Min-Ju Seo

Production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid by whole recombinant Escherichia coli cells expressing diol synthase from Aspergillus nidulans.

Diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells expressing diol synthase from A. nidulans converted linoleic acid to a product that was identified as 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The recombinant cells and the purified enzyme showed the highest activity for linoleic acid among the fatty acids tested. The optimal reaction conditions for the production of 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid from linoleic acid using whole recombinant E. coli cells expressing diol synthase were pH 7.5, 35°C, 250 rpm, 5 g l−1 linoleic acid, 23 g l−1 cells, and 20% (v/v) dimethyl sulfoxide in a 250-ml baffled flask. Under these optimized conditions, whole recombinant cells expressing diol synthase produced 4.98 g l−1 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid for 150 min without detectable byproducts, with a conversion yield of 99% (w/w) and a productivity of 2.5 g l−1 h−1. This is the first report on the biotechnological production of dihydroxy fatty acid using whole recombinant cells expressing diol synthase.

Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus

Ginsenoside compound K (C-K) is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD)-type ginsenosides in red-ginseng extract (RGE), the α-l-arabinofuranoside-hydrolyzing α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf) was used along with the β-d-glucopyranoside/α-l-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly) because SS-bgly showed very low hydrolytic activity on the α-l-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.

Prostaglandin synthases: Molecular characterization and involvement in prostaglandin biosynthesis

Prostaglandins (PGs) belong to a subclass of eicosanoids and are classified based on the structures of the cyclopentane ring and their number of double bonds in their hydrocarbon structures. PGs are important lipid mediators that are involved in inflammatory response. The biosynthesis of diverse PGs from unsaturated C20 fatty acids containing at least three double bonds such as dihomo-γ-linoleic acid (20:3Δ8Z,11Z,14Z), arachidonic acid (20:4Δ5Z,8Z,11Z,14Z), and eicosapentaenoic acid (20:5Δ5Z,8Z,11Z,14Z,17Z) is enables by various PG synthases, including prostaglandin H synthase (PGHS), 15-hydroxyprostaglandin dehydrogenase (15-HPGD), PGES, PGDS, PGFS, PGIS, and thromboxane A synthase (TXAS). This review summarizes the biochemical properties, reaction mechanism, and active site details of PG synthases. Because PGs are involved in the immune system, an understanding of PG synthases is important in the design of new anti-inflammatory drugs. The biosynthesis of PGs in various organisms, such as mammals, corals, florideae (a class of red algae), yeast, and fungi, is also introduced. The expression of PG synthases in the microbial systems for the synthesis of PGs is discussed. Now, the biosynthesis of PGs from glucose or glycerol is possible using metabolically engineered cells expressing both unsaturated fatty acid-producing enzymes and PG synthases.

Gene cloning of an efficiency oleate hydratase from Stenotrophomonas nitritireducens for polyunsaturated fatty acids and its application in the conversion of plant oils to 10‐hydroxy fatty acids

Hydroxy fatty acids are used as precursors of lactones and dicarboxylic acids, as starting materials of polymers, and as additives in coatings and paintings. Stenotrophomonas nitritireducens efficiently converts cis‐9 polyunsaturated fatty acids (PUFAs) to 10‐hydroxy fatty acids. However, gene encoding enzyme involved in this conversion has not been identified to date. We purified a putative fatty acid double‐bond hydratase from S. nitritireducens by ultrafiltration and HiPrep DEAE FF and Resource Q ion exchange chromatographies. Peptide sequences of the purified enzyme were obtained by liquid chromatography‐mass spectrometry/mass spectrometry (LC‐MS/MS) analysis. Sequence of the partial gene encoding this putative fatty acid double‐bond hydratase was determined by degenerate polymerase chain reaction (PCR) based on the peptide sequences. The remaining gene sequence was identified by rapid amplification of cDNA ends using cDNA of S. nitritireducens as a template, and the full‐length gene was cloned subsequently. The expressed enzyme was identified as an oleate hydratase by determining its kinetic parameters toward unsaturated fatty acids. S. nitritireducens oleate hydratase showed higher activity toward PUFAs compared with other available oleate hydratases. This suggested that the enzyme could be used effectively to convert plant oils to 10‐hydroxy fatty acids because these oils contained unsaturated fatty acids such as oleic acid (OA) and linoleic acid (LA) and PUFAs such as α‐linolenic acid and/or γ‐linolenic acid. The enzyme converted soybean oil and perilla seed oil hydrolyzates containing 10 mM total unsaturated fatty acids, including OA, LA, and ALA, to 8.87 and 8.70 mM total 10‐hydroxy fatty acids, respectively, in 240 min. To our knowledge, this is the first study on the biotechnological conversion of PUFA‐containing oils to hydroxy fatty acids. Biotechnol. Bioeng. 2017;114: 74–82.

Characterization of a novel 8R,11S-linoleate diol synthase from Penicillium chrysogenum by identification of its enzymatic products

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus.

Comparison of Biochemical Properties of the Original and Newly Identified Oleate Hydratases from Stenotrophomonas maltophilia

ABSTRACT Oleate hydratases (OhyAs) catalyze the conversion of unsaturated fatty acids to 10-hydroxy fatty acids, which are used as precursors of important industrial compounds, including lactones and ω-hydroxycarboxylic and α,ω-dicarboxylic acids. The genes encoding OhyA and a putative fatty acid hydratase in Stenotrophomonas maltophilia were identified by genomic analysis. The putative fatty acid hydratase was purified and identified as an oleate hydratase (OhyA2) based on its substrate specificity. The activity of OhyA2 as a holoenzyme was not affected by adding cofactors, whereas the activity of the original oleate hydratase (OhyA1) showed an increase. Thus, all characterized OhyAs were categorized as either OhyA1 or OhyA2 based on the activities of holoenzymes upon adding cofactors, which were determined by the type of the fourth conserved amino acid of flavin adenine dinucleotide (FAD)-binding motif. The hydration activities of S. maltophilia OhyA2 toward unsaturated fatty acids, including oleic acid, palmitoleic acid, linoleic acid, α-linolenic acid, and γ-linolenic acid, were greater than those of OhyA1. Moreover, the specific activity of S. maltophilia OhyA2 toward unsaturated fatty acids, with the exception of γ-linolenic acid, was the highest among all reported OhyAs. IMPORTANCE All characterized OhyAs were categorized as OhyA1s or OhyA2s based on the different properties of the reported and newly identified holo-OhyAs in S. maltophilia upon the addition of cofactors. OhyA2s showed higher activities toward polyunsaturated fatty acids (PUFAs), including linoleic acid, α-linolenic acid, and γ-linolenic acid, than those of OhyA1s. This suggests that OhyA2s can be used more effectively to convert plant oils to 10-hydroxy fatty acids because plant oils contain not only oleic acid but also PUFAs. The hydration activity of the newly identified OhyA2 from S. maltophilia toward oleic acid was the highest among the activity levels reported so far. Therefore, this enzyme is an efficient biocatalyst for the conversion of plant oils to 10-hydroxy fatty acids, which can be further converted to important industrial materials.

Production of 8,11-dihydroxy and 8-hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11-linoleate diol synthase from Penicillium chrysogenum

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11‐Linoleate diol synthase (8,11‐LDS) catalyzes the conversion of unsaturated fatty acid to 8‐hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11‐dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11‐LDS for the production of 8,11‐dihydroxy‐9,12(Z,Z)‐octadecadienoic acid (8,11‐DiHODE), 8,11‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid (8,11‐DiHOTrE), 8‐hydroxy‐9(Z)‐hexadecenoic acid (8‐HHME), and 8‐hydroxy‐9(Z)‐octadecenoic acid (8‐HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α‐linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11‐DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11‐DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8‐HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8‐HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11‐DiHODE, 8,11‐DiHOTrE, 8‐HHME, and 8‐HOME.

Molecular characterization of Penicillium oxalicum 6R,8R-linoleate diol synthase with new regiospecificity

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn886, Arg707, and Arg934, are catalytic importance and belong to the catalytic groove. Phe794 and Gln889 were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.

Biotransformation of fatty acid-rich tree oil hydrolysates to hydroxy fatty acid-rich hydrolysates by hydroxylases and their feasibility as biosurfactants

The fatty acid compositions of 10 types of tree oils were analyzed and Camellia japonica (CJ), Tetradium daniellii (TD), and Hovenia dulcis (HD) tree oils were selected to be oleic acid (OA)-, linoleic acid (LA)-, and α-linoleic acid (ALA)-rich tree oils, respectively. Recombinant Escherichia coli expressing 10-hydratase and 7,8-diol synthase converted 31.7 and 15.6 g/L unsaturated fatty acids (UFAs) in OA-rich oil hydrolysates to 21.7 g/L 10-monohydroxy fatty acid (monoHFA) and 13.3 g/L 7,8-diHFA, respectively. The cells expressing 13-hydratase, 13-lipoxygenase, 5,8-diol synthase, and 8,11-diol synthase converted 42.8, 28.5, 10.0, and 20.0 g/L UFAs in LA-rich oil hydrolysates to 28.2 g/L 13-monoHFA, 11.8 g/L 13-monoHFA, 7.2 g/L 5,8-diHFA, and 8.9 g/L 8,11-diHFA, respectively. The cells expressing 8,11-diol synthase converted containing 17.5 g/L UFAs in ALA-rich oil hydrolysate to 7.5 g/L 8,11-diHFA. The average emulsifying activities of diHFArich and monoHFA-rich tree oil hydrolysates were 13.9- and 4.3-fold higher than those of tree oil hydrolysates, respectively. Thus, HFA-rich tree oil hydrolysates derived from tree oils can be applied as biosurfactants, and the fatty acid-rich residue as by-product obtained from the tree refinery process may be recycled into biosurfactants.

An amino acid at position 512 in β-glucosidase from Clavibacter michiganensis determines the regioselectivity for hydrolyzing gypenoside XVII.

A recombinant β-glucosidase from Clavibacter michiganensis specifically hydrolyzed the outer and inner glucose linked to the C-3 position in protopanaxadiol (PPD)-type ginsenosides and the C-6 position in protopanaxatriol (PPT)-type ginsenosides except for the hydrolysis of gypenoside LXXV (GypLXXV). The enzyme converted gypenoside XVII (GypXVII) to GypLXXV by hydrolyzing the inner glucose linked to the C-3 position. The substrate-binding residues obtained from the GypXVII-docked homology models of β-glucosidase from C. michiganensis were replaced with alanine, and the amino acid residue at position 512 was selected because of the changed regioselectivity of W512A. Site-directed mutagenesis for the amino acid residue at position 512 was performed. W512A and W512K hydrolyzed the inner glucose linked to the C-3 position and the outer glucose linked to the C-20 position of GypXVII to produce GypLXXV and F2. W512R hydrolyzed only the outer glucose linked to the C-20 position of GypXVII to produce F2. However, W512E and W512D exhibited no activity for GypXVII. Thus, the amino acid at position 512 is a critical residue to determine the regioselectivity for the hydrolysis of GypXVII. These wild-type and variant enzymes produced diverse ginsenosides, including GypXVII, GypLXXV, F2, and compound K, from ginsenoside Rb1. To the best of our knowledge, this is the first report of the alteration of regioselectivity on ginsenoside hydrolysis by protein engineering.