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-β-D-glucuronopyranoside (1) and chlorflavonin-7-O-4 -O-methyl-β-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

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

Decoding and Reprogramming Fungal Iterative Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases (NRPSs) assemble a large group of structurally and functionally diverse natural products. While the iterative catalytic mechanism of bacterial NRPSs is known, it remains unclear how fungal NRPSs create products of desired length. Here we show that fungal iterative NRPSs adopt an alternate incorporation strategy. Beauvericin and bassianolide synthetases have the same C1-A1-T1-C2-A2-MT-T2a-T2b-C3 domain organization. During catalysis, C3 and C2 take turns to incorporate the two biosynthetic precursors into the growing depsipeptide chain that swings between T1 and T2a/T2b with C3 cyclizing the chain when it reaches the full length. We reconstruct the total biosynthesis of beauvericin in vitro by reacting C2 and C3 with two SNAC-linked precursors and present a domain swapping approach to reprogramming these enzymes for peptides with altered lengths. These findings highlight the difference between bacterial and fungal NRPS mechanisms and provide a framework for the enzymatic synthesis of non-natural nonribosomal peptides

Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases

BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme that also synthesizes beauvericins

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 to 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\u27s 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

An Efficient Process for Co-Production of γ-Aminobutyric Acid and Probiotic \u3ci\u3eBacillus subtilis\u3c/i\u3e Cells

This study was to establish an integrated process for the co-production of γ-aminobutyric acid (GABA) and live probiotics. Six probiotic bacteria were screened and Bacillus subtilis ATCC 6051 showed the highest GABA-producing capacity. The optimal temperature and initial pH value for GABA production in B. subtilis were found to be 30 °C and 8.0, respectively. A variety of carbon and nitrogen sources were tested, and potato starch and peptone were the preferred carbon and nitrogen sources for GABA production, respectively. The concentrations of carbon source, nitrogen source and substrate (sodium L-glutamate) were then optimized using the response surface methodology. The GABA titer and concentration of viable cells of B. subtilis reached 19.74 g/L and 6.0 × 108 cfu/mL at 120 h. The GABA titer represents the highest production of GABA in B. subtilis. This work thus demonstrates a highly efficient co-production process for GABA and probiotic B. subtilis cells

Identification of New Glutamate Decarboxylases from \u3cem\u3eStreptomyces\u3c/em\u3e for Efficient Production of γ-Aminobutyric Acid in Engineered \u3cem\u3eEscherichia coli\u3c/em\u3e

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 Streptomycesstrains, 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. coliBL21(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 Vmax, Km, kcat and kcat/Km values were investigated and calculated through in vitro reactions. SsGAD and ScGAD showed high biocatalytic efficiency with kcat/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

Isolation and Selective Glycosylation of Antisalmonellal Anthraquinones From the Stem Bark of Morinda Lucida Benth. (Rubiaceae)

In this work we report the isolation, identification and antibacterial activity of two anthraquinones, 2-hydroxy-1-methoxyanthraquinone (1) and 2,5-dihydroxy-1-methoxy-6-methoxymethylanthraquinone (2), from the stem bark of Morinda lucida. These two natural products were selectively converted into two new glycosylated derivatives, 2-hydroxy-1-methoxyanthraquinone-4′-O-methyl-2-O-β-d-glucopyranoside (3) and 2,5-dihydroxy-1-methoxy-6-methoxymethylanthraquinone-4′-O-methyl-2-O-β-d-glucopyranoside (4) by the filamentous fungus Beauveria bassiana ATCC 7159. Structure elucidation was accomplished based on the 1D and 2D NMR, IR and mass spectra. The glycosylated compounds 3 and 4 showed higher in vitro antibacterial activity against Salmonella enterica subsp. enterica sérovars Typhimurim (MIC of 8 μg/mL each) than the corresponding aglycons 1 and 2 (MIC of 16 μg/mL and 32 μg/mL, respectively). These results indicated that microbial glycosylation is an effective approach to modify natural products for enhanced biological activities