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The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids inLactobacillus plantarumas substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids. Oxidative cleavage of oxo unsaturated fatty acid into dicarboxylic acid by laccase. [ABSTRACT FROM PUBLISHER]

Hydroxy fatty acid dehydrogenase, which is involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a, was cloned, expressed, purified, and characterized. The enzyme preferentially catalyzed NADH-dependent hydrogenation of oxo fatty acids over NAD + -dependent dehydrogenation of hydroxy fatty acids. In the dehydrogenation reaction, fatty acids with an internal hydroxy group such as 10-hydroxy- cis -12-octadecenoic acid, 12-hydroxy- cis -9-octadecenoic acid, and 13-hydroxy- cis -9-octadecenoic acid served as better substrates than those with α- or β-hydroxy groups such as 3-hydroxyoctadecanoic acid or 2-hydroxyeicosanoic acid. The apparent K m value for 10-hydroxy- cis -12-octadecenoic acid (HYA) was estimated to be 38 μM with a k cat of 7.6 × 10 −3 s −1 . The apparent K m value for 10-oxo- cis -12-octadecenoic acid (KetoA) was estimated to be 1.8 μM with a k cat of 5.7 × 10 −1 s −1 . In the hydrogenation reaction of KetoA, both ( R )- and ( S )-HYA were generated, indicating that the enzyme has low stereoselectivity. This is the first report of a dehydrogenase with a preference for fatty acids with an internal hydroxy group. [ABSTRACT FROM AUTHOR]

Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy- cis -12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis -9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent K m value for linoleic acid was estimated to be 92 μM, with a k cat of 2.6∙10 −2 s −1 and a Hill factor of 3.3. The apparent K m value for HYA was estimated to be 98 μM, with a k cat of 1.2∙10 −3 s −1 . [ABSTRACT FROM AUTHOR]

Through the screening of about 300 strains of lactic acid bacteria, Pediococcus sp. AKU 1080 was selected as a strain with the ability to hydrate linoleic acid ( cis-9, cis-12-octadecadienoic acid) to three hydroxy fatty acids, i.e., 10-hydroxy- cis-12-octadecenoic acid, 13-hydroxy- cis-9-octadecenoic acid, and 10,13-dihydroxyoctadecanoic acid. The strain hydrated one of two cis double bonds at Δ9 and Δ12 positions to produce 10-hydroxy- cis-12-octadecenoic acid and 13-hydroxy- cis-9-octadecenoic acid, respectively, then further hydrated these two mono-hydroxy fatty acids to 10,13-dihydroxyoctadecanoic acid. The growing cells of this strain were applied to the production of 13-hydroxy- cis-9-octadecenoic acid, that is potential as polymer substrates and functional foods but its specific and efficient production was not established. Under the optimum conditions, 2.3 mg/mL of 13-hydroxy- cis-9-octadecenoic acid was produced from 12.3 mg/mL of linoleic acid with 0.04 mg/mL 10-hydroxy- cis-12-octadecenoic acid (HYA) and 0.05 mg/mL 10,13-dihydroxyoctadecanoic acid in the cultivation medium. Specific production of 13-hydroxy- cis-9-octadecenoic acid was attained using cell-free extracts of the strain as the catalyst. Under the optimum conditions, 0.4 mg/mL of 13-hydroxy- cis-9-octadecenoic acid was produced from 2.0 mg/mL of linoleic acid without HYA and 10,13-dihydroxyoctadecanoic acid. Practical applications: Hydroxy fatty acids are useful as starting materials for industrial chemicals, functional foods, and pharmaceuticals. Regioselective introduction of hydroxyl group to unsaturated fatty acids by microorganisms was applied to hydroxy fatty acid production. Especially, specific production of 13-hydroxy- cis-9-octadecenoic acid, which is useful for the production of 13-oxo-fatty acids with anti-obesity activity, was established in this work. [ABSTRACT FROM AUTHOR]

Lipid engineering related to biological functions has made remarkable progress in the fields of microbial production of functional lipids, metabolic engineering of microorganisms, elucidation of physiological functions of rare lipids, lipid-related enzyme engineering, and lipid analysis techniques. Various rare lipids are produced by utilizing microorganisms and their enzymes. It is also becoming clear that the rare lipids produced by intestinal bacteria contribute significantly to human health. Technological advances related to identification of lipid structures and quantification of lipids have led to such discoveries in the field of lipid engineering. This article reviews the latest findings that are attracting attention in the field of lipid engineering related to biological functions. [ABSTRACT FROM AUTHOR]

Microorganisms in animal gut produce unusual fatty acids from the ingested diet. Two types of hydroxy fatty acids (HFAs), 10-hydroxy- cis -12-octadecenoic acid (HYA) and 10-hydroxy-octadecanoic acid (HYB), are linoleic acid (LA) metabolites produced by Lactobacillus plantarum. In this study, we investigated the metabolism of these HFAs in mammalian cells. When Chinese hamster ovary (CHO) cells were cultured with HYA, approximately 50% of the supplemented HYA disappeared from the dish within 24 h. On the other hand, the amount of HYA that disappeared from the dish of peroxisome (PEX)-deficient CHO cells was lower than 20%. Significant amounts of C2– and C4-chain-shortened metabolites of HYA were detected in culture medium of HYA-supplemented CHO cells, but not in medium of PEX-deficient cells. These results suggested that peroxisomal β-oxidation is involved in the disappearance of HYA. The PEX-dependent disappearance was observed in the experiment with HYB, but not with LA. We also found that HYA treatment up-regulates peroxisomal β-oxidation activity of human gastric MKN74 cells and intestinal Caco-2 cells. These results indicate a possibility that HFAs produced from gut bacteria affect lipid metabolism of host via modulation of peroxisomal β-oxidation activity. • Hydroxy fatty acids (HFAs) from gut microbiota were β-oxidized in peroxisome. • Most of incorporated HFAs were eliminated rather than acylated to cellular lipids. • HYA up-regulated peroxisomal β-oxidation activity in gastrointestinal cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Serine 218 in FA-HY1 hydratase binding site affects regioselectivity of hydration. • Site-saturation mutagenesis rendered mutants with altered regioselectivity towards EPA. • A novel 15-hydroxy EPA product obtained with moderate regioselectivity. • Over 60 % conversion level for EPA can be achieved with a mutant whole cell catalyst. • Structural, kinetic and regression analysis provides the basis for observed changes. 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. [ABSTRACT FROM AUTHOR]