Biotransformation of docosahexaenoic acid into 10R,17S-dihydroxydocosahexaenoic acid as protectin DX 10-epimer by serial reactions of arachidonate 8R- and 15S-lipoxygenases.

Protectins, 10,17-dihydroxydocosahexaenoic acids (10,17-DiHDHAs), are belonged to specialized pro-resolving mediators (SPMs). Protectins are generated by polymorphonuclear leukocytes in humans and resolve inflammation and infection in trace amounts. However, the quantitative production of protectin DX 10-epimer (10-epi-PDX, 10R,17S-4Z,7Z,11E,13Z,15E,19Z-DiHDHA) has been not attempted to date. In this study, 10-epi-PDX was quantitatively produced from docosahexaenoic acid (DHA) by serial whole-cell biotransformation of Escherichia coli expressing arachidonate (ARA) 8R-lipoxygenase (8R-LOX) from the coral Plexaura homomalla and E. coli expressing ARA 15S-LOX from the bacterium Archangium violaceum. The optimal bioconversion conditions to produce 10R-hydroxydocosahexaenoic acid (10R-HDHA) and 10-epi-PDX were pH 8.0, 30 °C, 2.0 mM DHA, and 4.0 g/L cells; and pH 8.5, 20 °C, 1.4 mM 10R-HDHA, and 1.0 g/L cells, respectively. Under these optimized conditions, 2.0 mM (657 mg/L) DHA was converted into 1.2 mM (433 mg/L) 10-epi-PDX via 1.4 mM (482 mg/L) 10R-HDHA by the serial whole-cell biotransformation within 90 min, with a molar conversion of 60% and volumetric productivity of 0.8 mM/h (288 mg/L/h). To the best of our knowledge, this is the first quantitative production of 10-epi-PDX. Our results contribute to the efficient biocatalytic synthesis of SPMs.

PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

The Potential Role of Thyroid Hormone Therapy in Neural Progenitor Cell Differentiation and Its Impact on Neurodevelopmental Disorders.

Thyroid hormone (T3) plays a vital role in brain development and its dysregulation can impact behavior, nervous system function, and cognitive development. Large case-cohort studies have associated abnormal maternal T3 during early pregnancy to epilepsy, autism, and attention deficit hyperactivity disorder (ADHD) in children. Recent experimental findings have also shown T3's influence on the fate of neural precursor cells and raise the question of its convergence with embryonic neural progenitors. Our objective was to investigate how T3 treatment affects neuronal development and functionality at the cellular level. In vitro experiments using neural precursor cells (NPCs) measured cell growth and numbers after exposure to varying T3 concentrations. Time points included week 0 (W0) representing NPCs treated with 100 nM T3 for 5 days, and differentiated cortical neurons assessed at weeks 3 (W3), 6 (W6), and 8 (W8). Techniques such as single-cell calcium imaging and whole-cell patch clamp were utilized to evaluate neuronal activity and function. IHC staining detected mature neuron markers, and RNA sequencing enabled molecular profiling. W6 and W8 neurons exhibited higher action potential frequencies, with W6 showing increased peak amplitudes and shortened inter-spike intervals by 50%, indicating enhanced activity. Transcriptomic analysis revealed that W6 T3-treated neurons formed a distinct cluster, suggesting accelerated maturation. Comparison with the whole transcriptome further unveiled a correlation between W6 neurons treated with T3 and neuronal regulatory elements associated with autism and ADHD. These findings provide insights into T3's impact on neuronal development and potential mechanisms of T3 dysregulation and neurodevelopmental disorders.

Efficient biotransformation of docosahexaenoic acid-rich oils into the lipid mediator resolvin D5 by cells expressing 15S-lipoxygenase using a bioreactor.

Resolvin D5 (RvD5), 7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (DHA) is a specialized pro-resolving mediator (SPM) generated in human macrophages. It is implicated in the resolution of inflammation and synthesized using an inefficient chemical process. Here, DHA-enriched oil hydrolysate was prepared from oils by lipase with resin treatment and solvent extraction. The reaction factors on the biotransformation of oil hydrolysate into RvD5 were optimized using Escherichia coli expressing arachidonate double-oxygenating 15S-lipoxygenase. After optimization, the cells converted 5.0 mM (1.64 g/L) DHA in oil hydrolysate into 4.0 mM (1.44 g/L) RvD5 in a bioreactor for 3.0 h, which was 15-fold higher than that in a flask before optimization, and RvD5 with a purity of > 97% was prepared from reaction solution by treatments of resins. This is the first trial for the production of C22-dihydroxy fatty acid using a bioreactor. This study will contribute to the large-scale production of SPMs from oils.

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.

Deletion of TRPC6, an Autism Risk Gene, Induces Hyperexcitability in Cortical Neurons Derived from Human Pluripotent Stem Cells.

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder linked to numerous rare, inherited, and arising de novo genetic variants. ASD often co-occurs with attention-deficit hyperactivity disorder and epilepsy, which are associated with hyperexcitability of neurons. However, the physiological and molecular mechanisms underlying hyperexcitability in ASD remain poorly understood. Transient receptor potential canonical-6 (TRPC6) is a Ca2+-permeable cation channel that regulates store-operated calcium entry (SOCE) and is a candidate risk gene for ASD. Using human pluripotent stem cell (hPSC)-derived cortical neurons, single-cell calcium imaging, and electrophysiological recording, we show that TRPC6 knockout (KO) reduces SOCE signaling and leads to hyperexcitability of neurons by increasing action potential frequency and network burst frequency. Our data provide evidence that reduction of SOCE by TRPC6 KO results in neuronal hyperexcitability, which we hypothesize is an important contributor to the cellular pathophysiology underlying hyperactivity in some ASD.

Production of ginsenoside compound K from American ginseng extract by fed-batch culture of Aspergillus tubingensis.

Compound K (C-K), one of the most bioactive ginsenoside, is produced by hydrolyzing the glycoside moieties of protopanaxadiol (PPD)-type glycosylated ginsenosides in the ginseng extract. To enhance the biotransformation of PPD-type ginsenosides in American ginseng extract (AGE) into C-K, the optimization of the feed type, concentration, and period for the carbon source sucrose and the reactant AGE was performed in fed-batch fermentation of Aspergillus tubingensis using a fermenter. The concentration (3.94 g/L) and productivity (27.4 mg/L/h) of C-K after feed optimization in fed-batch fermentation increased 3.1-fold compared to those (1.29 g/L and 8.96 mg/L/h) in batch fermentation, and a molar conversion of 100% was achieved. To the best of our knowledge, this is the first trial of fed-batch fermentation to convert ginseng extract into deglycosylated ginsenoside and the highest reported C-K concentration and productivity using ginseng extract via fermentation. After ethanol and resin treatments, C-K solids with purities of 59% and 96% were obtained from the fermentation broth as food- and pharmaceutical-grade products, respectively.

Requirement of Cholesterol for Calcium-Dependent Vesicle Fusion by Strengthening Synaptotagmin-1-Induced Membrane Bending.

Cholesterol is essential for neuronal activity and function. Cholesterol depletion in the plasma membrane impairs synaptic transmission. However, the molecular mechanisms by which cholesterol deficiency leads to defects in vesicle fusion remain poorly understood. Here, it is shown that cholesterol is required for Ca2+ -dependent native vesicle fusion using the in vitro reconstitution of fusion and amperometry to monitor exocytosis in chromaffin cells. Purified native vesicles are crucial for the reconstitution of physiological Ca2+ -dependent fusion, because vesicle-mimicking liposomes fail to reproduce the cholesterol effect. Intriguingly, cholesterol has no effect on the membrane binding of synaptotagmin-1, a Ca2+ sensor for ultrafast fusion. Cholesterol strengthens local membrane deformation and bending induced by synaptotagmin-1, thereby lowering the energy barrier for Ca2+ -dependent fusion to occur. The data provide evidence that cholesterol depletion abolishes Ca2+ -dependent vesicle fusion by disrupting synaptotagmin-1-induced membrane bending, and suggests that cholesterol is an essential lipid regulator for Ca2+ -dependent fusion.

Biotransformation of Platycosides, Saponins from Balloon Flower Root, into Bioactive Deglycosylated Platycosides.

Platycosides, saponins from balloon flower root (Platycodi radix), have diverse health benefits, such as antioxidant, anti-inflammatory, anti-tussive, anti-cancer, anti-obesity, anti-diabetes, and whitening activities. Deglycosylated platycosides, which show greater biological effects than glycosylated platycosides, are produced by the hydrolysis of glycoside moieties in glycosylated platycosides. In this review, platycosides are classified according to the chemical structures of the aglycone sapogenins and also divided into natural platycosides, including major, minor, and rare platycosides, depending on the content in Platycodi radix extract and biotransformed platycosides. The biological activities of platycosides are summarized and methods for deglycosylation of saponins, including physical, chemical, and biological methods, are introduced. The biotransformation of glycosylated platycosides into deglycosylated platycosides was described based on the hydrolytic pathways of glycosides, substrate specificity of glycosidases, and specific productivities of deglycosylated platycosides. Methods for producing diverse and/or new deglycosylated platycosides are also proposed.

Production of C20 9S- and C22 11S-hydroxy fatty acids by cells expressing Shewanella hanedai arachidonate 9S-lipoxygenase.

The putative lipoxygenase (LOX) from the proteobacterium Shewanella hanedai was determined to be an 82 kDa monomeric enzyme by SDS-PAGE and gel filtration chromatography analysis. LOX was identified as a single-dioxygenating arachidonate (ARA) 9S-LOX by analyzing ARA-derived bioconversion products using high-performance liquid chromatography with reverse-, normal-, and chiral-phase columns and evaluating kinetic parameters for C20- and C22-polyunsaturated fatty acids (PUFAs). The catalytic efficiency (kcat/Km) values of 9S-LOX from S. hanedai for ARA, eicosapentaenoic acid, and docosahexaenoic acid were 3.1-, 4.1-, and 2.5-fold higher, respectively, than those only reported 9S-LOX from Sphingopyxis macrogoltabida with double-dioxygenating activity. To promote the production of C20 9S- and C22 11S-hydroxy fatty acids (HFAs) using Escherichia coli expressing 9S-LOX from S. hanedai, bioconversion conditions, including temperature, pH, solvent type and its concentration, concentrations of cells, and substrate, were optimized to 25 °C, pH 8.5, 6% (v/v) dimethyl sulfoxide, 0.2 g/l cells, and 7 mM ARA as substrate in a 500 ml-Erlenmeyer baffled flask with 50 ml reaction solution with agitation at 200 rpm in the presence of 10 mM cysteine as a reduction agent, respectively. Under these conditions, 6.4 mM 9S-hydroxyeicosatetraenoic acid, 6.2 mM 9S-hydroxyeicosapentaenoic acid, and 5.9 mM 11S-hydroxydocosahexaenoic acid were produced in 30 min, 40 min, and 60 min with specific productivities of 1067 µmol/min/g, 775 µmol/min/g, and 492 µmol/min/g, volumetric productivities of 213 µM/min, 155 µM/min, and 98 µM/min, and conversion yields of 91.4%, 88.6%, and 84.3%, respectively. To date, these are the highest specific productivities reported for the bioconversion of C20- and C22-PUFAs into HFAs. KEY POINTS: • Lipoxygenase from Shewanella hanedai was identified as arachidonate 9S-lipoxygenase • Optimization led to increased production of C20 9S- and C22 11S-hydroxy fatty acids • We reported the highest specific productivities of C20- and C22-hydroxy fatty acids.

Characterization of Arachidonate 5S-Lipoxygenase from Danio rerio with High Activity for the Production of 5S- and 7S-Hydroxy Polyunsaturated Fatty Acids.

A recombinant putative lipoxygenase (LOX) from Danio rerio (zebrafish), ALOX3c protein with 6-histidine tag, was purified using affinity chromatography, with a specific activity of 17.2 U mg-1 for arachidonic acid (AA). The molecular mass of the native ALOX3c was 156 kDa composed of a 78-kDa dimer by gel-filtration chromatography. The product obtained from the conversion of AA was identified as 5S-hydroxyeicosatetraenoic acid (5S-HETE) by HPLC and LC-MS/MS analyses. The specific activity and catalytic efficiency of the LOX from D. rerio for polyunsaturated fatty acids (PUFAs) followed the order AA (17.2 U mg-1, 1.96 s-1 µM-1) > docosahexaenoic acid (DHA, 13.6 U mg-1, 0.91 s-1 µM-1) > eicosapentaenoic acid (EPA, 10.5 U mg-1, 0.65 s-1 µM-1) and these values for AA were the highest among the 5S-LOXs reported to date. Based on identified products and substrate specificity, the enzyme is an AA 5S-LOX. The enzyme exhibited the maximal activity at pH 8.0 and 20 °C with 0.1 mM Zn2+ in the presence of 10 mM cysteine. Under these reaction conditions, 6.88 U mL-1 D. rerio 5S-LOX converted 1.0 mM of AA, EPA, and DHA to 0.91 mM 5S-HETE, 0.72 mM 5S-hydroxyeicosapentaenoic acid (5S-HEPE), and 0.68 mM 7S-hydroxydocosahexaenoic acid (7S-HDHA) in 25, 30, and 25 min, corresponding to molar conversion rates of 91, 72, and 68% and productivities of 2.18, 1.44, and 1.63 mM h-1, respectively. To the best of our knowledge, this study is the first to describe the bioconversion into 5S-HETE, 5S-HEPE, and 7S-HDHA for the application of biotechnological production.

Characterization of a loss-of-function NSF attachment protein beta mutation in monozygotic triplets affected with epilepsy and autism using cortical neurons from proband-derived and CRISPR-corrected induced pluripotent stem cell lines.

We investigated whether a homozygous recessive genetic variant of NSF attachment protein beta (NAPB) gene inherited by monozygotic triplets contributed to their phenotype of early-onset epilepsy and autism. Induced pluripotent stem cell (iPSC) lines were generated from all three probands and both parents. The NAPB genetic variation was corrected in iPSC lines from two probands by CRISPR/Cas9 gene editing. Cortical neurons were produced by directed, in vitro differentiation from all iPSC lines. These cell line-derived neurons enabled us to determine that the genetic variation in the probands causes exon skipping and complete absence of NAPB protein. Electrophysiological and transcriptomic comparisons of cortical neurons derived from parents and probands cell lines indicate that loss of NAPB function contributes to alterations in neuronal functions and likely contributed to the impaired neurodevelopment of the triplets.

A rigorous in silico genomic interrogation at 1p13.3 reveals 16 autosomal dominant candidate genes in syndromic neurodevelopmental disorders.

Genome-wide chromosomal microarray is extensively used to detect copy number variations (CNVs), which can diagnose microdeletion and microduplication syndromes. These small unbalanced chromosomal structural rearrangements ranging from 1 kb to 10 Mb comprise up to 15% of human mutations leading to monogenic or contiguous genomic disorders. Albeit rare, CNVs at 1p13.3 cause a variety of neurodevelopmental disorders (NDDs) including development delay (DD), intellectual disability (ID), autism, epilepsy, and craniofacial anomalies (CFA). Most of the 1p13.3 CNV cases reported in the pre-microarray era encompassed a large number of genes and lacked the demarcating genomic coordinates, hampering the discovery of positional candidate genes within the boundaries. In this study, we present four subjects with 1p13.3 microdeletions displaying DD, ID, autism, epilepsy, and CFA. In silico comparative genomic mapping with three previously reported subjects with CNVs and 22 unreported DECIPHER CNV cases has resulted in the identification of four different sub-genomic loci harboring five positional candidate genes for DD, ID, and CFA at 1p13.3. Most of these genes have pathogenic variants reported, and their interacting genes are involved in NDDs. RT-qPCR in various human tissues revealed a high expression pattern in the brain and fetal brain, supporting their functional roles in NDDs. Interrogation of variant databases and interacting protein partners led to the identification of another set of 11 potential candidate genes, which might have been dysregulated by the position effect of these CNVs at 1p13.3. Our studies define 1p13.3 as a genomic region harboring 16 NDD candidate genes and underscore the critical roles of small CNVs in in silico comparative genomic mapping for disease gene discovery. Our candidate genes will help accelerate the isolation of pathogenic heterozygous variants from exome/genome sequencing (ES/GS) databases.

Biotransformation of C20- and C22-polyunsaturated fatty acids to 11S- and 13S-hydroxy fatty acids by Escherichia coli expressing 11S-lipoxygenase from Enhygromyxa salina.

PURPOSE: Peroxidation and reduction of 11S- and 13S-positions on C20 and C22 polyunsaturated fatty acids (PUFAs) by Escherichia coli expressing highly active arachidonate (ARA) 11S-lipoxygenase (11S-LOX) from Enhygromyxa salina with the reducing agent cysteine. RESULTS: The specific activity and catalytic efficiency of ARA 11S-LOX from E. salina were 4.1- and 91-fold higher than those of only reported ARA 11S-LOX from Myxococcus xanthus, respectively. The hydroxy fatty acids (HFAs) obtained by the biotransformation of ARA, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexanoic acid (DHA) by Escherichia coli expressing 11S-LOX from E. salina in the presence of cysteine were identified as 11S-hydroxyeicosatetraenoic acid (11S-HETE), 11S-hydroxyeicosapentaenoic acid (11S-HEPE), 13S-hydroxydocosapentaenoic acid (13S-HDPA), and 13S-hydroxydocosahexaenoic acid (13S-HDHA), respectively. The recombinant cells converted 3 mM of ARA, EPA, DPA, and DHA into 2.9 mM of 11S-HETE, 2.4 mM 11S-HEPE, 1. 9 mM 13S-HDPA, and 2.2 mM 13S-HDHA in 60, 80, 120, and 120 min, corresponding to productivities of 72.5, 40.4, 18.5, and 22.4 µM min-1 and conversion yields of 96.7, 80.0, 62.3, and 74.6%, respectively. CONCLUSIONS: We report the highest concentrations, conversion yields, and productivities of 11S- and 13S-hydroxy fatty acids from C20- and C22-PUFAs achieved via E. coli expressing highly active E. salina 11S-LOX.

Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers.

Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders.

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.

Increased Production of Ginsenoside Compound K by Optimizing the Feeding of American Ginseng Extract during Fermentation by Aspergillus tubingensis.

The ginsenoside compound K (C-K) is widely used in traditional medicines, nutritional supplements, and cosmetics owing to its diverse pharmacological activities. Although many studies on C-K production have been conducted, fermentation is reported to produce C-K with low concentration and productivity. In the present study, addition of an inducer and optimization of the carbon and nitrogen sources in the medium were performed using response surface methodology to increase the C-K production via fermentation by Aspergillus tubingensis, a generally recognized as safe fungus. The optimized inducer and carbon and nitrogen sources were 2 g/l rice straw, 10 g/l sucrose, and 10 g/l soy protein concentrate, respectively, and they resulted in a 3.1-fold increase in the concentration and productivity of C-K (0.22 g/l and 1.52 mg/l/h, respectively) compared to those used before optimization without inducer (0.071 g/l and 0.49 mg/l/h, respectively). The feeding methods of American ginseng extract (AGE), including feeding timing, feeding concentration, and feeding frequency, were also optimized. Under the optimized conditions, A. tubingensis produced 3.96 mM (2.47 g/l) C-K at 144 h by feeding two times with 8 g/l AGE at 48 and 60 h, with a productivity of 17.1 mg/l/h. The concentration and productivity of C-K after optimization of feeding methods were 11-fold higher than those before the optimization (0.22 g/l and 1.52 mg/l/h, respectively). Thus, the optimization for the feeding methods of ginseng extract is an efficient strategy to increase C-K production. To our knowledge, this is the highest reported C-K concentration and productivity via fermentation reported so far.

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.

Production of Deglucose-Apiose-Xylosylated Platycosides from Glycosylated Platycosides by Crude Enzyme from Aspergillus tubingensis.

Platycosides, Platycodi radix (Platycodon grandiflorus root) saponins, are used as food supplements and exert diverse pharmacological activities. Deglycosylation of saponins enhances their biological efficacy, and deglycosylated platycosides are produced mainly through enzymatic hydrolysis. However, the types of available deglycosylated platycosides remain limited because of a lack of hydrolyzing enzymes that can act on specific glycosides in glycosylated platycosides. In this study, a crude enzyme from Aspergillus tubingensis converted platycoside E (PE) and polygalacin D3 (PGD3) into deglucose-apiose-xylosylated (deGAX)-platycodin D (PD) and deGAX-polygalacin D (PGD), respectively. The products were identified through LC/MS analysis by specifically hydrolyzing all glucose residues at C-3, and apiose and xylose residues at C-28 of platycoside. The hydrolytic activity of the crude enzyme obtained after the cultivation of the fungus using citrus pectin and corn steep solid as carbon and nitrogen sources, respectively, in culture medium was increased compared with those using other carbon and nitrogen sources. The crude enzyme from A. tubingensis was the most effective in producing deGAX platycoside at pH 5.0 and 60°C. The crude enzyme produced 0.32 mg/ml deGAX-PD and 0.34 mg/ml deGAX-PGD from 1 mg/ml PE and 1 mg/ml PGD3 (at pH 5.0 and 60°C) for 12 and 10 h, with productivities of 32.0 and 42.5 mg/l/h and molar yields of 62.1 and 59.6%, respectively. To the best of our knowledge, this is the first study to produce deGAX platycosides from glycosylated platycosides.

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.