Kyoung-Rok Kim

Production of hydroxy fatty acids by microbial fatty acid-hydroxylation enzymes

Hydroxy fatty acids are widely used in chemical, food, and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of lubricants, emulsifiers, and stabilizers. They have antibiotic, anti-inflammatory, and anticancer activities and therefore can be applied for medicinal uses. Microbial fatty acid-hydroxylation enzymes, including P450, lipoxygenase, hydratase, 12-hydroxylase, and diol synthase, synthesize regio-specific hydroxy fatty acids. In this article, microbial fatty acid-hydroxylation enzymes, with a focus on region-specificity and diversity, are summarized and the production of mono-, di-, and tri-hydroxy fatty acids is introduced. Finally, the production methods of regio-specific and diverse hydroxy fatty acids, such as gene screening, protein engineering, metabolic engineering, and combinatory biosynthesis, are suggested.

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia.

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.

Production of 13S-hydroxy-9(Z)-octadecenoic acid from linoleic acid by whole recombinant cells expressing linoleate 13-hydratase from Lactobacillus acidophilus.

Linoleate 13-hydratase from Lactobacillus acidophilus LMG 11470 converted linoleic acid to hydroxyl fatty acid, which was identified as 13S-hydroxy-9(Z)-octadecenoic acid (13-HOD) by GC-MS and NMR. The expression of linoleate 13-hydratase gene in Escherichia coli was maximized by using pACYC plasmid and super optimal broth with catabolite repression (SOC) medium containing 40mM Mg(2+). To optimize induction conditions, recombinant cells were cultivated at 37°C, 1mM isopropyl-β-d-thiogalactopyranoside was added at 2h, and the culture was further incubated at 16°C for 18h. Recombinant cells expressing linoleate 13-hydratase from L. acidophilus were obtained under the optimized expression conditions and used for 13-HOD production from linoleic acid. The optimal reaction conditions were pH 6.0, 40°C, 0.25% (v/v) Tween 40, 25gl(-1) cells, and 100gl(-1) linoleic acid, and under these conditions, whole recombinant cells produced 79gl(-1) 13-HOD for 3h with a conversion yield of 79% (w/w), a volumetric productivity of 26.3gl(-1)h(-1), and a specific productivity of 1.05g g-cells(-1)h(-1). To the best of our knowledge, the recombinant cells produced hydroxy fatty acid with the highest concentration and productivity reported so far.

Microbial synthesis of plant oxylipins from γ-linolenic acid through designed biotransformation pathways.

Secondary metabolites of plants are often difficult to synthesize in high yields because of the large complexity of the biosynthetic pathways and challenges encountered in the functional expression of the required biosynthetic enzymes in microbial cells. In this study, the biosynthesis of plant oxylipins--a family of oxygenated unsaturated carboxylic acids--was explored to enable a high-yield production through a designed microbial synthetic system harboring a set of microbial enzymes (i.e., fatty acid double-bond hydratases, alcohol dehydrogenases, Baeyer-Villiger monooxygenases, and esterases) to produce a variety of unsaturated carboxylic acids from γ-linolenic acid. The whole cell system of the recombinant Escherichia coli efficiently produced (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic acid (15), (Z)-dec-4-enedioic acid (17), and (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2). This study demonstrated that various secondary metabolites of plants can be produced by implementing artificial biosynthetic pathways into whole-cell biocatalysis.

Unveiling of novel regio‐selective fatty acid double bond hydratases from Lactobacillus acidophilus involved in the selective oxyfunctionalization of mono‐ and di‐hydroxy fatty acids

The aim of this study is the first time demonstration of cis‐12 regio‐selective linoleate double‐bond hydratase. Hydroxylation of fatty acids, abundant feedstock in nature, is an emerging alternative route for many petroleum replaceable products thorough hydroxy fatty acids, carboxylic acids, and lactones. However, chemical route for selective hydroxylation is still quite challenging owing to low selectivity and many environmental concerns. Hydroxylation of fatty acids by hydroxy fatty acid forming enzymes is an important route for selective biocatalytic oxyfunctionalization of fatty acids. Therefore, novel fatty acid hydroxylation enzymes should be discovered. The two hydratase genes of Lactobacillus acidophilus were identified by genomic analysis, and the expressed two recombinant hydratases were identified as cis‐9 and cis‐12 double‐bond selective linoleate hydratases by in vitro functional validation, including the identification of products and the determination of regio‐selectivity, substrate specificity, and kinetic parameters. The two different linoleate hydratases were the involved enzymes in the 10,13‐dihydroxyoctadecanoic acid biosynthesis. Linoleate 13‐hydratase (LHT‐13) selectively converted 10 mM linoleic acid to 13S‐hydroxy‐9(Z)‐octadecenoic acid with high titer (8.1 mM) and yield (81%). Our study will expand knowledge for microbial fatty acid‐hydroxylation enzymes and facilitate the designed production of the regio‐selective hydroxy fatty acids for useful chemicals from polyunsaturated fatty acid feedstocks. Biotechnol. Bioeng. 2015;112: 2206–2213.

A 2.4 GHz SiGe bipolar power amplifier with integrated diode linearizer for WLAN IEEE 802.11b/g applications

A linear RF power amplifier with integrated reverse biased diode linearizer for IEEE 802.11b/g WLAN terminals is implemented with a 33 GHz-fT, 0.5-mum-SiGe bipolar technology. The proposed linearizer maintains the fixed base voltage of power stage HBTs. The power amplifier exhibits 24.5 dBm of 1-dB compression point (P1dB), with 36% of the power-added efficiency (PAE), and 17 dB of the power gain under 3.3 V power supply. The third-order IMD value is less than - 32.5 dBc at 3 dB back-off from P1dB for the frequency of 2.45 GHz. The fabricated chip size is as small as 1times0.7 mm2, including input/inter-stage matching network and all active bias circuits

Enhancement of retinal production by supplementing the surfactant Span 80 using metabolically engineered Escherichia coli

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.

A MMIC smart power amplifier with on-chip dynamic bias controller for WCDMA mobile communication

This work presents an InGaP/GaAs HBT smart MMIC power amplifier provided with a new dynamic bias controller and dual power stages in parallel. The proposed dynamic bias circuit controls the quiescent current as a function of input power with perfect freedom, and is implemented for a WCDMA MMIC power amplifier together with the parallel power stages for both linearity and efficiency. Maximum output power, PAE, and gain are simulated to have 31.3 dBm, 40.8%, and 23.9 dB, respectively in WCDMA conditions. The smart power amplifier exhibits an improvement of average power usage efficiency by 220 % while keeping the WCDMA linearity.

Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus

ABSTRACT A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus. The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)+. We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (k cat/Km ) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD+ converted 400 μM all-trans-retinal to 210 μM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all-trans-retinal to 20, 90, and 40 μM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. IMPORTANCE Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)+ as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid by BcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 in BcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

Selective Production of 9R-Hydroxy-10E,12Z,15Z-Octadecatrienoic Acid from α-Linolenic Acid in Perilla Seed Oil Hydrolyzate by a Lipoxygenase from Nostoc Sp. SAG 25.82

Hydroxy fatty acids (HFAs) derived from omega-3 polyunsaturated fatty acids have been known as versatile bioactive molecules. However, its practical production from omega-3 or omega-3 rich oil has not been well established. In the present study, the stereo-selective enzymatic production of 9R-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9R-HOTE) from α-linolenic acid (ALA) in perilla seed oil (PO) hydrolyzate was achieved using purified recombinant 9R-lipoxygenase (9R-LOX) from Nostoc sp. SAG 25.82. The specific activity of the enzyme followed the order linoleic acid (LA) > ALA > γ-linolenic acid (GLA). A total of 75% fatty acids (ALA and LA) were used as a substrate for 9R-LOX from commercial PO by hydrolysis of Candida rugosa lipase. The optimal reaction conditions for the production of 9R-HOTE from ALA using 9R-LOX were pH 8.5, 15°C, 5% (v/v) acetone, 0.2% (w/v) Tween 80, 40 g/L ALA, and 1 g/L enzyme. Under these conditions, 9R-LOX produced 37.6 g/L 9R-HOTE from 40 g/L ALA for 1 h, with a conversion yield of 94% and a productivity of 37.6 g/L/h; and the enzyme produced 34 g/L 9R-HOTE from 40 g/L ALA in PO hydrolyzate for 1 h, with a conversion yields of 85% and a productivity of 34 g/L/h. The enzyme also converted 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from 40 g/L LA for 1.0 h, with a conversion yield of 95% and a productivity of 38.4 g/L. This is the highest productivity of HFA from both ALA and ALA-rich vegetable oil using LOX ever reported. Therefore, our result suggests an efficient method for the production of 9R-HFAs from LA and ALA in vegetable oil using recombinant LOX in biotechnology.