Biotransformation of 2-keto-4-hydroxybutyrate via aldol condensation using an efficient and thermostable carboligase from Deinococcus radiodurans.

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min-1 mg-1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L-1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L-1 h-1 and a specific productivity of 357.6 mg mg-enzyme-1 h-1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L-1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L-1 h-1 and 583.4 mg mg-enzyme-1 h-1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde.

Biotransformation of Polyunsaturated Fatty Acids to Trioxilins by Lipoxygenase from Pleurotus sajor-caju.

A lipoxygenase from Pleurotus sajor-caju (PsLOX) was cloned, expressed in Escherichia coli, and purified as a soluble protein with a specific activity of 629 µmol/min/mg for arachidonic acid (AA). The native PsLOX exhibited a molecular mass of 146 kDa, including a 73-kDa homodimer, as estimated by gel-filtration chromatography. The major products converted from polyunsaturated fatty acids (PUFAs), including AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were identified as trioxilins (TrXs), namely 13,14,15-TrXB3 , 13,14,15-TrXB4 , and 15,16,17-TrXB5 , respectively, through high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The enzyme displayed its maximum activity at pH 8.0 and 20 °C. Under these conditions, the specific activity and catalytic efficiency of PsLOX for PUFAs exhibited the following order: AA>EPA>DHA. Based on HPLC analysis and substrate specificity, PsLOX was identified as an arachidonate 15-LOX. PsLOX efficiently converted 10 mM of AA, EPA, and DHA to 8.7 mM of 13,14,15-TrXB3 (conversion rate: 87 %), 7.9 mM of 13,14,15-TrXB4 (79 %), and 7.2 mM of 15,16,17-TrXB5 (72 %) in 15, 20, and 20 min, respectively, marking the highest conversion rates reported to date. Collectively, our results demonstrate that PsLOX is an efficient TrXs-producing enzyme.

Screening of lactic acid bacteria with anti-adipogenic effect and potential probiotic properties from grains.

A total of 187 lactic acid bacteria were isolated from four types of grains collected in South Korea. The bacterial strains were assigned as members of Levilactobacillus brevis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, Lactococcus taiwanensis, Pediococcus pentosaceus, and Weissella paramesenteroides based on the closest similarity using 16S rRNA gene sequence analysis. The strains belonging to the same species were analyzed using RAPD-PCR, and one or two among strains showing the same band pattern were selected. Finally, 25 representative strains were selected for further functional study. Inhibitory effects of lipid accumulation were observed in the strains tested. Pediococcus pentosaceus K28, Levilactobacillus brevis RP21 and Lactiplantibacillus plantarum RP12 significantly reduced lipid accumulation and did not show cytotoxicity in C3H10T1/2 cells at treatment of 1-200 µg/mL. The three LAB strains decreased significantly expression of six adipogenic marker genes, PPARγ, C/EBPα, CD36, LPL, FAS and ACC, in C3H10T1/2 adipocytes. The three strains survived under strong acidity and bile salt conditions. The three strains showed adhesion to Caco-2 cells similar to a reference strain LGG. The resistance of the three strains to several antibiotics was also assessed. Strains RP12 and K28 were confirmed not to produce harmful enzymes based on API ZYM kit results. Based on these results, strains K28, RP21 and RP12 isolated from grains had the ability to inhibit adipogenesis in adipocytes and potentially be useful as probiotics.

One-Pot Biosynthesis of 2-Keto-4-hydroxybutyrate from Cheap C1 Compounds Using Rationally Designed Pyruvate Aldolase and Methanol Dehydrogenase.

One-carbon chemicals (C 1s) are potential building blocks as they are cheap, sustainable, and abiotic components. Methanol-derived formaldehyde can be another versatile building block for the production of 2-keto-4-hydroxyacid derivatives that can be used for amino acids, hydroxy carboxylic acids, and chiral aldehydes. To produce 2-keto-4-hydroxybutyrate from C 1s in an environment-friendly way, we characterized an aldolase from Pseudomonas aeruginosa PAO1 (PaADL), which showed much higher catalytic activity in condensing formaldehyde and pyruvate than the reported aldolases. By applying a structure-based rational approach, we found a variant (PaADLV121A/L241A) that exhibited better catalytic activities than the wild-type enzyme. Next, we constructed a one-pot cascade biocatalyst system by combining PaADL and a methanol dehydrogenase (MDH) and, for the first time, effectively produced 2-keto-4-hydroxybutyrate as the main product from pyruvate and methanol via an enzymatic reaction. This simple process applied here will help design a green process for the production of 2-keto-4-hydroxyacid derivatives.

Devosia litorisediminis sp. nov., isolated from a sand dune.

A Gram-negative, aerobic, non-motile, and rod-shaped bacterial strain, designated BSSL-BM10T, was isolated from sand of a dune that was collected from the Yellow Sea, Republic of Korea. It was subjected to a polyphasic taxonomic study. 16S rRNA gene sequence analysis showed that strain BSSL-BM10T fell phylogenetically within the radiation comprising type strains of Devosia species. The 16S rRNA gene sequence of strain BSSL-BM10T shared sequence similarities of 98.2% with the type strain of D. naphthalenivorans and 93.5-97.7% with type strains of other Devosia species. ANI and dDDH values between strain BSSL-BM10T and type strains of 18 Devosia species were 71.0-78.4% and 18.8-21.5%, respectively. The DNA G + C content of strain BSSL-BM10T was 60.9% based on its genomic sequence data. Strain BSSL-BM10T contained Q-10 as the predominant ubiquinone and 11-methyl C18:1 ω7c, C18:1 ω7c, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), and C16:0 as its major fatty acids. Major polar lipids of strain BSSL-BM10T were phosphatidylglycerol and two unidentified glycolipids. Strain BSSL-BM10T showed distinguishable phenotypic properties with its phylogenetic and genetic distinctiveness separated from recognized Devosia species. Based on data presented in this study, strain BSSL-BM10T should be placed in the genus Devosia. The name Devosia litorisediminis sp. nov. is proposed for strain BSSL-BM10T (= KACC 21633T = NBRC 115152T).

Molecular Properties of ß-Carotene Oxygenases and Their Potential in Industrial Production of Vitamin A and Its Derivatives.

ß-Carotene 15,15'-oxygenase (BCO1) and ß-carotene 9',10'-oxygenase (BCO2) are potential producers of vitamin A derivatives, since they can catalyze the oxidative cleavage of dietary provitamin A carotenoids to retinoids and derivative such as apocarotenal. Retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it, and are essential nutrients for humans and highly valuable in the food and cosmetics industries. ß-carotene oxygenases (BCOs) from various organisms have been overexpressed in heterogeneous bacteria, such as Escherichia coli, and their biochemical properties have been studied. For the industrial production of retinal, there is a need for increased production of a retinal producer and biosynthesis of retinal using biocatalyst systems improved by enzyme engineering. The current review aims to discuss BCOs from animal, plants, and bacteria, and to elaborate on the recent progress in our understanding of their functions, biochemical properties, substrate specificity, and enzyme activities with respect to the production of retinoids in whole-cell conditions. Moreover, we specifically propose ways to integrate BCOs into retinal biosynthetic bacterial systems to improve the performance of retinal production.

Production of 8,11-dihydroxy fatty acids from oleic and palmitoleic acids by Escherichia coli cells expressing variant 6,8-linoleate diol synthases from Penicillium oxalicum.

Recombinant Escherichia coli cells expressing 8,11-linoleate diol synthase (LDS) from Penicillium chrysogenum convert oleic and palmitoleic acids to 8-hydroperoxy-9(Z)-octadecenoic acid (HPOME) and 8-hydroperoxy-9(Z)-hexadecenoic acid (HPHME) only, respectively. However, recombinant E. coli cells expressing Q889A variant 6,8-LDS from Penicillium oxalicum as an 8,11-LDS converted oleic and palmitoleic acids to 8,11-dihydroxy-9(Z)-octadecenoic acid (DiHOME) and 8,11-dihydroxy-9(Z)-hexadecenoic acid (DiHHME), respectively, which were identified using liquid chromatography-tandem mass spectrometry analysis. To select suitable variants for producing these compounds, position 889 of 6,8-LDS from P. oxalicum was substituted with other amino acids, and recombinant E. coli cells expressing Q889L and Q889A variants were chosen as the best biocatalysts for producing 8,11-DiHOME and 8,11-DiHHME, respectively. The optimal conditions for producing 8,11-DiHOME or 8,11-DiHHME using cells expressing Q889L or Q889A variant 6,8-LDS were pH 6.5 and 30 °C with 5% (v/v) dimethyl sulfoxide, 60 g L-1 cells, and 10 g L-1 oleic acid or 7.5 g L-1 palmitoleic acid, respectively. Under these conditions, 10.7 g L-1 8,11-DiHOME and 8.1 g L-1 8,11-DiHHME were produced for 1.5 h with molar yields of 96.4% and 96.2% and productivities of 7.1 and 5.4 g L-1  h-1 , respectively. The molar yields and concentrations of 8,11-DiHOME and 8,11-DiHHME were highest among those of other reported DiHOMEs and DiHHMEs. To the best of our knowledge, this is the first quantitative production of 8,11-DiHOME and 8,11-DiHHME.

In Vitro One-Pot 3-Hydroxypropanal Production from Cheap C1 and C2 Compounds.

One- or two-carbon (C1 or C2) compounds have been considered attractive substrates because they are inexpensive and abundant. Methanol and ethanol are representative C1 and C2 compounds, which can be used as bio-renewable platform feedstocks for the biotechnological production of value-added natural chemicals. Methanol-derived formaldehyde and ethanol-derived acetaldehyde can be converted to 3-hydroxypropanal (3-HPA) via aldol condensation. 3-HPA is used in food preservation and as a precursor for 3-hydroxypropionic acid and 1,3-propanediol that are starting materials for manufacturing biocompatible plastic and polytrimethylene terephthalate. In this study, 3-HPA was biosynthesized from formaldehyde and acetaldehyde using deoxyribose-5-phosphate aldolase from Thermotoga maritima (DERATma) and cloned and expressed in Escherichia coli for 3-HPA production. Under optimum conditions, DERATma produced 7 mM 3-HPA from 25 mM substrate (formaldehyde and acetaldehyde) for 60 min with 520 mg/L/h productivity. To demonstrate the one-pot 3-HPA production from methanol and ethanol, we used methanol dehydrogenase from Lysinibacillus xylanilyticus (MDHLx) and DERATma. One-pot 3-HPA production via aldol condensation of formaldehyde and acetaldehyde from methanol and ethanol, respectively, was investigated under optimized reaction conditions. This is the first report on 3-HPA production from inexpensive alcohol substrates (methanol and ethanol) by cascade reaction using DERATma and MDHLx.

Improved production of deglucosylated platycodin D from saponins from balloon flower leaf by a food-grade enzyme using high hydrostatic pressure.

Platycosides, saponins contained in balloon flower, which have been used as food health supplements for respiratory diseases, have diverse pharmacological effects. Platycosides exhibit better pharmacological activity by hydrolyzing their own sugars. However, to date, there have been no studies on the production of deglucosylated platycodin D suitable for food applications. In this study, Pluszyme 2000P, which was derived from Aspergillus niger, a food-grade microorganism, was used to completely convert platycoside E into deglucosylated platycodin D. For an efficient and economical production of deglucosylated platycodin D, the productivity was improved approximately 2.4 times by application of high hydrostatic pressure and the discarded balloon flower leaf was used as a substrate. As a result, deglucosylated platycodin D was produced with the highest concentration (3.49 mg/mL) and productivity (581.7 mg/L/h) reported so far. Our results contribute to functional saponin production and the related food industries.

Oral Administration of Latilactobacillus sakei ADM14 Improves Lipid Metabolism and Fecal Microbiota Profile Associated With Metabolic Dysfunction in a High-Fat Diet Mouse Model.

Effects of Latilactobacillus sakei ADM14 on changes in lipid metabolism and fecal microbiota composition were studied in high-fat diet (HFD) mouse model. The mice were divided into three groups: normal diet (ND), high-fat diet (HD), and HFD plus L. sakei ADM14 (HDA). Oral administration of L. sakei ADM14 daily for 10weeks decreased body weight gain, fat tissue mass, and liver weight in mice and reduced the size of histologically stained liver adipocytes. In addition, serum total cholesterol, triglycerides, and blood glucose decreased significantly. Latilactobacillus sakei ADM14 regulated the expression of genes related to lipid metabolism in epididymal adipose tissue and liver and induced changes in the composition of fecal microbiota, thereby improving energy harvests and changing metabolic disorder-related taxa. A significant decrease (p<0.05) in the Firmicutes to Bacteroidetes ratio was found in the HDA group compared to the HD group, particularly due to the difference in the relative abundance of the Bacteroidetes between the two groups over 10weeks. Differences in proportions of some taxa reported to have correlation with obesity were also found between HD and HDA groups. These results suggest that L. sakei ADM14 can have a positive effect on metabolic disorders such as obesity and fatty liver through effective regulation of host lipid metabolism and gut microbiota.