Identification of the Biosynthetic Gene Cluster for Himeic Acid A: A Ubiquitin‐Activating Enzyme (E1) Inhibitor in Aspergillus japonicus MF275

Himeic acid A, which is produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by Agrobacterium‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the him biosynthetic gene cluster for himeic acid in A. japonicus MF275 has been identified.     

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4‐Oxoproline Synthase

X‐type actinomycins (Acms) contain 4‐hydroxyproline (Acm X₀) or 4‐oxoproline (Acm X₂) in their β‐pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4‐hydroxyproline‐ or 4‐oxoproline‐containing Acm halves. In turn, we show—using an artificial Acm half analogue (PPL 1) with proline in its peptide chain—their conversion into the 4‐hydroxyproline‐ and 4‐oxoproline‐containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host‐encoded ferredoxin reductase in a NADH (NADPH)‐dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4′′‐oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.     

Strictly Conserved Residues in Euphorbia tirucalli β‐Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation–π Interaction and CH–π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.     

A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit–Activating Enzyme CahJ

Cahuitamycins are biofilm inhibitors assembled by a convergent nonribosomal peptide synthetase pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Here, the CahJ protein was probed structurally and functionally to guide the formation of new analogues by mutasynthetic studies. This analysis enabled the in vivo production of a new cahuitamycin congener through targeted precursor incorporation.     

Exploiting the Catalytic Diversity of Short‐Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope

Numerous short‐chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH‐dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine‐reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine‐reducing enzymes.     

Antimicrobial nanocomposites and electrospun coatings based on poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) and copper oxide nanoparticles for active packaging and coating applications

Active biodegradable poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHBV) melt mixed nanocomposites and bilayer structures containing copper oxide (CuO) nanoparticles were developed and characterized. The bilayer structures consisted of a bottom layer of compression molded PHBV3 (3% mol valerate) coated with an active electrospun fibers mat made with CuO nanoparticles and PHBV18 (18% valerate) derived from microbial mixed cultures and cheese whey. The results showed that the water vapor permeability increased with the CuO addition while the oxygen barrier properties were slightly enhanced by the addition of 0.05 wt % CuO nanoparticles to nanocomposite films but a negligible effect was registered for the bilayer structures. However, the mechanical properties were modified by the addition of CuO nanoparticles. Interestingly, by incorporating highly dispersed and distributed CuO nanoparticles in a coating by electrospinning, a lower metal oxide loading was required to exhibit significant bactericidal and virucidal performance against the food‐borne pathogens Salmonella enterica, Listeria monocytogenes, and murine norovirus. The biodisintegration tests of the samples under composting conditions showed that even the 0.05% CuO‐coated structures biodegraded within 35 days. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45673.     

Synthesis of C‐Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies‐Based Biosynthetic Pathway

Carminic acid is a C‐glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C‐glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane‐bound enzymes.     

Biosynthesis of essential oils in aromatic plants: A review

In aromatic plants species, biosynthesis of essential oils occurs through two complex natural biochemical pathways involving different enzymatic reactions. Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the universal precursors of essential oil biosynthesis and are produced by the cytosolic enzymatic MVA (mevalonic acid) pathway or by plastidic and enzymatic 1-deoxy-d-xylolose-5-phosphate (DXP) pathway, also called the 2-C-methylerythritol-4-phosphate (MEP) pathway. In the particular plant cell part, prenyl diphosphate synthases condense isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) further to form prenyl diphosphates, which are used as substrates for geranyl diphosphate (GPP; C₁₀) or for fernesyl diphosphate (FPP; C₁₅). Essential oils are final terpenoid products and are formed by a huge group of enzymes known as terpene synthases (TPS). Essential oils are important secondary metabolites of plants and have been used not only in different industries but also in ethnobotanical medicines for centuries. Hence, considerable research has been undertaken to understand the essential oil biosynthetic pathways. This review will be a valuable source of information in the field of natural products, as we give detailed insights about biosynthesis of essential oils in plants and thus indicate also new unexplored horizons for further research.     

Artificial reconstruction of two cryptic angucycline antibiotic biosynthetic pathways

Genome-sequencing projects have revealed that Streptomyces bacteria have the genetic potential to produce considerably larger numbers of natural products than can be observed under standard laboratory conditions. Cryptic angucycline-type aromatic polyketide gene clusters are particularly abundant. Sequencing of two such clusters from Streptomyces sp. PGA64 and H021 revealed the presence of several open reading frames that could be involved in processing the basic angucyclic carbon skeleton. The pga gene cluster contains one putative FAD-dependant monooxygenase (pgaE) and a putatively bifunctional monooxygenase/short chain alcohol reductase (pgaM), whereas the cab cluster contains two similar monooxygenases (cabE and cabM) and an independent reductase (cabV). In this study we have reconstructed the biosynthetic pathways for aglycone synthesis by cloning and sequentially expressing the angucycline tailoring genes with genes required for the synthesis of the unmodified angucycline metabolite-UWM6-in Streptomyces lividans TK24. The expression studies unequivocally showed that, after the production of UWM6, the pathways proceed through the action of the similar monooxygenases PgaE and CabE, followed by reactions catalysed by PgaM and CabMV. Analysis of the metabolites produced revealed that addition of pgaE and cabE genes directs both pathways to a known shunt product, rabelomycin, whereas expression of all genes from a given pathway results in the production of the novel angucycline metabolites gaudimycin A and B. However, one of the end products is most probably further modified by endogenous S. lividans TK24 enzymes. These experiments demonstrate that genes that are either inactive or cryptic in their native host can be used as biosynthetic tools to generate new compounds.