Effective melanin degradation by a synergistic laccase-peroxidase enzyme complex for skin whitening and other practical applications.

Melanin is major cause of dark skin, which is regarded as social status in eastern Asia. As a result, researchers in cosmetic industries are developing skin whitening agents. Melanin can be decolorized by many oxidative enzymes. Laccase (CueO) from Escherichia coli and dye-decolorizing peroxidase (DyP) from Bacillus subtilis were merged with the dockerin domain of endoglucanase B from Clostridium cellulovorans. Scaffoldin has great potential to exert structural benefits that enable complementary enzyme effects. The carbohydrate binding module (CBM) in scaffoldin was replaced with the melanin binding peptide (MBP) to increase melanin binding and thereby enhance melanin degradation. The modified scaffoldin exhibits a nearly 64% increase in specific binding to melanin over that of the native scaffoldin. Laccase was used to degrade melanin via the production of hydrogen peroxide, which produced synergistic activity with peroxidase. The activity of the optimized complex was approximately 6.4-fold greater than that of laccase alone. This enzyme complex can also reduce the number of melanin granules in corneocytes. Based on these results, a recombinant enzyme complex is suitable for use in melanin degradation by next generation whitening agents in the skin cosmetics industry.

Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive.

Taurine is a biologically and physiologically valuable food additive. However, commercial taurine production mainly relies on environmentally harmful chemical synthesis. Herein, for the first time in bacteria, we attempted to produce taurine in metabolically engineered Corynebacterium glutamicum. The taurine-producing strain was developed by introducing cs, cdo1, and csad genes. Interestingly, while the control strain could not produce taurine, the engineered strains successfully produced taurine via the newly introduced metabolic pathway. Furthermore, we investigated the effect of a deletion strain of the transcriptional repressor McbR gene on taurine production. As a result, sulfur accumulation and l-cysteine biosynthesis were reinforced by the McbR deletion strain, which further increased the taurine production by 2.3-fold. Taurine production of the final engineered strain Tau11 was higher than in other previously reported strains. This study demonstrated a potential approach for eco-friendly biosynthesis as an alternative to the chemical synthesis of a food additive.

Biosynthesis of organic photosensitizer Zn-porphyrin by diphtheria toxin repressor (DtxR)-mediated global upregulation of engineered heme biosynthesis pathway in Corynebacterium glutamicum.

Zn-porphyrin is a promising organic photosensitizer in various fields including solar cells, interface and biomedical research, but the biosynthesis study has been limited, probably due to the difficulty of understanding complex biosynthesis pathways. In this study, we developed a Corynebacterium glutamicum platform strain for the biosynthesis of Zn-coproporphyrin III (Zn-CP III), in which the heme biosynthesis pathway was efficiently upregulated. The pathway was activated and reinforced by strong promoter-induced expression of hemAM (encoding mutated glutamyl-tRNA reductase) and hemL (encoding glutamate-1-semialdehyde aminotransferase) genes. This engineered strain produced 33.54 ± 3.44 mg/l of Zn-CP III, while the control strain produced none. For efficient global regulation of the complex pathway, the dtxR gene encoding the transcriptional regulator diphtheria toxin repressor (DtxR) was first overexpressed in C. glutamicum with hemAM and hemL genes, and its combinatorial expression was improved by using effective genetic tools. This engineered strain biosynthesized 68.31 ± 2.15 mg/l of Zn-CP III. Finally, fed-batch fermentation allowed for the production of 132.09 mg/l of Zn-CP III. This titer represents the highest in bacterial production of Zn-CP III reported to date, to our knowledge. This study demonstrates that engineered C. glutamicum can be a robust biotechnological model for the production of photosensitizer Zn-porphyrin.

Synergistic effect of the enzyme complexes comprising agarase, carrageenase and neoagarobiose hydrolase on degradation of the red algae.

In the practice of converting red algae biomass into biofuel or valuable biomaterials, the critical step is the decomposition process of the agarose to give fermentable monomeric sugars. In this study, we selected three enzymes such as agarase, carrageenase and neoagarobiose hydrolase to inducible the simultaneous hydrolysis of the major substrates such as agar and carrageenan constituting the pretreated red algae, and expressed the chimeric enzymes and formed a complexes through optimization of addition ratio. As a result, hydrolysis by enzyme complexes showed a maximum sugar release of 679 mg L-1 with 67.9% saccharification yield from G. verrucosa natural substrate. The difference in the reducing sugar by the enzyme complexes was 3.6-fold higher than that of the monomer enzyme (cAgaB yield 188.6 mg L-1). The synergistic effect of producing sugars from red algae biomass through these enzyme complexes can be a very important biological tools aimed at bioenergy production.

Integration of Bacterial Expansin on Agarolytic Complexes to Enhance the Degrading Activity of Red Algae by Control of Gelling Properties.

Expansin act by loosening hydrogen bonds in densely packed polysaccharides. This work characterizes the biological functions of expansin in the gelling and degradation of algal polysaccharides. In this study, the bacterial expansin BpEX from Bacillus pumilus was fused with the dockerin module of a cellulosome system for assembly with agarolytic complexes. The assembly of chimeric expansin caused an indicative enhancement in agarase activity. The enzymatic activities on agar substrate and natural biomass were 3.7-fold and 3.3-fold higher respectively than that of agarase as a single enzyme. To validate the effect on the agar degradation, the regulation potential of parameters related to gel rheology by bacterial expansin was experimentally investigated to indicate that the bacterial expansin lowered the gelling temperature and viscosity of agar. Thus, these results demonstrated the possibility of advancing more efficient strategies for utilizing agar as oligo sugar source in the biorefinery field that uses marine biomass as feedstocks.

Enhancing Fatty Acid Production of Saccharomyces cerevisiae as an Animal Feed Supplement.

Saccharomyces cerevisiae is used for edible purposes, such as human food or as an animal feed supplement. Fatty acids are also beneficial as feed supplements, but S. cerevisiae produces small amounts of fatty acids. In this study, we enhanced fatty acid production of S. cerevisiae by overexpressing acetyl-CoA carboxylase, thioesterase, and malic enzyme associated with fatty acid metabolism. The enhanced strain pAMT showed 2.4-fold higher fatty acids than the wild-type strain. To further increase the fatty acids, various nitrogen sources were analyzed and calcium nitrate was selected as an optimal nitrogen source for fatty acid production. By concentration optimization, 672 mg/L of fatty acids was produced, which was 4.7-fold higher than wild-type strain. These results complement the low level fatty acid production and make it possible to obtain the benefits of fatty acids as an animal feed supplement while, simultaneously, maintaining the advantages of S. cerevisiae.

Bio-Based Production of Dimethyl Itaconate From Rice Wine Waste-Derived Itaconic Acid.

Dimethyl itaconate is an important raw material for copolymerization, but it is not synthesized from itaconic acid by organisms. Moreover, Corynebacterium glutamicum is used as an important industrial host for the production of organic acids, but it does not metabolize itaconic acid. Therefore, the biosynthetic route toward dimethyl itaconate from itaconic acid is highly needed. In this study, a biological procedure for dimethyl itaconate production is developed from rice wine waste-derived itaconic acid using the engineered C. glutamicum strain. The first step is to investigate the effect of the co-overexpression of the codon-optimized cis-aconitic acid decarboxylase (CadA*) and a transcriptional regulator of genes involved in acetic acid metabolism (RamA) on itaconic acid production. The second step is to convert itaconic acid into dimethyl itaconate by lipase-catalyzed esterification. The CadA* and RamA-overexpressing CG4 strain increases the itaconic acid concentration under N-starvation with glucose and acetic acid compared with the concentration produced in the base mCGXII medium with glucose. Furthermore, the rice wine waste-derived itaconic acid is successfully converted into dimethyl itaconate using lipase from Rhizomucor miehei and a methanol substrate. This study is the first trial for bio-based production of dimethyl itaconate from rice wine waste-derived itaconic acid.

Metabolic Design of Corynebacterium glutamicum for Production of l-Cysteine with Consideration of Sulfur-Supplemented Animal Feed.

l-Cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. However, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. In this study, we constructed recombinant Corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. The aims of this work were to investigate the effect of the combined overexpression of serine acetyltransferase (CysE), O-acetylserine sulfhydrylase (CysK), and the transcriptional regulator CysR on l-cysteine production. The CysR-overexpressing strain accumulated approximately 2.7-fold more intracellular sulfide than the control strain (empty pMT-tac vector). Moreover, in the resulting CysEKR recombinant strain, combinatorial overexpression of genes involved in l-cysteine production successfully enhanced its production by approximately 3.0-fold relative to that in the control strain. This study demonstrates a biotechnological model for the production of animal feed supplements such as l-cysteine using metabolically engineered C. glutamicum.

BLT2 is a pro-tumorigenic mediator during cancer progression and a therapeutic target for anti-cancer drug development.

Cancer is a leading cause of death worldwide and has been linked to inflammation. Leukotriene B4 (LTB4) is synthesized from arachidonic acid via the 5-lipoxygenase pathway and is a potent chemoattractant for inflammatory cells. LTB4 was recently shown to be associated with the pathogenesis of inflammatory diseases, including cancer. Of the two known LTB4 receptors, BLT1 and BLT2, the biological roles of the low-affinity LTB4 receptor 2, BLT2, have only recently been elucidated. This review focuses on recent discoveries regarding BLT2 and its roles in cancer progression and the downstream signaling mechanisms of the BLT2-linked signaling cascade in cancer cells. We believe that these findings will facilitate the development of new cancer treatments.

New biotransformation process for production of the fragrant compound γ-dodecalactone from 10-hydroxystearate by permeabilized Waltomyces lipofer cells.

A new biotransformation process for the production of the flavor lactone was developed by using permeabilized Waltomyces lipofer, which was selected as an efficient γ-dodecalactone-producing yeast among 10 oleaginous yeast strains. The optimal reaction conditions for γ-dodecalactone production by permeabilized W. lipofer cells were pH 6.5, 35°C, 200 rpm, 0.7 M Tris, 60 g/liter of 10-hydroxystearic acid, and 30 g/liter of cells. Under these conditions, nonpermeabilized cells produced 12 g/liter of γ-dodecalactone after 30 h, with a conversion yield of 21% (wt/wt) and a productivity of 0.4 g/liter/h, whereas permeabilized cells obtained after sequential treatments with 50% ethanol and 0.5% Triton X-100 produced 46 g/liter of γ-dodecalactone after 30 h, with a conversion yield of 76% (wt/wt) and a productivity of 1.5 g/liter/h. These values were 3.7- and 3.8-fold higher than those obtained using nonpermeabilized cells. These are the highest reported concentration, conversion yield, and productivity for the production of the bioflavor lactone.

Lipoxygenases: potential starting biocatalysts for the synthesis of signaling compounds.

Lipoxygenases (LOXs) have attracted a great deal of attention as potential starting biocatalysts for synthesizing signaling compounds. Significant advances during the past decade include the discovery of regiospecific LOXs and structural investigation for their diverse regiospecificity. Eight regiospecific (5-, 8-, 9-, 10-, 11-, 12-, 13-, and 15-) LOXs catalyze positional-specific dioxygenation of polyunsaturated fatty acids, forming positional-specific hydroperoxy fatty acids that are further metabolized into signaling compounds. The LOX-derived signaling compounds can be applied not only for clinical uses but also for industrial uses. For example, animal lipoxin LXA4, plant jasmonic acids, plant green leaf volatiles, and bacterial lactones have been used as anti-inflammatory agents, anti-pest agents, flavors, and food additives, respectively. Prostaglandins, as controllers of hormone regulation and cell growth, are also suggested as LOX-derived compounds in corals.

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 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis.

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850 min(-1) and a K (m) of 540 µM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35 °C, 4% (v/v) ethanol, 2,500 U ml(-1) (8.3 mg ml(-1)) of enzyme, and 40 g l(-1) oleic acid. Under these conditions, 40 g l(-1) (142 mM) oleic acid was converted into 40 g l(-1) (133 mM) 10-hydroxystearic acid for 150 min, with a molar yield of 94% and a productivity of 16 g l(-1) h(-1), and olive oil hydrolyzate containing 40 g l(-1) oleic acid was converted into 40 g l(-1) 10-hydroxystearic acid for 300 min, with a productivity of 8 g l(-1) h(-1).

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus.

A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC-MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k(cat)/K(m)) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG((A/S))X((15-21))E((D))), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.

Antioxidant activity of glyceollins derived from soybean elicited with Aspergillus sojae.

The extract of soybean exposed to biotic elicitors such as food-grade fungus is known to have antioxidant activity. Glyceollins were major bioactive compounds present in soybean elicited by fungi and shown to have antifungal and anticancer activities. The purpose of present study was to evaluate the antioxidant activities of glyceollins by measuring ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, singlet oxygen quenching, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, hydroxyl radical scavenging activity, and lipid peroxidation inhibition. In addition, the antioxidant potential of glyceollins were measured by a fluorescent probe, 2',7'-dichlorofluorescin diacetate (DCFDA), and dihydroethidium (DHE) in mouse hepatoma hepa1c1c7 cells in which they were insulted with H2O2 to generate reactive oxygen species (ROS). Glyceollins showed a strong reducing power and inhibited lipid peroxidation, with significant scavenging activities of radicals including singlet oxygen, superoxide anion, ABTS, and DPPH. We also found that glyceollins significantly suppressed H2O2-induced ROS production in hepa1c1c7 cells. Therefore, glyceollins deserve further study as natural antioxidants and nutraceuticals.