Sustained silencing peanut allergy by xanthopurpurin is associated with suppression of peripheral and bone marrow IgE-producing B cell.

Introduction: Peanut allergy is an immunoglobulin E (IgE) mediated food allergy. Rubia cordifolia L. (R. cordifolia), a Chinese herbal medicine, protects against peanut-induced anaphylaxis by suppressing IgE production in vivo. This study aims to identify IgE-inhibitory compounds from the water extract of R. cordifolia and investigate the underlying mechanisms using in vitro and in vivo models. Methods: Compounds were isolated from R. cordifolia water extract and their bioactivity on IgE production was assessed using a human myeloma U266 cell line. The purified active compound, xanthopurpurin (XPP), was identified by LC-MS and NMR. Peanut-allergic C3H/HeJ mice were orally administered with or without XPP at 200µg or 400µg per mouse per day for 4 weeks. Serum peanut-specific IgE levels, symptom scores, body temperatures, and plasma histamine levels were measured at challenge. Cytokines in splenocyte cultures were determined by ELISA, and IgE + B cells were analyzed by flow cytometry. Acute and sub-chronic toxicity were evaluated. IL-4 promoter DNA methylation, RNA-Seq, and qPCR analysis were performed to determine the regulatory mechanisms of XPP. Results: XPP significantly and dose-dependently suppressed the IgE production in U266 cells. XPP significantly reduced peanut-specific IgE (>80%, p <0.01), and plasma histamine levels and protected the mice against peanut-allergic reactions in both early and late treatment experiments (p < 0.05, n=9). XPP showed a strong protective effect even 5 weeks after discontinuing the treatment. XPP significantly reduced the IL-4 level without affecting IgG or IgA and IFN-γ production. Flow cytometry data showed that XPP reduced peripheral and bone marrow IgE + B cells compared to the untreated group. XPP increased IL-4 promoter methylation. RNA-Seq and RT-PCR experiments revealed that XPP regulated the gene expression of CCND1, DUSP4, SDC1, ETS1, PTPRC, and IL6R, which are related to plasma cell IgE production. All safety testing results were in the normal range. Conclusions: XPP successfully protected peanut-allergic mice against peanut anaphylaxis by suppressing IgE production. XPP suppresses murine IgE-producing B cell numbers and inhibits IgE production and associated genes in human plasma cells. XPP may be a potential therapy for IgE-mediated food allergy.

Microbial glycosylation of antitubercular agent chlorflavonin.

Flavonoids have shown health-benefiting properties, such as antioxidative and anti-inflammatory activities, and are commonly used as nutraceuticals and pharmaceuticals. Although flavonoids are predominantly identified from plants, several filamentous fungal species have also been reported to produce bioactive flavonoids, including chlorflavonin from Aspergillus candidus, a novel halogenated flavonoid with potent antifungal and antitubercular (anti-TB) activities. Unfortunately, the low water-solubility of this molecule may hinder its bioavailability. Glycosylation is an effective method to enhance the polarity of natural products and alter their physicochemical properties. This work focuses on the development of novel water-soluble chlorflavonin derivatives to combat the threat of drug-resistant tuberculosis. In this study, we first increased the production titer of chlorflavonin in A. candidus NRRL 5214 by optimizing the fermentation and purification processes. Next, chlorflavonin-5-O-ß-d-glucuronopyranoside (1) and chlorflavonin-7-O-4″-O-methyl-ß-d-glucopyranoside (2) were produced from chlorflavonin using Streptomyces chromofuscus ATCC 49982 and Beauveria bassiana ATCC 7159, respectively. Compared to chlorflavonin (4.38 ± 0.54 mg/L in water), the water solubility of the two new glycosides was determined to be 117.86 ± 4.81 mg/L (1) and 124.34 ± 9.13 mg/L (2), respectively. This study provides a promising method to create water-soluble glycosides of chlorflavonin for the development of novel anti-TB drugs.

Berberine-containing natural-medicine with boiled peanut-OIT induces sustained peanut-tolerance associated with distinct microbiota signature.

Background: Gut microbiota influence food allergy. We showed that the natural compound berberine reduces IgE and others reported that BBR alters gut microbiota implying a potential role for microbiota changes in BBR function. Objective: We sought to evaluate an oral Berberine-containing natural medicine with a boiled peanut oral immunotherapy (BNP) regimen as a treatment for food allergy using a murine model and to explore the correlation of treatment-induced changes in gut microbiota with therapeutic outcomes. Methods: Peanut-allergic (PA) mice, orally sensitized with roasted peanut and cholera toxin, received oral BNP or control treatments. PA mice received periodic post-therapy roasted peanut exposures. Anaphylaxis was assessed by visualization of symptoms and measurement of body temperature. Histamine and serum peanut-specific IgE levels were measured by ELISA. Splenic IgE+B cells were assessed by flow cytometry. Fecal pellets were used for sequencing of bacterial 16S rDNA by Illumina MiSeq. Sequencing data were analyzed using built-in analysis platforms. Results: BNP treatment regimen induced long-term tolerance to peanut accompanied by profound and sustained reduction of IgE, symptom scores, plasma histamine, body temperature, and number of IgE+ B cells (p <0.001 vs Sham for all). Significant differences were observed for Firmicutes/Bacteroidetes ratio across treatment groups. Bacterial genera positively correlated with post-challenge histamine and PN-IgE included Lachnospiraceae, Ruminococcaceae, and Hydrogenanaerobacterium (all Firmicutes) while Verrucromicrobiacea. Caproiciproducens, Enterobacteriaceae, and Bacteroidales were negatively correlated. Conclusions: BNP is a promising regimen for food allergy treatment and its benefits in a murine model are associated with a distinct microbiota signature.

Engineered production of bioactive polyphenolic O-glycosides.

Polyphenolic compounds (such as quercetin and resveratrol) possess potential medicinal values due to their various bioactivities, but poor water solubility hinders their health benefits to humankind. Glycosylation is a well-known post-modification method to biosynthesize natural product glycosides with improved hydrophilicity. Glycosylation has profound effects on decreasing toxicity, increasing bioavailability and stability, together with changing bioactivity of polyphenolic compounds. Therefore, polyphenolic glycosides can be used as food additives, therapeutics, and nutraceuticals. Engineered biosynthesis provides an environmentally friendly and cost-effective approach to generate polyphenolic glycosides through the use of various glycosyltransferases (GTs) and sugar biosynthetic enzymes. GTs transfer the sugar moieties from nucleotide-activated diphosphate sugar (NDP-sugar) donors to sugar acceptors such as polyphenolic compounds. In this review, we systematically review and summarize the representative polyphenolic O-glycosides with various bioactivities and their engineered biosynthesis in microbes with different biotechnological strategies. We also review the major routes towards NDP-sugar formation in microbes, which is significant for producing unusual or novel glycosides. Finally, we discuss the trends in NDP-sugar based glycosylation research to promote the development of prodrugs that positively impact human health and wellness.

Inhibition of pathologic immunoglobulin E in food allergy by EBF-2 and active compound berberine associated with immunometabolism regulation.

Introduction: Food allergy is a significant public health problem with limited treatment options. As Food Allergy Herbal Formula 2 (FAHF-2) showed potential as a food allergy treatment, we further developed a purified version named EBF-2 and identified active compounds. We investigated the mechanisms of EBF-2 on IgE-mediated peanut (PN) allergy and its active compound, berberine, on IgE production. Methods: IgE plasma cell line U266 cells were cultured with EBF-2 and FAHF-2, and their effects on IgE production were compared. EBF-2 was evaluated in a murine PN allergy model for its effect on PN-specific IgE production, number of IgE+ plasma cells, and PN anaphylaxis. Effects of berberine on IgE production, the expression of transcription factors, and mitochondrial glucose metabolism in U266 cells were evaluated. Results: EBF-2 dose-dependently suppressed IgE production and was over 16 times more potent than FAHF-2 in IgE suppression in U266 cells. EBF-2 significantly suppressed PN-specific IgE production (70%, p<0.001) and the number of IgE-producing plasma cells in PN allergic mice, accompanied by 100% inhibition of PN-induced anaphylaxis and plasma histamine release (p<0.001) without affecting IgG1 or IgG2a production. Berberine markedly suppressed IgE production, which was associated with suppression of XBP1, BLIMP1, and STAT6 transcription factors and a reduced rate of mitochondrial oxidation in an IgE-producing plasma cell line. Conclusions: EBF-2 and its active compound berberine are potent IgE suppressors, associated with cellular regulation of immunometabolism on IgE plasma cells, and may be a potential therapy for IgE-mediated food allergy and other allergic disorders.

Genus Tetrastigma: A review of its folk uses, phytochemistry and pharmacology.

The genus Tetrastigma belongs to the Vitaceae family and contains over 100 species. This paper reviewed folk uses, chemical constituents, pharmacological activities, and clinical applications of the medicinal plants in the genus Tetrastigma. In addition, the paper also discussed the current problems for the further studies. Up to now, more than 240 compounds were reported from the genus Tetrastigma, covering 74 flavonoids, 14 terpenoids, 19 steroids, 21 phenylpropanoids, 14 alkaloids and others constituents. Among them, flavonoids are the major and the characteristic chemical constituents in this genus. Modern pharmacological studies and clinical practice showed that the extracts and chemical constituents of Tetrastigma species possessed wide pharmacological activities including antitumor, antioxidative, hepatoprotective, antiviral, anti-inflammatory, and analgesic activities. The information summarized in this paper provides valuable clues for new drug discovery and an incentive to expand the research of genus Tetrastigma.

Creating diverse glycosides of 2'-hydroxyflavone through microbial glycosylation.

Four new 2'-hydroxyflavone glycosides, namely hydroxyflavone-2'-O-ß-D-glucuronide (1), hydroxyflavone-2'-O-α-L-rhamnoside (2), hydroxyflavone-2'-O-ß-D-glucoside (3), and hydroxyflavone-2'-O-4″-O-methyl-ß-D-glucoside (4), were biosynthesized through microbial glycosylation using Streptomyces coeruleorubidus NRRL B-2569, Streptomyces toxytricini NRRL 15443, Escherichia coli BL21(DE3)/pWZ8, and Beauveria bassiana ATCC 7159, respectively. Compounds 1-4 were structurally characterized through extensive analysis of 1D and 2D NMR spectroscopic data. The water solubility of glycosylated products 1-4 were enhanced by 7 to 15 times compared to the substrate 2'-hydroxyflavone. Moreover, antioxidant assays revealed that compounds 1 and 2 exhibited stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than the substrate, decreasing the logIC50 by 68.7% and 80.7%, respectively. Therefore, this research provides several effective biocatalysts that can be used for structural modification of flavonoids for enhanced water solubility and biological activities.

Time-dependent dual beneficial modulation of interferon-γ, interleukin 5, and Treg cytokines in asthma patient peripheral blood mononuclear cells by ganoderic acid B.

Th2 cytokines play a dominant role in the pathogenesis of allergic asthma. Interferon gamma (IFN-γ), a Th1 cytokine, links to therapeutic mechanisms of allergic asthma. Interleukin (IL)-10, a regulatory cytokine, is involved in the induction of immune tolerance. We previously demonstrated that Anti-Asthma Simplified Herbal Medicine Intervention (ASHMI) suppressed Th2 and increased IFN-γ in patients with asthma and in animal models, but its bioactive compound is unknown. Ganoderic acid beta (GAB) was isolated from Ganoderma lucidum (one herb in ASHMI). Human peripheral blood mononuclear cells (PBMCs) from adult patients with asthma were cultured with GAB or dexamethasone (Dex) in the presence of environmental allergens. The cytokine levels of IL-10, IFN-γ, IL-5, transcription factors T-bet, Foxp-3, and GATA3 were measured. Following 3-day culture, GAB, but not Dex, significantly increased IL-10 and IFN-γ levels by allergic patients' PBMCs. Following 6-day treatment, GAB inhibited IL-5 production, but IL-10 and IFN-γ remained high. Dex suppressed production of all three cytokines. GAB suppressed GATA3 and maintained Foxp-3 and T-bet gene expression, while Dex significantly suppressed GATA3 and T-bet expression. GAB simultaneously increased IL-10, IFN-γ associated with induction of T-bet and Foxp3, while suppressing IL-5, which was associated with suppression of GATA3, demonstrating unique beneficial cytokine modulatory effect, which distinguishes from Dex's overall suppression.

Identification of a novel glucuronyltransferase from Streptomyces chromofuscus ATCC 49982 for natural product glucuronidation.

Glycosylation is an effective way to increase the polarity of natural products. UDP-glucuronyltransferases (UGTs) are commonly observed and extensively studied in phase II drug metabolism. However, UGTs in microorganisms are not well studied, which hampered the utilization of this type of enzyme in microbial glucuronidation of natural products. Screening of five actinomycete strains showed that Streptomyces chromofuscus ATCC 49982 can convert diverse plant polyphenols into more polar products, which were characterized as various glucuronides based on their spectral data. Analysis of the genome of this strain revealed a putative glucuronidation gene cluster that contains a UGT gene (gcaC) and two UDP-glucuronic acid biosynthetic genes (gcaB and gcaD). The gcaC gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Incubation of the purified enzyme with resveratrol and UDP-glucuronic acid led to the production of resveratrol-4'-O-ß-D-glucuronide and resveratrol-3-O-ß-D-glucuronide, allowing GcaC to be characterized as a flexible UGT. The optimal in vitro reaction pH and temperature for GcaC are 7.5 and 30 °C, respectively. Its activity can be stimulated by Ca2+, Mg2+, and Mn2+, whereas Zn2+, Cu2+, and Fe2+ showed inhibitory effects. Furthermore, GcaC has a broad substrate specificity, which can glucuronidate various substrates besides resveratrol, including quercetin, ferulic acid, vanillic acid, curcumin, vanillin, chrysin, zearalenone, and apigenin. The titers of resveratrol-4'-O-ß-D-glucuronide and resveratrol-3-O-ß-D-glucuronide in E. coli-GcaC were 78.381 ± 0.366 mg/L and 14.991 ± 0.248 mg/L from 114.125 mg/L resveratrol within 3 h. Therefore, this work provides an effective way to produce glucuronides of resveratrol and other health-benefitting natural products. KEY POINTS: • A novel versatile microbial UDP-glucuronyltransferase was discovered and characterized from Streptomyces chromofuscus ATCC 49982. • The UDP-glucuronyltransferase was expressed in Escherichia coli and can convert resveratrol into two glucuronides both in vitro and in vivo. • The UDP-glucuronyltransferase has a highly flexible substrate specificity and is an effective tool to prepare mono- or diglucuronides of bioactive molecules.

A highly versatile fungal glucosyltransferase for specific production of quercetin-7-O-ß-D-glucoside and quercetin-3-O-ß-D-glucoside in different hosts.

Glycosylation is an effective way to improve the water solubility of natural products. In this work, a novel glycosyltransferase gene (BbGT) was discovered from Beauveria bassiana ATCC 7159 and heterologously expressed in Escherichia coli. The purified enzyme was functionally characterized through in vitro enzymatic reactions as a UDP-glucosyltransferase, converting quercetin to five monoglucosylated and one diglucosylated products. The optimal pH and temperature for BbGT are 35 ℃ and 8.0, respectively. The activity of BbGT was stimulated by Ca2+, Mg2+, and Mn2+, but inhibited by Zn2+. BbGT enzyme is flexible and can glycosylate a variety of substrates such as curcumin, resveratrol, and zearalenone. The enzyme was also expressed in other microbial hosts including Saccharomyces cerevisiae, Pseudomonas putida, and Pichia pastoris. Interestingly, the major glycosylation product of quercetin in E. coli, P. putida, and P. pastoris was quercetin-7-O-ß-D-glucoside, while the enzyme dominantly produced quercetin-3-O-ß-D-glucoside in S. cerevisiae. The BbGT-harboring E. coli and S. cerevisiae strains were used as whole-cell biocatalysts to specifically produce the two valuable quercetin glucosides, respectively. The titer of quercetin-7-O-ß-D-glucosides was 0.34 ± 0.02 mM from 0.83 mM quercetin in 24 h by BbGT-harboring E. coli. The yield of quercetin-3-O-ß-D-glucoside was 0.22 ± 0.02 mM from 0.41 mM quercetin in 12 h by BbGT-harboring S. cerevisiae. This work thus provides an efficient way to produce two valuable quercetin glucosides through the expression of a versatile glucosyltransferase in different hosts. KEY POINTS: • A highly versatile glucosyltransferase was identified from B. bassiana ATCC 7159. • BbGT converts quercetin to five mono- and one di-glucosylated derivatives in vitro. • Different quercetin glucosides were produced by BbGT in E. coli and S. cerevisiae.

Formononetin isolated from Sophorae flavescentis inhibits B cell-IgE production by regulating ER-stress transcription factor XBP-1.

Rationale: IgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI (anti-asthma herbal medicine intervention) suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown. Objective: We sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production. Methods: The compounds in Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR. Results: A single compound identified as formononetin was isolated from Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2-20 µg/ml (p < 0.05-0.001 vs. untreated cells) with an IC50 value of 3.43 µg/ml. There was no cytotoxicity at any tested concentration. Formononetin significantly decreased XBP-1, and IgE-heavy chain gene expression compared with untreated cells (p < 0.001). Conclusion: Formononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases.

A Computational Metabolic Model for Engineered Production of Resveratrol in Escherichia coli.

Although engineered microbial production of natural compounds provides a promising alternative method to plant production and extraction, the process can be inefficient and ineffective in terms of time and cost. To render microbial systems profitable and viable, the process must be optimized to produce as much product as possible. To this end, this work illustrates the construction of a new probabilistic computational model to simulate the microbial production of a well-known cardioprotective molecule, resveratrol, and the implementation of the model to enhance the yield of the product in Escherichia coli. This model identified stilbene synthase as the limiting enzyme and informed the effects on changes in concentration and source of this enzyme. These parameters, when employed in a laboratory system, were able to improve the titer from 62.472 mg/L to 172.799 mg/L, demonstrating the model's ability to produce a useful simulation of a dynamic microbial resveratrol production system.

Identification and Characterization of an Efficient Phenylalanine Ammonia-Lyase from Photorhabdus luminescens.

A putative aromatic amino acid ammonia-lyase gene (named Pl-pal) was discovered in Photorhabdus luminescens DSM 3368. BLAST and phylogenetic analyses predicted that this enzyme is a histidine ammonia-lyase, whereas sequence alignment suggested that it is more likely a phenylalanine ammonia-lyase (PAL). This gene was amplified from P. luminescens and expressed in Escherichia coli BL21(DE3). The function of Pl-PAL (58 kDa) was characterized by in vitro enzymatic reactions with L-phenylalanine (L-Phe), L-tyrosine (L-Tyr), L-histidine (L-His), and L-tryptophan (L-Trp). Pl-PAL can convert L-Phe and L-Tyr to trans-cinnamic acid and p-coumaric acid, respectively, but had no function on L-His and L-Trp. The optimum temperature and pH were determined to be 40 °C and 11.0, respectively. Under the optimal conditions, Pl-PAL had a kcat/Km value of 0.52 s-1 mM-1 with L-Phe as the substrate, while only 0.013 s-1 mM-1 for L-Tyr. Therefore, the primary function of Pl-PAL was determined to be PAL. The Pl-pal-harboring E. coli strain was used as a whole-cell biocatalyst to produce trans-cinnamic acid from L-Phe. The overall molar conversion rate and productivity were 65.98% and 228.10 mg L-1 h-1, respectively, after the cells were repeatedly utilized 7 times. This work thus provides a promising strain for industrial production of trans-cinnamic acid.

Discovery of a novel analogue of FR901533 and the corresponding biosynthetic gene cluster from Streptosporangium roseum No. 79089.

FR901533 (1, also known as WS79089B), WS79089A (2), and WS79089C (3) are polycyclic aromatic natural products with promising inhibitory activity to endothelin-converting enzymes. In this work, we isolated five tridecaketide products from Streptosporangium roseum No. 79089, including 1-3, benaphthamycin (4) and a novel FR901533 analogue (5). The structure of 5 was characterized based on spectroscopic data. Compared with the major product 2, the new compound 5 has an additional hydroxyl group at C-12 and an extra methyl group at the 13-OH. The configuration of C-19 of these compounds was determined to be R using Mosher's method. A putative biosynthetic gene cluster for compounds 1-5 was discovered by analyzing the genome of S. roseum No. 79089. This 38.6-kb gene cluster contains 38 open reading frames, including a minimal polyketide synthase (wsaA-C), an aromatase (wsaD), three cyclases (wsaE, F, and W), and a series of tailoring enzymes such as monooxygenases (wsaO1-O7) and methyltransferases (wsaM1 and M2). Disruption of the ketosynthase gene (wsaA) in this gene cluster abolished the production of 1-5, confirming that this gene cluster is indeed responsible for the biosynthesis of 1-5. A type II polyketide biosynthetic pathway was proposed for this group of natural endothelin-converting enzyme inhibitors. KEY POINTS: • Five aromatic tridecaketides were isolated from Streptosporangium roseum No. 79089. • A novel FR901533 analogue, 12-hydroxy-13-O-methyl-WS79089A, was characterized. • The absolute configuration of C-19 of FR901533 and analogues was determined. • The biosynthetic gene cluster of FR901533 and analogues was discovered.

Characterization of two new aromatic amino acid lyases from actinomycetes for highly efficient production of p-coumaric acid.

p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceuticals. It can be synthesized from deamination of L-tyrosine by tyrosine ammonia lyase (TAL). In this work, we discovered two aromatic amino acid lyase genes, Sas-tal and Sts-tal, from Saccharothrix sp. NRRL B-16348 and Streptomyces sp. NRRL F-4489, respectively, and expressed them in Escherichia coli BL21(DE3). The two enzymes were functionally characterized as TAL. The optimum reaction temperature for Sas-TAL and Sts-TAL is 55 °C and 50 °C, respectively; while, the optimum pH for both TALs is 11. Sas-TAL had a kcat/Km value of 6.2 µM-1 min-1, while Sts-TAL had a much higher efficiency with a kcat/Km value of 78.3 µM-1 min-1. Both Sts-TAL and Sas-TAL can also take L-phenylalanine as the substrate to yield trans-cinnamic acid, and Sas-TAL showed much higher phenylalanine ammonia lyase activity than Sts-TAL. Using E. coli/Sts-TAL as a whole-cell biocatalyst, the productivity of p-CA reached 2.88 ± 0.12 g (L h)-1, which represents the highest efficiency for microbial production of p-CA. Therefore, this work not only reports the identification of two new TALs from actinomycetes, but also provides an efficient way to produce the industrially valuable material p-CA.

Efficient production of gamma-aminobutyric acid by engineered Saccharomyces cerevisiae with glutamate decarboxylases from Streptomyces.

Gamma-aminobutyric acid (GABA) is an industrially valuable natural product. This study was aimed to establish an efficient food-grade production process of GABA by engineering Saccharomyces cerevisiae that is generally recognized as safe (GRAS). GABA can be produced by catalytic decarboxylation of l-glutamate (l-Glu) by glutamate decarboxylase (GAD, EC4.1.1.15). Two GADs, SsGAD from Streptomyces sp. MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982, were heterologously expressed in S. cerevisiae BJ5464. The engineered yeast strains were used as whole-cell biocatalysts for GABA production. S. cerevisiae BJ5464/SsGAD exhibited significantly higher efficient catalytic activity than that of S. cerevisiae BJ5464/ScGAD. The optimal bioconversion system consisted of a cell density of OD600 30, 0.1 M l-Glu, and 0.28 mM pyridoxal phosphate in 0.2 M Na2 HPO4 -citric acid buffer with pH 5.4, and the reactions were performed at 50 °C for 12 H. S. cerevisiae BJ5464/SsGAD cells can be reused, and the accumulated GABA titer reached 62.6 g/L after 10 batches with an overall molar conversion rate of 60.8 mol%. This work thus provides an effective production process of GABA using engineered yeast for food and pharmaceutical applications.

Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase.

BACKGROUND: Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture. RESULTS: The MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and N-desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the N-methyltransferase activity of the MT domain as both the isolated domain (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme. CONCLUSIONS: The embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products.

Discovery and engineering of an endophytic Pseudomonas strain from Taxus chinensis for efficient production of zeaxanthin diglucoside.

BACKGROUND: Endophytic microorganisms are a rich source of bioactive natural products. They are considered as promising biofertilizers and biocontrol agents due to their growth-promoting interactions with the host plants and their bioactive secondary metabolites that can help manage plant pathogens. Identification of new endophytes may lead to the discovery of novel molecules or provide new strains for production of valuable compounds. RESULTS: In this study, we isolated an endophytic bacterium from the leaves of Taxus chinensis, which was identified as Pseudomonas sp. 102515 based on the 16S rRNA gene sequence and physiological characteristics. Analysis of its secondary metabolites revealed that this endophytic strain produces a major product zeaxanthin diglucoside, a promising antioxidant natural product that belongs to the family of carotenoids. A carotenoid (Pscrt) biosynthetic gene cluster was amplified from this strain, and the functions of PsCrtI and PsCrtY in the biosynthesis of zeaxanthin diglucoside were characterized in Escherichia coli BL21(DE3). The entire Pscrt biosynthetic gene cluster was successfully reconstituted in E. coli BL21(DE3) and Pseudomonas putida KT2440. The production of zeaxanthin diglucoside in Pseudomonas sp. 102515 was improved through the optimization of fermentation conditions such as medium, cultivation temperature and culture time. The highest yield under the optimized conditions reached 206 mg/L. The engineered strain of P. putida KT2440 produced zeaxanthin diglucoside at 121 mg/L in SOC medium supplemented with 0.5% glycerol at 18 °C, while the yield of zeaxanthin diglucoside in E. coli BL21(DE3) was only 2 mg/L. To further enhance the production, we introduced an expression plasmid harboring the Pscrt biosynthetic gene cluster into Pseudomonas sp. 102515. The yield in this engineered strain reached 380 mg/L, 85% higher than the wild type. Through PCR, we also discovered the presence of a turnerbactin biosynthetic gene cluster in Pseudomonas sp. 102515. Because turnerbactin is involved in nitrogen fixation, this endophytic strain might have a role in promoting growth of the host plant. CONCLUSIONS: We isolated and identified an endophytic strain of Pseudomonas from T. chinensis. A zeaxanthin diglucoside biosynthetic gene cluster was discovered and characterized in this bacterium. Through fermentation and genetic engineering, the engineered strain produced zeaxanthin diglucoside at 380 ± 12 mg/L, representing a promising strain for the production of this antioxidant natural product. Additionally, Pseudomonas sp. 102515 might also be utilized as a plant-promoting strain for agricultural applications.

Identification of new glutamate decarboxylases from Streptomyces for efficient production of γ-aminobutyric acid in engineered Escherichia coli.

BACKGROUND: Gamma (γ)-Aminobutyric acid (GABA) as a bioactive compound is used extensively in functional foods, pharmaceuticals and agro-industry. It can be biosynthesized via decarboxylation of monosodium glutamate (MSG) or L-glutamic acid (L-Glu) by glutamate decarboxylase (GAD; EC4.1.1.15). GADs have been identified from a variety of microbial sources, such as Escherichia coli and lactic acid bacteria. However, no GADs from Streptomyces have been characterized. The present study is aimed to identify new GADs from Streptomyces strains and establish an efficient bioproduction platform for GABA in E. coli using these enzymes. RESULTS: By sequencing and analyzing the genomes of three Streptomyces strains, three putative GADs were discovered, including StGAD from Streptomyces toxytricini NRRL 15443, SsGAD from Streptomyces sp. MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982. The corresponding genes were cloned from these strains and heterologously expressed in E. coli BL21(DE3). The purified GAD proteins showed a similar molecular mass to GadB from E. coli BL21(DE3). The optimal reaction temperature is 37 °C for all three enzymes, while the optimum pH values for StGAD, SsGAD and ScGAD are 5.2, 3.8 and 4.2, respectively. The kinetic parameters including V max , Km, k cat and k cat /Km values were investigated and calculated through in vitro reactions. SsGAD and ScGAD showed high biocatalytic efficiency with k cat /Km values of 0.62 and 1.21 mM- 1·s- 1, respectively. In addition, engineered E. coli strains harboring StGAD, SsGAD and ScGAD were used as whole-cell biocatalysts for production of GABA from L-Glu. E. coli/SsGAD showed the highest capability of GABA production. The cells were repeatedly used for 10 times, with an accumulated yield of 2.771 kg/L and an average molar conversion rate of 67% within 20 h. CONCLUSIONS: Three new GADs have been functionally characterized from Streptomyces, among which two showed higher catalytic efficiency than previously reported GADs. Engineered E. coli harboring SsGAD provides a promising cost-effective bioconversion system for industrial production of GABA.