Biosynthesis of Natural Products

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs, and (ii) how to rapidly connect genes to biosynthesized small molecules. Here we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes co-localized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty ...

Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug mycophenolic acid. The fungal NPs biosynthetic genes are always arranged within one single biosynthetic gene cluster (BGC). However, a rare but fascinating phenomenon that a crosstalk between two separate BGCs is indispensable to some fungal dimeric NPs biosynthesis has attracted increasing attention. The hybridization of two separate BGCs not only increases the structural complexity and chemical diversity of fungal NPs, but also expands the scope of bioactivities. More importantly, the underlying mechanism for this hybridization process is poorly understood and needs further exploration, especially the determination of BGCs for each building block construction and the identification of enzyme(s) catalyzing the two biosynthetic precursors coupling processe...

Background: Umbelliferone, also known as 7-hydroxycoumarin, is a phenolic metabolite found in many familiar plants. Its derivatives have been shown to have various pharmacological and chemo-preventive effects on human health. A uridine diphosphate glycosyltransferase YjiC from Bacillus licheniformis DSM 13, a cytochrome P450BM3 (CYP450 BM3) variant namely mutant 13 (M13) from Bacillus megaterium, and an O-methyltransferase from Streptomyces avermitilis (SaOMT2) were used for modifications of umbelliferone. Results: Three umbelliferone derivatives (esculetin, skimmin, and herniarin) were generated through enzymatic and whole cell catalysis. To improve the efficiencies of biotransformation, different media, incubation time and concentration of substrate were optimized and the production was scaled up using a 3-L fermentor. The maximum yields of esculetin, skimmin, and herniarin were 337.10 μM (67.62%), 995.43 μM (99.54%), and 37.13 μM (37.13%), respectively. The water solubility of esculetin and skimmin were 1.28-folds and 3.98-folds as high as umbelliferone, respectively, whereas herniarin was 1.89-folds less soluble than umbelliferone. Moreover, the antibacterial and anticancer activities of herniarin showed higher than umbelliferone, esculetin and skimmin.

Given the upsurge of new technologies based on nanomaterials, the development of sustainable methods
to obtain functional nanostructures has become an imperative task. In this matter, several recent
researches have shown that the biodegradable natural antioxidants of several plant extracts can be used
simultaneously as reducing and stabilizing agents in the wet chemical synthesis of metallic nanoparticles,
opening new opportunities to design greener synthesis. However, the challenge of these new techniques
is to produce stable colloidal nanoparticles with controlled particle uniformity, size, shape and
aggregation state, in similar manner than the well-established synthetic methods. In the present work,
colloidal metallic silver nanoparticles have been synthesized using silver nitrate and extracts of Illicium
verum (star anise) seeds at room temperature in a facile one-step procedure. The resulting products were
colloidal suspensions of two populations of silver nanoparticles, one of them with particle sizes of few
nanometers and the other with particles of tens of nm. Strikingly, the variation of the AgNO
/extract
weight ratio in the reaction medium yielded to the variation of the spatial distribution of the nanoparticles:
high AgNO
/extract concentration ratios yielded to randomly dispersed particles, whereas for lower
AgNO
3
3
/extract ratios, the biggest particles appeared coated with the finest nanoparticles. This biosynthesized
colloidal system, with controlled particle aggregation states, presents plasmonic and SERS properties
with potential applications in molecular sensors and nanophotonic devices.

In this research the kinetics of cellulose accumulation in growing cotton fibers due to biosynthesis has been studied. The kinetic curve had initial slow stage (up to 15 days after flowering, AF), fast stage (15 to 35 days AF) and final very slow stage (above 35 days AF). To describe such complex kinetics, equation of Avrami-Kolmogorov-Erofeev (AKE) was used: Ct/Cm = 1 – exp [-K (t – to) n ], where Ct is amount of cellulose accumulated in fibers for time t AF; Cm is maximum amount of cellulose in mature cotton fibers; K is effective rate constant; n is effective order of the process; t is time AF and to is induction period. The calculated parameters of AKE-equation are: to= 7 days AF, K= 5.94 x 10-3 and n=1.72. Since n>1, the process of cellulose biosynthesis is not limited by diffusion of monomers. The kinetic curve calculated by AKE-equation coincide with the experimental points, which confirms the adequacy this equation for describing the biosynthesis process of natural cellulose. Structural studies have shown, that crystalline structure of cellulose in immature fibers is low-ordered. However, with increase in duration of cotton maturation, additional crystallization of cellulose occurs.

Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformati...

Phytocannabinoids are the active substances responsible for the medicinal and psychotropic effects of Cannabis sativa. Although the bioactivity of cannabis and its preparations have been known for millennia, several steps in the biosynthetic pathway leading to phytocannabinoids remain unclear. Phytocannabinoids are prenylated resorcylic acids which are formed in specialized plant organs called glandular trichomes. Following the analysis of a pre-generated cannabis trichome cDNA library, a type III polyketide synthase (tetraketide synthase; TKS) was identified and assayed, yielding three major compounds, hexanoyl triacetic acid lactone (HTAL), pentyl diacetic acid lactone (PDAL), and olivetol, yet no resorcylic acid was detected. This lack of resorcylic acid in enzyme assays has instigated the characterization of TKS and a search for putative cyclases in the cannabis trichome cDNA library, and involved protein pulldown, co-immunoprecipitation, and co-assay experiments. These experiments led to the discovery of a novel polyketide cyclase protein named olivetolic acid cyclase (OAC) responsible for the proper cyclization of a polyketide intermediate produced by TKS. This thesis shows that TKS assays conducted with OAC produce olivetolic acid (OA), an intermediate required during the biosynthesis of cannabinoids. The TKS/OAC spatial relationship was also investigated following the creation of fluorescent fusion proteins which show that the enzymes co-localized in vivo when viewed with confocal microscopy. Furthermore, yeast two-hybrid assays using TKS and OAC were performed to establish whether the enzymes physically interact. Finally, an attempt to determine the responsible amino acids involved in OAC’s mechanism was conducted by comparing the activity of single point OAC mutants with the wild-type OAC. Based on the available data, mechanisms for the production of HTAL, PDAL, olivetol, and OA are proposed.

Two new siderophores belonging to the hydroxamate class, Legonoxamine A (1) and B (2) have been isolated from the soil bacterium, Streptomyces sp. MA37, together with one known compound, desferriox-amine B (3). Their structures were elucidated based on spectroscopic methods including 1D, 2D NMR, MS, as well as by comparison with the relevant literatures. To our knowledge, this is the first report describing a siderophore containing the N-hydroxyl phenylacetyl cadaverine (HPAC) moiety in the structure. Based on bioinformatics analysis and previous knowledge of the biosynthesis of the hydroxamate-type siderophore, the biosynthetic gene cluster (lgo) responsible for the production of 1-3 was identified in the annotated genome of the producing strain. The supplementation of phenylacetate and benzoate analogues with meta substitution into the cultures of Streptomyces sp. MA37 resulted in the production of new legonoxamine A derivatives as observed in LC-HR-ESIMS, suggesting that the legonoxamine biosynthetic pathway has a good degree of natural flexibility of accepting unnatural precursors with different functional groups.

Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or “orphaned” secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs, and (ii) how to rapidly connect genes to biosynthesized small molecules. Here we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes co-localized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and auto-resistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized, but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.

Resumen Context: The genus Kniphofia Moench, with species close to 70, is traditionally used to treat wide ranges of ailments including menstrual pains, infertility, abdominal cramps, wounds, malaria, chest complaint and hepatitis B. Kniphofia isoetifolia, an endemic species in Ethiopia, is known for wounds healing applications in Southern Ethiopia. Aims: To investigate the roots of Kniphofia isoetifolia and evaluate the extracts and its constituents for antibacterial activity. Methods: The roots of Kniphofia isoetifolia was extracted with chloroform/methanol (1:1 v/v), and the resulting crude extract was partitioned between EtOAc/H2O. The ethyl acetate extract afforded four compounds after chromatographic purification, and their identification was based on spectroscopic analyses. The extracts and pure compounds were evaluated for in vitro antibacterial activity on four bacterial strains namely Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis.

Cores of aromatic polyketides are essential for their biological activities. Most type II polyketide synthases (PKSs) biosynthesize these core structures involving the minimal PKS, a PKS-associated ketoreductase (KR) and aromatases/cyclases (ARO/CYCs). Oxygenases (OXYs) are rarely involved. BE-7585A is an anticancer polyketide with an angucyclic core. 13 C isotope labeling experiments suggest that its angucyclic core may arise from an oxidative rearrangement of a linear anthracyclinone. Here, we present the crystal structure and functional analysis of BexE, the oxygenase proposed to catalyze this key oxidative rearrangement step that generates the angucyclinone framework. Biochemical assays using various linear anthracyclinone model compounds combined with docking simulations narrowed down the substrate of BexE to be an immediate precursor of aklaviketone, possibly 12-deoxy-aklaviketone. The structural analysis, docking simulations, and biochemical assays provide insights into the role of BexE in BE-7585A biosynthesis and lay the groundwork for engineering such framework-modifying enzymes in type II PKSs.

Silver-based nanostructures were prepared through reduction/oxidation reactions of aqueous silver nitrate solutions mediated by extracts of red fruits of the piquin pepper (Capsicum annuum var. aviculare)at room temperature. Detailed morphological and microstructural studies using X-ray diffraction, conventional and high-resolution transmission electron microscopy and selected area electron diffraction revealed that the product was constituted by three kinds of nanoparticles. One of them was composed of twinned metallic silver nanoparticles with a size of few nanometers. Other kind of particles was ultrafine disk-like single crystals of silver 4,4-dimethyldiazoaminobenzene, being in our best knowledge the first time that this compound is reported in the form of nanoparticles. Both kinds of nanoparticles experienced processes of self-assembly and subsequent grain growth to form the third kind of nanoparticles. Such resulting nanostructures are monocrystalline and flattened metallic silver nanoparticles that have diameters around tens of nanometers, the [112] direction perpendicular to the particle plane, and are coated by a surface organometallic layer and residues of biomolecules. The ultraviolet-visible spectrum of the biosynthesized product showed a surface plasmon resonance (SPR) extinction band with an
absorbance maximum at around 400 nm, thereby confirming the presence of fine Ag particles. Studies carried out by Fourier transform infrared spectroscopy indicated that the principal active compounds responsible of the reduction of the Ag ions are proteins and capsaicin (through the amino groups) and phenolic compounds (through hydroxyl groups).

Streptomyces chrysomallus and Streptomyces parvulus produce novel C-demethylactinomycins besides their normal
actinomycins when fed with 3-hydroxyanthranilic acid (3-HA). The 3-HA is incorporated into pentapeptide lactone precursors in
competition with the regular precursor 4-methyl-3-hydroxyanthranilic acid (4-MHA). The resultant 3-HA pentapeptide lactones can
condense with each other, as well as with the continuously formed 4-MHA pentapeptide lactones giving C-demethylactinomycins
lacking one or both methyl groups in their phenoxazinone chromophores. In case of C-demethylactinomyins lacking one methyl
group, the condensation was shown to be regiospecific directing the 3-HA portion almost exclusively to the a-side of the
phenoxazinone chromophore. As 3-HA is a weaker substrate for the 4-MHA-incorporating enzyme actinomycin synthetase I than
4-MHA, C-demethylactinomycins never exceeded 7–8% of total actinomycin formed. Surprisingly, C-demethylactinomycins
(up to 0.8%) were also found in the actinomycin mixtures of unsupplemented streptomycete cultures after longer cultivation
times, indicating the natural presence of 3-HA. Feeding with 3-hydroxykynurenine (3-HK) induced also formation of
C-demethylactinomycins indicating that 3-HK is source of 3-HA. Analysis of tryptophan metabolites in the intracellular pools of
the streptomycetes using 5-3H-tryptophan as radiotracer revealed formation of 4-MHA, but not of 3-HA. This indicates that
intracellular 3-HK is almost exclusively converted to 3-hydroxy-4-methylkynurenine (4-MHK), which has been identified previously
as direct precursor of 4-MHA. However, small amount of 3-HK leaking out from the 4-MHA pathway can be prematurely converted
to 3-HA all along the cultivation of the streptomycetes resulting in the formation of natural C-demethylactinomycins.

A cryptic trans-acyltransferase polyketide synthase biosynthetic gene cluster sdl (80 kb) from Streptomyces sp. B59 was cloned and transferred into a heterologous host Streptomyces albus J1074, resulting in a class of polycyclic macrolide shuangdaolides A−D (1−4) and dumulmycin (5). Heterologous expression and gene inactivation experiments allowed the identification of two biosynthetic intermediates, 6 and 7, suggesting an unusual multidomain SDR oxidoreductase SdlR in charge of the formation of a rare 2-hydroxycyclopentenone moiety in this class of compounds.

Background: Umbelliferone, also known as 7-hydroxycoumarin, is a phenolic metabolite found in many familiar plants. Its derivatives have been shown to have various pharmacological and chemo-preventive effects on human health. A uridine diphosphate glycosyltransferase YjiC from Bacillus licheniformis DSM 13, a cytochrome P450BM3 (CYP450 BM3) variant namely mutant 13 (M13) from Bacillus megaterium, and an O-methyltransferase from Streptomyces avermitilis (SaOMT2) were used for modifications of umbelliferone. Results: Three umbelliferone derivatives (esculetin, skimmin, and herniarin) were generated through enzymatic and whole cell catalysis. To improve the efficiencies of biotransformation, different media, incubation time and concentration of substrate were optimized and the production was scaled up using a 3-L fermentor. The maximum yields of esculetin, skimmin, and herniarin were 337.10 μM (67.62%), 995.43 μM (99.54%), and 37.13 μM (37.13%), respectively. The water solubility of es...