Production Of Hydroxy Fatty Acids Research Articles

Systematic screening for the biocatalytic hydration of fatty acids from different oily substrates by Elizabethkingia meningoseptica oleate hydratase through a Design-of-experiments approach

The edible plant oils production is associated with the release of different types of by-products. The latter represent cheap and available substrates to produce valuable compounds, such as flavours and fragrances, biologically active compounds and bio-based polymers. Elizabethkingia meningoseptica Oleate hydratases (Em_OhyA) can selectively catalyze the conversion of unsaturated fatty acids, specifically oleic acid, into hydroxy fatty acids, which find different industrial applications. In this study, Design-of-experiment (DoE) strategy was used to screen and identify conditions for reaching high yields in the reaction carried out by Escherichia coli whole-cell carrying the recombinant enzyme Em_OhyA using Waste Cooking Oils (WCO)-derived free fatty acids (FFA) as substrate. The identified reaction conditions for high oleic acid conversion were also tested on untreated triglycerides-containing substrates, such as pomace oil, sunflower oil, olive oil and oil mill wastewater (OMW), combining the triglyceride hydrolysis by the lipase from Candida rugosa and the E. coli whole-cell containing Em_OhyA for the production of hydroxy fatty acids. When WCO, sunflower oil and OMW were used as substrate, the one-pot bioconversion led to an increase of oleic acid conversion compared to the standard reaction. This work highlights the efficiency of the DoE approach to screen and identify conditions for an enzymatic reaction for the production of industrially-relevant products.

Exploring the fatty acid double bond hydration activities of Lacticaseibacillus rhamnosus strains

The fatty acid double bond hydratases (FADBH) are involved in the detoxification of unsaturated fatty acids and the production of multi-functional hydroxy fatty acids. Thereby, we have investigated the fatty acid hydration activities of Lacticaseibacillus rhamnosus strains including the ones isolated from the oral microbiome. Interestingly, the fatty acid hydration activities were different among L. rhamnosus strains; one of the isolated oral strains (DM065) exhibited 0.14 μmol/g dry cells/min (U/g dry cells) for hydration of linoleic acid, whereas the well-known probiotics, L. rhamnosus GG (LGG) showed 1.66 U/g dry cells. With an aim to examine the critical factors to determine microbial hydration activities, the FADBH of LGG (OhyLr_LGG) was cloned, engineered to have maltose-binding protein as a soluble fusion partner, and subjected to kinetic and mutation studies. The catalytic efficiencies of OhyLr_LGG for oleic and linoleic acid were 0.102 and 0.066 s−1μM−1, respectively. These values are comparable to those of OhyA from Stenotrophomonas maltophilia (Sm_OhyA2), which was one of the most active enzymes. In addition, the T545K mutation of OhyLr_LGG led to a 76 % reduction in the linoleic acid hydration activity (0.016 s−1μM−1). Since the OhyLr_DM065 possesses K545, the T545K substitution could be one of the major reasons for the lower hydration activities of the DM065 strain. Thereby, it was concluded that the amino acid sequence of FADBH would be one of the key factors to determine the production of hydroxy fatty acids of Lactobacillus probiotics.

Functional Genome Analyses Reveal the Molecular Basis of Oil Accumulation in Developing Seeds of Castor Beans.

Castor (Ricinus communis L.) seeds produce abundant ricinoleic acid during seed maturation, which is important for plant development and human demands. Ricinoleic acid, as a unique hydroxy fatty acid (HFA), possesses a distinct bond structure that could be used as a substitute for fossil fuels. Here, we identified all homologous genes related to glycolysis, hydroxy fatty acid biosynthesis, and triacylglycerol (TAG) accumulation in castor seeds. Furthermore, we investigated their expression patterns globally during five seed development stages. We characterized a total of 66 genes involved in the glycolysis pathway, with the majority exhibiting higher expression levels during the early stage of castor bean seed development. This metabolic process provided abundant acetyl-CoA for fatty acid (FA) biosynthesis. Subsequently, we identified 82 genes involved in the processes of de novo FA biosynthesis and TAG assembly, with the majority exhibiting high expression levels during the middle or late stages. In addition, we examined the expression patterns of the transcription factors involved in carbohydrate and oil metabolism. For instance, RcMYB73 and RcERF72 exhibited high expression levels during the early stage, whereas RcWRI1, RcABI3, and RcbZIP67 showed relatively higher expression levels during the middle and late stages, indicating their crucial roles in seed development and oil accumulation. Our study suggests that the high HFA production in castor seeds is attributed to the interaction of multiple genes from sugar transportation to lipid droplet packaging. Therefore, this research comprehensively characterizes all the genes related to glycolysis, fatty acid biosynthesis, and triacylglycerol (TAG) accumulation in the castor and provides novel insight into exploring the genetic mechanisms underlying seed oil accumulation in the endosperm of castor beans.

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens.

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.

Regio- and stereoselective biocatalytic hydration of fatty acids from waste cooking oils en route to hydroxy fatty acids and bio-based polyesters

The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds. In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefinery feedstocks, which contribute to a reduced impact on the environment. Frequently, this waste is wrongly disposed of into municipal sewage systems, thereby creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs, which were intended for the production of biofuels, were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst containing the recombinantly produced Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA) was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the production of (R)− 10-hydroxystearic acid methyl ester that was further purified with a high purity (> 90%), at gram scale. The purified (R)− 10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction to produce the corresponding polyester. This work highlights the potential of waste products to obtain bio-based hydroxy fatty acids and polyesters through a biorefinery approach.

The role of peroxiredoxin 6 in biosynthesis of FAHFAs.

Peroxiredoxin 6 (Prdx6) is a multifunctional enzyme, a unique member of the peroxiredoxin family, with an important role in antioxidant defense. Moreover, it has also been linked with the biosynthesis of anti-inflammatory and anti-diabetic lipids called fatty acid esters of hydroxy fatty acids (FAHFAs) and many diseases, including cancer, inflammation, and metabolic disorders. Here, we performed metabolomic and lipidomic profiling of subcutaneous adipose tissue from mouse models with genetically modified Prdx6. Deletion of Prdx6 resulted in reduced levels of FAHFAs containing 13-hydroxylinoleic acid (13-HLA). Mutation of Prdx6 C47S impaired the glutathione peroxidase activity and reduced FAHFA levels, while D140A mutation, responsible for phospholipase A2 activity, showed only minor effects. Targeted analysis of oxidized phospholipids and triacylglycerols in adipocytes highlighted a correlation between FAHFA and hydroxy fatty acid production by Prdx6 or glutathione peroxidase 4. FAHFA regioisomer abundance was negatively affected by the Prdx6 deletion, and this effect was more pronounced in longer and more unsaturated FAHFAs. The predicted protein model of Prdx6 suggested that the monomer-dimer transition mechanism might be involved in the repair of longer-chain peroxidized phospholipids bound over two monomers and that the role of Prdx6 in FAHFA synthesis might be restricted to branching positions further from carbon 9. In conclusion, our work linked the peroxidase activity of Prdx6 with the levels of FAHFAs in adipose tissue.

Enhanced production of hydroxy fatty acids in Arabidopsis seed through modification of multiple gene expression

BackgroundCastor (Ricinus communis L.) seeds contain unusual fatty acid, hydroxy fatty acid (HFA) used as a chemical feedstock for numerous industrial products. Castor cultivation is limited by the potent toxin ricin in its seeds and other poor agronomic traits, so it is advantageous to develop a suitable HFA-producing crop. Significant research efforts have been made to produce HFA in model Arabidopsis, but the level of HFA produced in transgenic Arabidopsis is much less than the level found in castor seeds which produce 90% HFA in seed oil.ResultsWe designed a transformation construct that allowed co-expression of five essential castor genes (named pCam5) involved in HFA biosynthesis, including an oleateDelta12-hydroxylase (FAH12), diacylglycerol (DAG) acyltransferase 2 (DGAT2), phospholipid: DAG acyltransferase 1–2 (PDAT1-2), phosphatidylcholine (PC): DAG cholinephosphotransferase (PDCT) and Lyso-PC acyltransferase (LPCAT). Transgenic ArabidopsispCam5 lines produced HFA counting for 25% in seed oil. By knocking out ArabidopsisFatty acid elongase 1 (AtFAE1) in pCam5 using CRISPR/Cas9 technology, the resulted pCam5-atfae1 lines produced over 31% of HFA. Astonishingly, the pCam5-atfae1 line increased seed size, weight, and total oil per seed exceeding wild type by 40%. Seed germination, seedling growth and seed mucilage content of pCam5-atfae1 lines were not affected by the genetic modification.ConclusionsOur results provide not only insights for future research uncovering mechanisms of HFA synthesis in seed, but also metabolic engineering strategies for generating safe HFA-producing crops.

Production of Hydroxy Fatty Acids, Precursors of γ-Hexalactone, Contributes to the Characteristic Sweet Aroma of Beef.

Aroma is an essential factor for meat quality. The meat of Japanese Black cattle exhibits fine marbling and a rich and sweet aroma with a characteristic lactone composition. The mechanism of lactone formation associated with beef aroma has not been elucidated. In this study, we examined the precursors of γ-hexalactone, an indicator of the sweet aroma of beef and identified the mechanism underlying γ-hexalactone production. A low-temperature vacuum system was used to prepare beef tallow from Japanese Black cattle and Holstein cattle. The odor components were identified using headspace–gas chromatography. The analysis revealed that γ-hexalactone, γ-dodecalactone, δ-tetradecalactone, and δ-hexadecalactone were present as sweet aroma components of beef tallow prepared from marbling and muscle. Since we previously reported that γ-hexalactone formation correlates with linoleic acid content in beef, we analyzed ten oxidized fatty acids derived from linoleic acid by liquid chromatography–triple quadrupole mass spectrometry and detected two hydroxy-octadecadienoic acids (9S-HODE and 13S-HODE) in beef tallow. Significant differences in arachidonic acid 15-lipoxygenase and cyclooxygenase protein expression levels among subcutaneous fat, intramuscular fat, and muscle tissue were observed. Our results suggest that the combination of linoleic acid and the expression of lipid oxidase derived from beef muscle and intramuscular fat produce hydroxy fatty acids that result in a sweet aroma.

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.

Microbial production of hydroxy fatty acids utilizing crude glycerol

Strain D2 was isolated from a natural sample as a crude glycerol-assimilating microorganism. The ITS-5.8S rDNA sequence of strain D2 was most similar to that of Fusarium solani deposited in the NCBI database. Strain D2 accumulated 10-hydroxy-cis-12-octadecenoic acid (HYA), 10-hydroxyoctadecanoic acid (HYB), and 10-oxooctadecanoic acid (KetoB) in a medium containing crude glycerol, as a carbon source, and yeast extract, named CG medium. The growth and HYB production of strain D2 depended on the crude glycerol concentration in the medium. Strain D2 produced 2.20 g/L (40% of total fatty acids) of HYB on cultivation in CG medium containing 8% crude glycerol. When strain D2 was cultivated in CG medium containing 6% crude glycerol, the yield of HYB on cultivation under 4 days-shaking and 3 days-static conditions reached 1.19 g/L, which was 2.2 times higher than that under 7 days-shaking conditions and accounted for 53% of total fatty acids. The fungus was found not only to efficiently produce fatty acids utilizing crude glycerol, but also to be the first filamentous fungus to produce hydroxy and oxo fatty acids such as HYB, HYA, and KetoB.

A multigene approach secures hydroxy fatty acid production in Arabidopsis.

A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from more than a dozen wild species, progress has been limited because expression of these enzymes in transgenic plants produces only low yields of the desired products. For example, fatty acid hydroxylase 12 (FAH12) from castor (Ricinus communis) produces only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis (Arabidopsis thaliana), compared with 90% HFAs in castor seeds. The transgenic plants also have reduced oil content and seed vigor. Here, we review experiments that have provided for steady increased HFA accumulation and oil content. This research has led to exciting new discoveries of enzymes and regulatory processes in the pathways of both seed oil synthesis and lipid metabolism in other parts of the plant. Recent investigations have revealed that HFA-accumulating seeds are unable to rapidly mobilize HFA-containing triacylglycerol (TAG) storage lipid after germination to provide carbon and energy for seedling development, resulting in reduced seedling establishment. These findings present a new opportunity to investigate a different, key area of lipid metabolism-the pathways of TAG lipolysis and β-oxidation in germinating seedlings.

Effects of the engineering of a single binding pocket residue on specificity and regioselectivity of hydratases from Lactobacillus Acidophilus

Fatty acid hydratase (FAH) mediated hydroxy fatty acid (HFA) production is a promising enzymatic route that demands diversification of hydration position to access a broader range of high-value HFAs. FA-HY1 is a promiscuous FAH from Lactobacillus Acidophilus, whereas its homolog from the same organism, FA-HY2, is strict in substrate scope and regioselectivity. Our earlier work demonstrated that three amino acid mutations at the carboxylate end of the substrate (T391/H393/I378 in FA-HY2) shift regioselectivity of FA-HY2 towards that of FA-HY1. Here, we explore alanine 216 of FA-HY2 as a hot-spot residue at the omega end of the substrate. A quadruple mutant (T391S/H393S/I378 P/A216S) demonstrates further shift in regioselectivity towards FA-HY1. Moreover, site-saturation mutagenesis of this residue in FA-HY1 (S218) led to novel variants exhibiting significant changes in regioselectivity for EPA (eicosapentaenoic acid) as substrate, where, unlike wild-type enzyme, 15-OH product was the dominant product (63:37 for wild-type vs. 26:74 for S218I mutant; 12-OH:15-OH). Alterations in conversion levels that indicate pronounced correlation to the exchanged residue type were also detected. A likely explanation for the observed differences is provided based on structural, statistical and kinetic analysis.

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78–100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

Production of hydroxy fatty acids and its effects on photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

Microalgal lipids and fatty acids are important components for achieving biofuel because of their potential high productivity. Although fatty acids that have a hydroxy group adjacent to the end of the acyl chain might be an important chemical feedstock, most algae do not accumulate it. To produce (ω−1)-hydroxy fatty acids from 3-hydroxybutylyl-CoA, an intermediate for polyhydroxybutylate biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, we expressed a gene for the promiscuous 3-ketoacyl-ACP synthase III from Alicyclobacillus acidocalderius (aaKASIII) by the cpc560 promoter. To supply 3-hydroxybutyryl-CoAs for aaKASIII, the phaC gene for polyhydroxybutylate polymerase was deleted, and the phaAB genes for 3-hydroxybutyryl-CoA synthesis from Cupriavidus necator were overexpressed. The genetically modified strain synthesized 15-hydroxyhexadecanoic acid, 17-hydroxyoctadecanoic acid, and 17-hydroxyoctadec-9-enoic acid, and accumulated approximately 2.1 mol% of (ω−1)-hydroxy fatty acids in total fatty acids under illumination with 70 μmol photons m−2 s−1, although its growth was severely retarded. Under weak light (35 μmol photons m−2 s−1) conditions, the strain grew as well as the wild-type and showed lower hydroxy fatty acids (0.04 mol%) accumulation than that at higher illumination levels. The photosynthetic activity of this strain was lower than that of wild-type cells, suggesting that high light conditions enhanced hydroxy fatty acids production and inhibited photosynthesis. (ω−1)-Hydroxy fatty acids were not predominantly observed in the galactolipids from thin-layer chromatography, which are the major lipid classes in cyanobacteria. To the best of our knowledge, this is the first report on photoautotrophic production of fatty acids possessing a functional group near the end of the acyl chain in cyanobacteria.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase‐like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

Identification of a novel enzyme from E. pacifica that acts as an eicosapentaenoic 8R-LOX and docosahexaenoic 10R-LOX

North Pacific krill (Euphausia pacifica) contain 8R-hydroxy-eicosapentaenoic acid (8R-HEPE), 8R-hydroxy-eicosatetraenoic acid (8R-HETE) and 10R-hydroxy-docosahexaenoic acid (10R-HDHA). These findings indicate that E. pacifica must possess an R type lipoxygenase, although no such enzyme has been identified in krill. We analyzed E. pacifica cDNA sequence using next generation sequencing and identified two lipoxygenase genes (PK-LOX1 and 2). PK-LOX1 and PK-LOX2 encode proteins of 691 and 686 amino acids, respectively. Recombinant PK-LOX1 was generated in Sf9 cells using a baculovirus expression system. PK-LOX1 metabolizes eicosapentaenoic acid (EPA) to 8R-HEPE, arachidonic acid (ARA) to 8R-HETE and docosahexaenoic acid (DHA) to 10R-HDHA. Moreover, PK-LOX1 had higher activity for EPA than ARA and DHA. In addition, PK-LOX1 also metabolizes 17S-HDHA to 10R,17S-dihydroxy-docosahexaenoic acid (10R,17S-DiHDHA). PK-LOX1 is a novel lipoxygenase that acts as an 8R-lipoxygenase for EPA and 10R-lipoxygenase for DHA and 17S-HDHA. Our findings show PK-LOX1 facilitates the enzymatic production of hydroxy fatty acids, which are of value to the healthcare sector.

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, 150rpm. Under optimized reaction conditions, maximum TOD production reached 340.3mg/100mL of the culture. However, requirement of nutritional factors by KNU-2B for production of TOD were considerably different from those by PR3 strain.

Efficient Transformation of Linoleic Acid into 13(S)-Hydroxy-9,11-(Z,E)-octadecadienoic Acid Using Putative Lipoxygenases from Cyanobacteria

Enzymatic dioxygenation of unsaturated fatty acids by lipoxygenases (LOXs) represents a useful method in the transformation of bioresources for hydroperoxy and hydroxy fatty acids production. Diffi...

Characteristics of bifidobacterial conjugated fatty acid and hydroxy fatty acid production and its potential application in fermented milk

This is the first study to elucidate bifidobacterial conjugated fatty acid and hydroxy fatty acid production as well as its potential in fermented milk. One hundred and fifty-two bifidobacteria belonging to seven species were assayed in this study. The results showed that only Bifidobacterium breve (B. breve), Bifidobacterium longum (B. longum), Bifidobacterium pesudocatenulatum (B. pseudocatenulatum) and Bifidobacterium dentium (B. dentium) generated CLA and CLNA, while nearly all the strains could convert free LA into 10-HOE. B. breve showed the highest CLA- and CLNA-generation ability (the highest conversion rate, 90.1%, for B. breve CCFM683), while B. pseudocatenulatum revealed higher 10-HOE-producing ability. Further study demonstrated that 10-HOE competition could influence the CLA generation in Bifidobacterium. Furthermore, B. breve CCFM683, as the highest CLA- and CLNA-producer, could increase the content of CLA and CLNA to 0.14 mg/mL and 0.065 mg/mL in fermented milk, presenting as a potentially ideal starter culture increasing conjugated fatty acid contents in milk.

Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity.

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C18 polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.