fatty acid from is S. S. M. S. A. expression of a gene in Arabidopsis seeds accumulation of PubMed Scopus Google Scholar, E.B. Dyer J.M. fatty acids to high levels in of and Arabidopsis 2006; PubMed Scopus Google and of the fatty acid from to are the or the production of this fatty acid in transgenic plants production of fatty acids in transgenic Plant Biol. PubMed Scopus Google Scholar, E.B. Shockey J.M. Dyer J.M. oilseeds for production of industrial and in fatty acid Opin. Plant Biol. PubMed Scopus (177) Google Scholar, P.D. Browse J. pathway of triacylglycerol synthesis in Arabidopsis a for the accumulation of fatty acids in transgenic J. 2011; PubMed Scopus Google Scholar). are the of the in fatty acid in the the and expression of a fatty acid in Arabidopsis and was to a high level of fatty acids in in developing and seeds of transgenic plants E.B. Dyer J.M. fatty acids to high levels in of and Arabidopsis 2006; PubMed Scopus Google Scholar). the transgenic Pichia the genes ricinoleic acid in free fatty acid of the and the level of this fatty acid in of the total fatty acids. an accumulation is as is that free fatty acids are to In the has been in transgenic plants producing fatty acids. This that Pichia are highly to free fatty acids and the have a than transgenic plants to ricinoleic acid from to the free fatty acid by or the or from the by accumulation of the hydroxyl fatty acid. this is a for improved production of fatty acids in transgenic plants on the of or to the fatty acids from to the free fatty acid from these fatty acids are to for However, was that a from castor bean was recently with a hydroxylase in resulted in production of an level of ricinoleic acid with transgenic plants with the hydroxylase S. G. Browse J. A from castor the of hydroxy fatty acids from in transgenic Arabidopsis Physiol. PubMed Scopus (42) Google Scholar). It is that the is not to with ricinoleic acid in transgenic was to in yeast by the in In plants highly to the for the factors that can the of the free fatty acid the biosynthesis for transgenic plant production of fatty acids in the P. and M. for of the and the of the for and diacylglycerol acyltransferase from Claviceps fatty acid hydroxylase from Claviceps diacylglycerol acyltransferase fatty acid free ricinoleic acid triacylglycerol

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