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Abstract 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. Graphical Abstract

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.

In the human body, docosahexaenoic acid (DHA) contained in fish oil is converted to trace amounts of specialized pro-resolving mediators (SPMs) as the principal bioactive metabolites for their pharmacological effects. Protectin Dx (PDX), an SPM, is an important medicinal compound with biological activities such as modulation of endogenous antioxidant systems, inflammation pro-resolving action, and inhibition of influenza virus replication. Although it can be biotechnologically synthesized from DHA, it has not yet been produced quantitatively. Here, we found that 15S-lipoxygenase from Burkholderia thailandensis (BT 15SLOX) converted 10S-hydroxydocosahexaenoic acid (10S-HDHA) to PDX using enzymatic reactions, which was confirmed by LC-MS/MS and NMR analyses. Thus, whole-cell reactions of Escherichia coli cells expressing BT 15SLOX were performed in flasks to produce PDX from lipase-treated DHA-enriched fish oil along with E. coli cells expressing Mus musculus (mouse) 8S-lipoxygenase (MO 8SLOX) that converted DHA to 10S-HDHA. First, 1 mM DHA (DHA-enriched fish oil hydrolysate, DFOH) was obtained from 455 mg/L DHA-enriched fish oil by lipase for 1 h. Second, E. coli cells expressing MO 8SLOX converted 1 mM DHA in DFOH to 0.43 mM 10S-HDHA for 6 h. Finally, E. coli cells expressing BT 15SLOX converted 0.43 mM 10S-HDHA in MO 8SLOX-treated DFOH to 0.30 mM (108 mg/L) PDX for 5 h. Consequently, DHA-enriched fish oil at 455 mg/L was converted to 108 mg/L PDX after a total of 12 h (conversion yield: 24% (w/w); productivity: 4.5 mg/L/h). This study is the first report on the quantitative production of PDX via biotechnological approaches.

β-Carotene 15,15′-oxygenase (BCO1) and β-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. β-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.

d-Tagatose, a functional sweetener, is converted from d-galactose by l-arabinose isomerase, which catalyzes the conversion of l-arabinose to l-ribulose. In this study, the araA gene encoding l-arabinose isomerase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli, and the expressed enzyme was purified and characterized. The purified l-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for d-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 °C in the presence of Mn2+ and relative activity for pentoses and hexoses in the order l-arabinose > d-galactose > l-ribulose > d-xylulose > d-xylose > d-tagatose > d-glucose. The thermal stability of recombinant E. coli cells expressing l-arabinose isomerase from K. pneumoniae was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 °C, and 4 g/L cell concentration using 100 g/L d-galactose with 1 mM Mn2+. Under these conditions, 33.5 g/L d-tagatose was produced from d-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce d-tagatose using whole-cell reactions, extending its industrial application.

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.

Abstract 20(S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because β-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. β-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α-l-arabinopyranoside moiety in compound Y. Therefore, β-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α-l-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of β-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far.

Platycosides are the functional saponins present in balloon flowers that exert diverse biological effects, and which can be further improved by their deglycosylation. Deapiose-xylosylated platycodin D, which is absent in balloon flowers, can be generated only by cytolase PCL5 by acting on platycoside E. To improve cytolase PCL5-catalyzed production of deapiose-xylosylated platycodin D from platycoside E, we explored the use of high hydrostatic pressure (HHP). At an HHP of 150 MPa, the optimal temperature of cytolase PCL5 activity for converting platycoside E into deapiose-xylosylated platycodin D shifted from 50 to 55 °C, and increased the activity and stability of the enzyme by 5- and 4.9-fold, respectively. Under HHP, the enzyme completely converted 1 mM platycoside E into deapiose-xylosylated platycodin D within 4 h, with a 3.75-fold higher productivity than that under atmospheric pressure. Our results suggest that the application of HHP is a potential method for the economical production of platycosides and enzyme-catalyzed biotransformation of functional saponins.

Abstract Ginsenoside compound K has been used as a key nutritional and cosmetic component because of its anti-fatigue and skin anti-aging effects. β-Glycosidase from Sulfolobus solfataricus (SS-BGL) is known as the most efficient enzyme for compound K production. The hydrolytic pathway from ginsenoside Rb1 to compound K via Rd and F2 is the most important because Rb1 is the most abundant component in ginseng extract. However, the enzymatic conversion of ginsenoside Rd to F2 is a limiting step in the hydrolytic pathway because of the relatively low activity for Rd. A V209 residue obtained from error-prone PCR was related to Rd-hydrolyzing activity, and a docking pose showing an interaction with Val209 was selected from numerous docking poses. W361F was obtained by rational design using the docking pose that exhibited 4.2-fold higher activity, 3.7-fold higher catalytic efficiency, and 3.1-fold lower binding energy for Rd than the wild-type enzyme, indicating that W361F compensated for the limiting step. W361F completely converted Rb1 to compound K with a productivity of 843 mg l−1 h−1 in 80 min, and showed also 7.4-fold higher activity for the flavanone, hesperidin, than the wild-type enzyme. Therefore, the W361F variant SS-BGL can be useful for hydrolysis of other glycosides as well as compound K production from Rb1, and semi-rational design is a useful tool for enhancing hydrolytic activity of β-glycosidase.

Significant advances in enzyme discovery, protein and reaction engineering have transformed biocatalysis into a viable technology for the industrial scale manufacturing of chemicals. Multi-enzyme catalysis has emerged as a new frontier for the synthesis of complex chemicals. However, the in vitro operation of multiple enzymes simultaneously in one vessel poses challenges that require new strategies for increasing the operational performance of enzymatic cascade reactions. Chief among those strategies is enzyme co-immobilization. This review will explore how advances in synthetic biology and protein engineering have led to bioinspired co-localization strategies for the scaffolding and compartmentalization of enzymes. Emphasis will be placed on genetically encoded co-localization mechanisms as platforms for future autonomously self-organizing biocatalytic systems. Such genetically programmable systems could be produced by cell factories or emerging cell-free systems. Challenges and opportunities towards self-assembling, multifunctional biocatalytic materials will be discussed.

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.

The phytochemical oxyresveratrol has been shown to exert diverse biological activities including prevention of obesity. However, the exact reason underlying the anti-obese effects of oxyresveratrol is not fully understood. Here, we investigated the effects and mechanism of oxyresveratrol in adipocytes and high-fat diet (HFD)-fed obese mice. Oxyresveratrol suppressed lipid accumulation and expression of adipocyte markers during the adipocyte differentiation of 3T3-L1 and C3H10T1/2 cells. Administration of oxyresveratrol in HFD-fed obese mice prevented body-weight gains, lowered adipose tissue weights, improved lipid profiles, and increased glucose tolerance. The anti-obese effects were linked to increases in energy expenditure and higher rectal temperatures without affecting food intake, fecal lipid content, and physical activity. The increased energy expenditure by oxyresveratrol was concordant with the induction of thermogenic genes including Ucp1, and the reduction of white adipocyte selective genes in adipose tissue. Furthermore, Foxo3a was identified as an oxyresveratrol-induced gene and it mimicked the effects of oxyresveratrol for induction of thermogenic genes and suppression of white adipocyte selective genes, suggesting the role of Foxo3a in oxyresveratrol-mediated anti-obese effects. Taken together, these data show that oxyresveratrol increases energy expenditure through the induction of thermogenic genes in adipose tissue and further implicates oxyresveratrol as an ingredient and Foxo3a as a molecular target for the development of functional foods in obesity and metabolic diseases.

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.

Abstract 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.

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