Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis.

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Preparation of Ricinoleic Acid from Castor Oil:A Review.

Castor oil is a vegetable product extracted from Ricinus communis L (castor seed), which is primarily considered an important commercial value for the manufacturing of soaps, lubricants, coatings, etc. It is rich in hydroxylated fatty acids (ricinoleic acid, 89-92%) and is widely used in the cosmetic, pharmaceutical, oleochemical, and agricultural industries. This oil has also been confirmed as a bactericidal, anti-inflammatory, and antiherpetic agents, due to the ricinoleic acid having functional groups, such as -COOH, -OH, and -C=C-. Furthermore, it is converted into various acid derivative compounds with several applications. Therefore, this article reviewed some reaction stages to the preparation of ricinoleic acid from castor oil. Several methods or reaction pathways were employed in the preparation procedure, such as the Twitchell and Colgate-Emery processes, as well as the alkaline catalyzed, transesterification with methyl ricinoleic, and lipase-catalyzed hydrolysis, respectively. Although each of these preparation methods has advantages and disadvantages, the most effective technique was the hydrolysis through the use of the enzyme lipozyme TL IM. Besides being a green method, the conversion rate in the hydrolysis process was 96.2 ± 1.5.

Heterologous Synthesis and Secretion of Ricinoleic Acid in Starmerella bombicola with Sophorolipid as an Intermediate.

Ricinoleic acid (RA) is a long-chain hydroxy fatty acid produced from castor bean that is used in the manufacturing of a variety of industrial products. The demand for RA keeps increasing due to its broad applications. However, due to the presence of a potent toxin ricin, the native oilseed plant is not an ideal source for hydroxy fatty acid production. Although there have been significant efforts on engineering different microorganisms for heterologous production of RA, all had very limited success. The main reason for this is the exhibited toxicity of the intracellularly accumulated RA. To avoid this issue, we genetically modified a Starmerella bombicola strain by engineering its native sophorolipid production pathway to direct the synthesized RA bound with sophorolipid to be secreted out of the cell, followed by acid hydrolysis to recover RA. The engineered S. bombicola strain expressing the heterologous codon-optimized oleate hydroxylase-encoding gene from ergot fungus Claviceps purpurea resulted in a record production titer of RA at about 2.96 g/L. Thus, this work highlights a new strategy to produce a high level of hydroxy fatty acids in engineered yeast through a sophorolipid intermediate, enabling a new biocatalysis platform for the future.

Oleate Hydratase in Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 Catalyzes the Reversible Conversion between Linoleic Acid and Ricinoleic Acid.

Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. CLA is an intermediate product of the biohydrogenation pathway of linoleic acid (LA) in bacteria. Several bacterial species capable of efficiently converting LA into CLA have been widely reported in the literature, among them Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230. Over the last few years, a multicomponent enzymatic system consisting of three enzymes involved in the biohydrogenation process of LA has been proposed. Sequencing the genome of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. The OleH was cloned, expressed, purified, and characterized. Fatty acid measurements were made by an internal standard method using a gas chromatography-coupled flame ionization detector (GC-FID) system. It was found that the enzyme is a hydratase/dehydratase, leading to a reversible transformation between LA and RA. In addition, the results showed that L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. In conclusion, the OleH of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 catalyzes the initial stage of saturation metabolism of LA, although it has not converted the substrates directly into CLA. IMPORTANCE This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c9, t11 CLA isomer and stress tolerance by E. coli has been assisted. The results provide an understanding of the factors which influence OleH activity. L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH presented two putative fatty acid-binding sites. Recombinant OleH catalyzed both LA hydration and RA dehydration. OleH was shown to play a role in bacterial growth performance in the presence of LA.

Production of 7,10,12-trihydroxy-8(E)-octadecenoic acid from ricinoleic acid by Pseudomonas aeruginosa KNU-2B.

Microbial production of hydroxy fatty acids (HFAs) was widely studied because of important biological properties of HFAs. Among microorganisms producing HFAs, Pseudomonas aeruginosa PR3 was well known to produce various HFAs from different unsaturated fatty acids. Recently, a new variant species of P. aeruginosa PR3 was isolated and characterized, showing improved efficiency for producing 7,10-dihydroxy-8(E)-octadecenoic acid from oleic acid. In this study, we report the production of 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD) from ricinoleic acid by the newly isolated P. aeruginosa KNU-2B. TOD was efficiently produced from ricinoleic acid by KNU-2B with the maximum conversion yield of 56.7% under the optimum reaction conditions of pH 8.0 and 48-h incubation at 27 °C, 150 rpm. Under optimized reaction conditions, maximum TOD production reached 340.3 mg/100 mL of the culture. However, requirement of nutritional factors by KNU-2B for production of TOD were considerably different from those by PR3 strain.

Characterization of a PLDζ2 Homology Gene from Developing Castor Bean Endosperm.

Castor oil contains approximately 90% ricinoleic acid (RA) which is stored mainly in the form of tri-ricinoleic acid containing triacylglycerols (TAG). Ricinoleate is synthesized from oleate (18:1n-9) esterified to the sn-2 position of phosphatidylcholine (PtdCho) catalyzed by oleoyl-12-hydroxylase. PtdCho-derived diacylglycerol (DAG) is an important substrate pool for TAG synthesis, and the interconversion between PtdCho and DAG has been shown to play a critical role in channeling hydroxy fatty acids (HFA) to TAG. Although phospholipase D (PLD) has been reported to catalyze the hydrolysis of PtdCho to produce phosphatidic acid which can then be converted to DAG, its potential functions in the channeling of RA from PtdCho to DAG and the assembly of RA on TAG is largely unknown. In the present study, 11 PLD genes were identified from the Castor Bean Genome Database. Gene expression analysis indicated that RcPLD9 is expressed at relatively high levels in developing seeds compared to other plant tissues. Sequence and phylogenetic analyses revealed that RcPLD9 is a homolog of Arabidopsis PLDζ2. Overexpression of RcPLD9 in the Arabidopsis CL7 line producing C18-HFA resulted in RA content reductions in the polar lipid fraction (mainly PtdCho) and mono-HFA-TAG, but increased RA content in di-HFA-TAG. Since part of RA in di-HFA-TAG is derived from HFA-DAG, the results indicated that RcPLD9 facilitates the channeling of RA from PtdCho to DAG for its assembly on TAG in developing seeds.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

Enhanced Biotransformation Productivity of Gamma-Decalactone from Ricinoleic Acid Based on the Expanded Vermiculite Delivery System.

Natural gamma-decalactone (GDL) produced by biotransformation is an essential food additive with a peach-like aroma. However, the difficulty of effectively controlling the concentration of the substrate ricinoleic acid (RA) in water limits the biotransformation productivity, which is a bottleneck for industrialization. In this study, expanded vermiculite (E-V) was utilized as a carrier of RA to increase its distribution in the medium. E-V and three commonly used organic compounds were compared with respect to their effects on the biotransformation process, and the mechanism was revealed. Scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis indicated that RA was physically adsorbed onto the surface of and inside E-V instead of undergoing a chemical reaction, which increased the opportunity for interactions between microorganisms and the substrate. The highest concentration of GDL obtained in the medium with E-V was 6.2 g/l, which was 50% higher than that in the reference sample. In addition, the presence of E-V had no negative effect on the viability of the microorganisms. This study provides a new method for producing natural GDL through biotransformation on an industrial scale.

A survey of transcriptome complexity using PacBio single-molecule real-time analysis combined with Illumina RNA sequencing for a better understanding of ricinoleic acid biosynthesis in Ricinus communis.

BACKGROUND: Ricinus communis is a highly economically valuable oil crop plant from the spurge family, Euphorbiaceae. However, the available reference genomes are incomplete and to date studies on ricinoleic acid biosynthesis at the transcriptional level are limited. RESULTS: In this study, we combined PacBio single-molecule long read isoform and Illumina RNA sequencing to identify the alternative splicing (AS) events, novel isoforms, fusion genes, long non-coding RNAs (lncRNAs) and alternative polyadenylation (APA) sites to unveil the transcriptomic complexity of castor beans and identify critical genes related to ricinoleic acid biosynthesis. Here, we identified 11,285 AS-variants distributed in 21,448 novel genes and detected 520 fusion genes, 320 lncRNAs and 9511 (APA-sites). Furthermore, a total of 6067, 5983 and 4058 differentially expressed genes between developing beans of the R. communis lines 349 and 1115 with extremely different oil content were identified at 7, 14 and 21 days after flowering, respectively. Specifically, 14, 18 and 11 DEGs were annotated encoding key enzymes related to ricinoleic acid biosynthesis reflecting the higher castor oil content of 1115 compared than 349. Quantitative real-time RT-PCR further validated fifteen of these DEGs at three-time points. CONCLUSION: Our results significantly improved the existed gene models of R. communis, and a putative model of key genes was built to show the differences between strains 349 and 1115, illustrating the molecular mechanism of castor oil biosynthesis. A multi-transcriptome database and candidate genes were provided to further improve the level of ricinoleic acid in transgenic crops.

Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids.

Whole cell biocatalysts can be used to convert fatty acids into various value-added products. However, fatty acid transport across cellular membranes into the cytosol of microbial cells limits substrate availability and impairs membrane integrity, which in turn decreases cell viability and bioconversion activity. Because these problems are associated with the mechanism of fatty acid transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was generated to promote substrate endocytosis. Caveolin-1 ( CAV1) expression in Escherichia coli increased both the fatty acid transport rate and intracellular fatty acid concentrations via endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-hydroxyoctadec-9-enoic acid) to ( Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of hydrophobic substrates.

Tissue-specific differences in metabolites and transcripts contribute to the heterogeneity of ricinoleic acid accumulation in Ricinus communis L. (castor) seeds.

INTRODUCTION: Castor (Ricinus communis L.) seeds are valued for their production of oils which can comprise up to 90% hydroxy-fatty acids (ricinoleic acid). Castor oil contains mono-, di- and tri- ricinoleic acid containing triacylglycerols (TAGs). Although the enzymatic synthesis of ricinoleic acid is well described, the differential compartmentalization of these TAG molecular species has remained undefined. OBJECTIVES: To examine the distribution of hydroxy fatty acid accumulation within the endosperm and embryo tissues of castor seeds. METHODS: Matrix assisted laser desorption/ionization mass spectrometry imaging was used to map the distribution of triacylglycerols in tissue sections of castor seeds. In addition, the endosperm and embryo (cotyledons and embryonic axis) tissues were dissected and extracted for quantitative lipidomics analysis and Illumina-based RNA deep sequencing. RESULTS: This study revealed an unexpected heterogeneous tissue distribution of mono-, di- and tri- hydroxy-triacylglycerols in the embryo and endosperm tissues of castor seeds. Pathway analysis based on transcript abundance suggested that distinct embryo- and endosperm-specific mechanisms may exist for the shuttling of ricinoleic acid away from phosphatidylcholine (PC) and into hydroxy TAG production. The embryo-biased mechanism appears to favor removal of ricinoleic acid from PC through phophatidylcholine: diacylglycerol acyltransferase while the endosperm pathway appears to remove ricinoleic acid from the PC pool by preferences of phospholipase A (PLA2α) and/or phosphatidylcholine: diacylglycerol cholinephosphotransferase. CONCLUSIONS: Collectively, a combination of lipidomics and transcriptomics analyses revealed previously undefined spatial aspects of hydroxy fatty acid metabolism in castor seeds. These studies underscore a need for tissue-specific studies as a means to better understand the regulation of triacylglycerol accumulation in oilseeds.

Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils.

MAIN CONCLUSION: In vivo and in vitro analyses of Euphorbiaceae species' triacylglycerol assembly enzymes substrate selectivity are consistent with the co-evolution of seed-specific unusual fatty acid production and suggest that many of these genes will be useful for biotechnological production of designer oils. Many exotic Euphorbiaceae species, including tung tree (Vernicia fordii), castor bean (Ricinus communis), Bernardia pulchella, and Euphorbia lagascae, accumulate unusual fatty acids in their seed oils, many of which have valuable properties for the chemical industry. However, various adverse plant characteristics including low seed yields, production of toxic compounds, limited growth range, and poor resistance to abiotic stresses have limited full agronomic exploitation of these plants. Biotechnological production of these unusual fatty acids (UFA) in high yielding non-food oil crops would provide new robust sources for these valuable bio-chemicals. Previous research has shown that expression of the primary UFA biosynthetic gene alone is not enough for high-level accumulation in transgenic seed oils; other genes must be included to drive selective UFA incorporation into oils. Here, we use a series of in planta molecular genetic studies and in vitro biochemical measurements to demonstrate that lysophosphatidic acid acyltransferases from two Euphorbiaceae species have high selectivity for incorporation of their respective unusual fatty acids into the phosphatidic acid intermediate of oil biosynthesis. These results are consistent with the hypothesis that unusual fatty acid accumulation arose in part via co-evolution of multiple oil biosynthesis and assembly enzymes that cooperate to enhance selective fatty acid incorporation into seed oils over that of the common fatty acids found in membrane lipids.

Production of (Z)-11-(heptanoyloxy)undec-9-enoic acid from ricinoleic acid by utilizing crude glycerol as sole carbon source in engineered Escherichia coli expressing BVMO-ADH-FadL.

Production of (Z)-11-(heptanoyloxy)undec-9-enoic acid from recinoleic acid was achieved by whole-cell biotransformation by Escherichia coli, utilizing crude glycerol as the sole carbon source. Whole-cell biotransformation resulted in ∼93% conversion of the substrate ricinoleic acid to (Z)-11-(heptanoyloxy)undec-9-enoic acid. We replaced the inducer-dependent promoter system (T7 and Rhm promotors) with a constitutive promoter system. This resulted in successful expression of ADH, FadL, and E6-BVMO, without costly inducer addition. Efficacy evaluation of the whole-cell biotransformation by inducer-free system by five different E. coli strains revealed that the highest product titer was accumulated in E. coli BW25113 strain. The engineered inducer-free system using crude glycerol as the sole carbon source showed competitive performance with induction systems. Optimized conditions resulted in the accumulation of 7.38 ± 0.42 mM (Z)-11-(heptanoyloxy)undec-9-enoic acid, and when 10 mM substrate was used as feed concentration, the product titer reached 2.35 g/L. The inducer-free construct with constitutive promoter system that this study established, which utilizes the waste by-product crude glycerol, will pave the way for the economic synthesis of many industrially important chemicals, like (Z)-11-(heptanoyloxy)undec-9-enoic acid.

Improving catalytic activity of the Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the overproduction of (Z)-11-(heptanoyloxy)undec-9-enoic acid from ricinoleic acid.

Baeyer-Villiger monooxygenases (BVMOs) can be used for the biosynthesis of lactones and esters from ketones. However, the BVMO-based biocatalysts are not so stable under process conditions. Thereby, this study focused on enhancing stability of the BVMO-based biocatalysts. The biotransformation of ricinoleic acid into (Z)-11-(heptanoyloxy)undec-9-enoic acid by the recombinant Escherichia coli expressing the BVMO from Pseudomonas putida and an alcohol dehydrogenase from Micrococcus luteus was used as a model system. After thorough investigation of the key factors to influence stability of the BVMO, Cys302 was identified as an engineering target. The substitution of Cys302 to Leu enabled the engineered enzyme (i.e., E6BVMOC302L) to become more stable toward oxidative and thermal stresses. The catalytic activity of E6BVMOC302L-based E. coli biocatalysts was also greater than the E6BVMO-based biocatalysts. Another factor to influence biocatalytic performance of the BVMO-based whole-cell biocatalysts was availability of carbon and energy source during biotransformations. Glucose feeding into the reaction medium led to a marked increase of final product concentrations. Overall, the bioprocess engineering to improve metabolic stability of host cells in addition to the BVMO engineering allowed us to produce (Z)-11-(heptanoyloxy)undec-9-enoic acid to a concentration of 132 mM (41 g/L) from 150 mM ricinoleic acid within 8 h.

Effect of PelB signal sequences on Pfe1 expression and ω-hydroxyundec-9-enoic acid biotransformation in recombinant Escherichia coli.

ω-Hydroxyundec-9-enoic acid (ω-HUA) was reported as a valuable medium-chain fatty acid with industrial potentials. For bioconversion of ricinoleic acid to ω-HUA, in this study, an alcohol dehydrogenase (Adh) from Micrococcus luteus, a Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440 and an esterase (Pfe1) from Pseudomonas fluorescens SIK WI were overexpressed in Escherichia coli BL21(DE3). In order to enhance accessibility of Pfe1 to the (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA) substrate, a truncated PelB signal sequence without the recognition site of signal peptidase (tPelB) was attached to the N-terminus of Pfe1, resulting in the construction of E. coli AB-tPE strain expressing Adh, BVMO and the tPelB-Pfe1 fusion protein. A batch-type biotransformation of ricinoleic acid by E. coli AB-tPE resulted in 1.8- and 2.2-fold increases in ω-HUA conversion yield and productivity, respectively, relative to those for the control strain without the PelB sequence (AB-E). By fed-batch-type biotransformation with glycerol feeding, the AB-tPE strain produced 23.7 mM (equivalent to 4.7 g/L) of ω-HUA with 60.8%(mol/mol) of conversion yield and 1.2 mM/h of productivity, which were 13.2, 2.9, and 2.6 times higher than those in a batch-type biotransformation using the AB-E strain. In conclusion, combination of the truncated PelB-Pfe1 fusion and fed-batch process with glycerol feeding provided the highest efficiency of ω-HUA biotransformation, of which strategies might be applicable for biotransformation of hydrophobic substances.

Overexpression of Seipin1 Increases Oil in Hydroxy Fatty Acid-Accumulating Seeds.

While plant oils are an important source of food, plants also produce oils containing specialized fatty acids with chemical and physical properties valued in industry. Ricinoleic acid, a hydroxy fatty acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized fatty acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.

New insights on the metabolism of ricinoleic acid in ruminants.

Dairy goats were fed a total mixed ration with or without the inclusion of castor oil [40 g/kg of dry matter (DM)] to study the metabolism of ricinoleic acid (12-OH,cis-9-18:1). Ten goats, at 39.7 ± 4.0 d in milk, were individually penned and allocated at random to the 2 experimental diets. Goats were manually milked twice a day. Milk fatty acids (FA) were analyzed as methyl esters and hydroxyl groups were derivatized in trimethylsilyl ethers. Apart from ricinoleic acid, 6 FA were only detected in the milk of the castor oil group. Ricinoleic acid composed 0.3% of total FA in milk of the castor oil group, whereas the hydroxy-FA (8-OH-14:0, 10-OH-16:0, and 12-OH-18:0) and oxo-FA (8-oxo-14:0, 10-oxo-16:0, and 12-oxo-18:0) reached 7.5% of total FA in milk. We anticipate that these FA were derived from the metabolism of ricinoleic acid, although it was not clear if they were produced in the rumen or in the tissues. To confirm that, we conducted in vitro batch incubations repeated for 3 consecutive weeks with castor oil (40 g/kg of DM) and strained rumen fluid from 2 fistulated sheep. To examine the products formed over time, incubation tubes were stopped at 0, 6, 12, 24, 48, and 72 h. The results of the in vitro experiment showed that ricinoleic acid was metabolized in the rumen at a slow rate and the main products formed were 12-OH-18:0 and 12-oxo-18:0, by hydrogenation of the cis-9 double bond, followed by oxidation of the hydroxyl group, respectively. Our results suggest that the 12-OH-18:0 and 12-oxo-18:0 escape rumen and are further metabolized through partial ß-oxidation in ruminant tissues. We propose that the 10-OH-16:0 and 8-OH-14:0 found in goat milk of the castor oil group are successive products of the ß-oxidation of 12-OH-18:0, and the 10-oxo-16:0 and 8-oxo-14:0 are successive products of the 12-oxo-18:0 in tissues. Overall, our results indicate that ricinoleic acid is extensively metabolized in the rumen and tissues, producing mainly oxo- and hydroxy-FA that are further excreted in milk.

Continuous supply of glucose and glycerol enhances biotransformation of ricinoleic acid to (E)-11-(heptanoyloxy) undec-9-enoic acid in recombinant Escherichia coli.

This study aimed at the development of biotransformation strategies with feeding of energy sources for bioconversion of ricinoleic acid to (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA), a key intermediate of brassylic acid, by recombinant Escherichia coli overexpressing an alcohol dehydrogenase from Micrococcus luteus and a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440. Feeding of glucose or glycerol facilitated both the preparation of high-density cell biocatalyst and supply of the NAD+ and NADPH cofactors. By the glucose feeding strategy, 30.8g/L of the engineered E. coli cells produced 29.7mM of 11-HOUA with 1.9mM/h of productivity, which were 1.8 and 1.6 times higher than the same biotransformation without the glucose feeding, respectively. Intermittent addition of glycerol increased 11-HOUA productivity by 16% compared to that by the glucose feeding. Finally, 34.5mM of 11-HOUA concentration, 77% conversion and 2.2mM/h productivity were obtained using 31.6g/L of cell biocatalyst along with the glycerol addition. It was concluded that supplementation of additional carbon sources in biotransformation process would be a potent strategy to increase the performance of fatty acid conversion.

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

Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps purpurea. In transgenic C. gracilis, RA content increased at low temperatures, reaching 2.2 pg/cell when cultured for 7 d at 15 °C, without affecting cell growth, and was enhanced (3.3 pg/cell) by the co-expression of a palmitic acid-specific elongase gene. Most of the accumulated RA was linked with monoestolide triacylglycerol (ME TAG), in which one RA molecule was esterified to the α position of the glycerol backbone and was further esterified at its hydroxy group with a fatty acid or second RA moiety, or 1-OH TAG, in which RA was esterified to the glycerol backbone. Overall, 80% of RA was accumulated as ME TAGs. Furthermore, exogenous RA-methyl ester suppressed the growth of wild-type diatoms in a dose-dependent manner and was rapidly converted to ME TAG. These results suggest that C. gracilis masks the hydroxyl group and accumulates RA as the less-toxic ME TAG.

Enhanced Enzymatic Preparation of Biodiesel Using Ricinoleic Acid as Acyl Donor: Optimization Using Response Surface Methodology.

Castor oil methyl ester is a kind of biodiesel from castor oil. However, in those previous methods for biodiesel preparation using castor oil as feedstock, glycerol was the main by-product, which had a strong blocking effect on the immobilized enzyme activity and affected the mass transfer of reaction system. For avoiding the negative effect of glycerol on the enzymatic esterification, biodiesel was prepared using ricinoleic acid (RA) as acyl donor. Enzyme screening was also studied, and the effects of reaction temperature, molar ratio of ricinoleic acid and methanol, enzyme load, and reaction time, on the preparation of castor methyl ester were also evaluated. Response surface methodology (RSM) was used to optimize the interaction effect of reaction variables (reaction temperature (30-70°C), enzyme load (2-7%; relative to the weight of total substrates), molar ratio of methanol to ricinoleic acid (2:1-10:1), and reaction time (0.5-2.5 h)) on the acid value (AV) and the degree of esterification (DE). Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of AV and DE. The optimum preparation conditions were as follows: reaction temperature, 48.2°C; enzyme load, 5.8%; molar ratio of methanol to ricinoleic acid, 5.56:1; reaction time, 2.36 h. Under these conditions, the AV and DE of the esterification reaction are 10.36±1.05 mgKOH/g and 94.03±0.60%, respectively. The relationship between initial reaction rate and temperature was also established, and the activation energy (Ea) of the enzymatic esterification is 33.87 KJ/mol.