Extender Units Research Articles

Alligamycin A, an antifungal β-lactone spiroketal macrolide from Streptomyces iranensis

Fungal infections pose a great threat to public health and there are only four main types of antifungal drugs, which are often limited with toxicity, drug-drug interactions and antibiotic resistance. Streptomyces is an important source of antibiotics, represented by the clinical drug amphotericin B. Here we report the discovery of alligamycin A (1) as an antifungal compound from the rapamycin-producer Streptomyces iranensis through genome-mining, genetics and natural product chemistry approaches. Alligamycin A harbors a unique chemical scaffold with 13 chiral centers, featuring a β-lactone moiety, a [6,6]-spiroketal ring, and an unreported 7-oxo-octylmalonyl-CoA extender unit incorporated by a potential crotonyl-CoA carboxylase/reductase. It is biosynthesized by a type I polyketide synthase which is confirmed through CRISPR-based gene editing. Alligamycin A displayed potent antifungal effects against numerous clinically relevant filamentous fungi, including resistant Aspergillus and Talaromyces species. β-Lactone ring is essential for the antifungal activity since alligamycin B (2) with disruption in the ring abolished the antifungal effect. Proteomics analysis revealed alligamycin A potentially disrupts the integrity of fungal cell walls and induces the expression of stress-response proteins in Aspergillus niger. Discovery of the potent antifungal candidate alligamycin A expands the limited antifungal chemical space.

Engineering of a Malonyl-CoA Ligase for Production of Fluorinated Polyketide Extender Units.

Enzymatic platforms for producing malonyl-CoA-based extender units required for polyketide biosynthesis are often based on malonyl-CoA ligases such as MatB from Rhizobium trifolii and Rhodopseudomonas palustris. However, despite broad interest in the fluorination of polyketides and prior success with engineering MatB homologs, the suitability of MatB for accessing the tertiary substituted fluoromethylmalonyl-CoA needed to produce flurithromycin and solithromycin has not yet been reported. Herein, we report the structure-guided engineering of a MatB homolog to optimize the production of fluoromethylmalonyl-CoA, resulting in a variant with increased conversion and providing a platform to produce a suitable building block mixture for fluorinated macrolide production. Additionally, the mutant demonstrated broad utility for various substituted malonyl-CoAs. The MatB mutant sets the stage to access fluorinated macrolides by coupling it with altered PKS machinery to install fluorinated malonyl-CoA into macrolide scaffolds.

Gatekeeping Activity of Collinear Ketosynthase Domains Limits Product Diversity for Engineered Type I Polyketide Synthases.

Engineered type I polyketide synthases (type I PKSs) can enable access to diverse polyketide pharmacophores and generate non-natural natural products. However, the promise of type I PKS engineering remains modestly realized at best. Here, we report that ketosynthase (KS) domains, the key carbon-carbon bond-forming catalysts, control which intermediates are allowed to progress along the PKS assembly lines and which intermediates are excluded. Using bimodular PKSs, we demonstrate that KSs can be exquisitely selective for the upstream polyketide substrate while retaining promiscuity for the extender unit that they incorporate. It is then the downstream KS that acts as a gatekeeper to ensure the fidelity of the extender unit incorporation by the upstream KS. We also demonstrate that these findings are not universally applicable; substrate-tolerant KSs do allow engineered polyketide intermediates to be extended. Our results demonstrate the utility for evaluating the KS-induced bottlenecks to gauge the feasibility of engineering PKS assembly lines.

Structural and computational insights into the substrate specificity of acyltransferase domains from modular polyketide synthases.

Polyketides are natural products synthesized by polyketide synthases (PKSs), where acyltransferase (AT) domains play a crucial role in selection of extender units. Engineering of AT domains enables the site-specific incorporation of non-natural extender units, leading to generation of novel derivatives. Here, we determined the crystal structures of AT domains from the fifth module of tylosin PKS (TylAT5) derived from Streptomyces fradiae and the eighth module of spinosad PKS (SpnAT8) derived from Saccharopolyspora spinosa, and combined them with molecular dynamics simulations and enzyme kinetic studies to elucidate the molecular basis of substrate selection. The ethylmalonyl-CoA-specific conserved motif TAGH of TylAT5 and the MMCoA-specific conserved motif YASH of SpnAT8 were identified within the substrate-binding pocket, and several key residues close to the substrate acyl moiety were located. Through site-directed mutagenesis of four residues near the active site, we successfully reprogrammed the specificity of these two AT domains toward malonyl-CoA. Mutations in TylAT5 enhanced its catalytic activity 2.6-fold toward malonyl-CoA, and mutations in SpnAT8 eliminated the substrate promiscuity. These results extend our understanding of AT substrate specificity and would benefit the engineering of PKSs.

Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.

ДОСЛІДЖЕННЯ ДИНАМІЧНИХ ПАРАМЕТРІВ Н-РОТОРІВ ДАР’Є ЗМІННОЇ КОНФІГУРАЦІЇ

Calculations of the natural frequencies and forms of oscillations of a typical structural assembly of the H-rotor design of Dareus were carried out. It includes a sliding traverse and a blade fixed at its end. The dependencies of the eigenfrequencies of the extendable traverse-blade unit on the number of the vibration form at different values of the traverse extension value, as well as the dependence of the eigenfrequencies on the degree of traverse extension for five forms of oscillations, were obtained.

Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

Flame morphology of hydrous ethanol combustion under EGR dilution for port fuel injection and direct injection in a spark ignition optical engine

The increasing popularity of plug-in hybrid vehicles has prompted investigation of options such as range extender units, which may include small engines powered by biofuels. To minimize energy consumption, the best technologies must be chosen, including the use of exhaust gas recirculation (EGR, as a charge dilution technique) and the best alternative for fuel injection systems. Therefore, a combustion evaluation was conducted on an optically accessible engine fueled with hydrous ethanol to determine the effects of different injection modes, such as direct injection (DI) and port fuel injection (PFI), combined with EGR. The study employed high-speed camera imaging to analyze flame morphological characteristics and understand their impact on in-cylinder thermodynamics and engine emissions. The DI mode presented more stability than PFI, although the dilution limit was lower for DI.

The energy efficiency of an extended range unit involving a polymer exchange membrane fuel cell stack

This article presents the results of experimental investigations on an advanced model of a polymer exchange membrane fuel cell (PEMFC) stack designed to serve as a charging unit for electric vehicle batteries or off-grid distributed power sources. The assembled 720 W PEMFC stack comprises two 360 W modules that can be electrically connected in series or parallel. A liquid cooling system for the PEMFC stack has also been constructed. The dependencies – voltage (U) versus current (I) and current (I) versus electrical power (P) – for single modules, as well as for the electrically connected two-module PEMFC stack, are determined. Additionally, the hydrogen utilization versus the electrical power (P) of the PEMFC stack is examined. The electrical efficiency of the PEMFC stack varies between 42% and 50%, depending on the electrical power. An adjustable DC/DC converter, operating in two modes (step-down or stepup), is proposed as a device to integrate a lithium-ion (Li-ion) battery pack with approximately 720 W PEMFC stack. The electrical architecture of the integrated system, comprising the PEMFC stack, DC/DC converter, and Li-ion battery pack, is investigated and discussed in this paper. It was found that the electrical efficiency of the proposed DC/DC converter varies depending on the electrical power, reaching a peak efficiency of 95%– 98%. The environmental benefit, in terms of reducing CO2 emissions when charging the battery, was also identified.

A first-in-class dual-chelator theranostic agent designed for use with imaging-therapy radiometal pairs of different elements.

A covalent adduct of DFOB and DOTA separated by a l-lysine residue (DFOB-l-Lys-N 6-DOTA) exhibited remarkable regioselective metal binding, with {1H}-13C NMR spectral shifts supporting Zr(iv) coordinating to the DFOB unit, and Lu(iii) coordinating to the DOTA unit. This first-in-class, dual-chelator theranostic design could enable the use of imaging-therapy radiometal pairs of different elements, such as 89Zr for positron emission tomography (PET) imaging and 177Lu for low-energy β--particle radiation therapy. DFOB-l-Lys-N 6-DOTA was elaborated with an amine-terminated polyethylene glycol extender unit (PEG4) to give DFOB-N 2-(PEG4)-l-Lys-N 6-DOTA (compound D2) to enable installation of a phenyl-isothiocyanate group (Ph-NCS) for subsequent monoclonal antibody (mAb) conjugation (mAb = HuJ591). D2-mAb was radiolabeled with 89Zr or 177Lu to produce [89Zr]Zr-D2-mAb or [177Lu]Lu-D2-mAb, respectively, and in vivo PET/CT imaging and in vivo/ex vivo biodistribution properties measured with the matched controls [89Zr]Zr-DFOB-mAb or [177Lu]Lu-DOTA-mAb in a murine LNCaP prostate tumour xenograft model. The 89Zr-immuno-PET imaging function of [89Zr]Zr-D2-mAb and [89Zr]Zr-DFOB-mAb showed no significant difference in tumour accumulation at 48 or 120 h post injection. [89Zr]Zr-D2-mAb and [177Lu]Lu-D2-mAb showed similar ex vivo biodistribution properties at 120 h post-injection. Tumour uptake of [177Lu]Lu-D2-mAb shown by SPECT/CT imaging at 48 h and 120 h post-injection supported the therapeutic function of D2, which was corroborated by similar therapeutic efficacy between [177Lu]Lu-D2-mAb and [177Lu]Lu-DOTA-mAb, both showing a sustained reduction in tumour volume (>80% over 65 d) compared to vehicle. The work identifies D2 as a trifunctional chelator that could expand capabilities in mixed-element radiometal theranostics to improve dosimetry and the clinical outcomes of molecularly targeted radiation.

Biosynthesis of Octacosamicin A: Uncommon Starter/extender Units and Product Releasing via Intermolecular Amidation.

Octacosamicin A is an antifungal metabolite featuring a linear polyene-polyol chain flanked by N-hydroxyguanidine and glycine moieties. We report here that sub-inhibitory concentrations of streptomycin elicited the production of octacosamicin A in Amycolatopsis azurea DSM 43854T . We identified the biosynthetic gene cluster (oca BGC) that encodes a modular polyketide synthase (PKS) system for assembling the polyene-polyol chain of octacosamicin A. Our analysis suggested that the N-hydroxyguanidine unit originates from a 4-guanidinobutyryl-CoA starter unit, while the PKS incorporates an α-hydroxyketone moiety using a (2R)-hydroxymalonyl-CoA extender unit. The modular PKS system contains a non-canonical terminal module that lacks thioesterase (TE) and acyl carrier protein (ACP) domains, indicating the biosynthesis is likely to employ an unconventional and cryptic off-loading mechanism that attaches glycine to the polyene-polyol chain via an intermolecular amidation reaction.

Functional aggregation of cell-free proteins enables fungal ice nucleation.

Biological ice nucleation plays a key role in the survival of cold-adapted organisms. Several species of bacteria, fungi, and insects produce ice nucleators (INs) that enable ice formation at temperatures above -10 °C. Bacteria and fungi produce particularly potent INs that can promote water crystallization above -5 °C. Bacterial INs consist of extended protein units that aggregate to achieve superior functionality. Despite decades of research, the nature and identity of fungal INs remain elusive. Here, we combine ice nucleation measurements, physicochemical characterization, numerical modeling, and nucleation theory to shed light on the size and nature of the INs from the fungus Fusarium acuminatum. We find ice-binding and ice-shaping activity of Fusarium IN, suggesting a potential connection between ice growth promotion and inhibition. We demonstrate that fungal INs are composed of small 5.3 kDa protein subunits that assemble into ice-nucleating complexes that can contain more than 100 subunits. Fusarium INs retain high ice-nucleation activity even when only the ~12 kDa fraction of size-excluded proteins are initially present, suggesting robust pathways for their functional aggregation in cell-free aqueous environments. We conclude that the use of small proteins to build large assemblies is a common strategy among organisms to create potent biological INs.

Discovery and Biosynthesis of the Cytotoxic Polyene Terpenomycin in Human Pathogenic Nocardia.

Nocardia are opportunistic human pathogens that can cause a range of debilitating and difficult to treat infections of the lungs, brain, skin, and soft tissues. Despite their close relationship to the well-known secondary metabolite-producing genus, Streptomyces, comparatively few natural products are known from the Nocardia, and even less is known about their involvement in the pathogenesis. Here, we combine chemistry, genomics, and molecular microbiology to reveal the production of terpenomycin, a new cytotoxic and antifungal polyene from a human pathogenic Nocardia terpenica isolate. We unveil the polyketide synthase (PKS) responsible for terpenomycin biosynthesis and show that it combines several unusual features, including "split", skipped, and iteratively used modules, and the use of the unusual extender unit methoxymalonate as a starter unit. To link genes to molecules, we constructed a transposon mutant library in N. terpenica, identifying a terpenomycin-null mutant with an inactivated terpenomycin PKS. Our findings show that the neglected actinomycetes have an unappreciated capacity for the production of bioactive molecules with unique biosynthetic pathways waiting to be uncovered and highlights these organisms as producers of diverse natural products.

Analysis of evaporation and autoignition of droplet clouds with a unit cell model considering transient evaporating boundary layer

Liquid clouds (sprays) are commonly encountered in nature and industrial applications, where droplets are subject to complex interactions. In this paper, an extended unit cell model is proposed to analyze the evaporation and autoignition of monodisperse, single-component, and quiescent droplet clouds. The extended model incorporates the transient development of the evaporating boundary layer attached to the droplet surface. Due to the inclusion of this gas-phase transient effect, the current theory can be applied to both dilute and dense clouds across a wide range of ambient pressures. In the absence of chemical reactions, the effects of multiple parameters, such as droplet spacing and ambient pressure, on the evaporation rate and lifetime of droplets are examined. The critical condition for fuel vapor saturation in the gas phase is re-obtained. Based on this, we further investigate the autoignition of droplet clouds exposed to hot oxidizing environments and derive the corresponding ignition criterion. Effects of various factors, including droplet size, droplet spacing, ambient pressure, initial oxygen concentration, and reaction order, on critical ignition temperatures are also considered. In comparison with transient numerical results, it is found that the current theoretical solutions predict well for a broad variety of parameters.

Optimization of Milbemycin Component Ratio by Coordinating Acyl-Coenzyme A Supply Pathways in Streptomyces bingchenggensis

Milbemycins are a group of macrolide pesticides with great potential in the agricultural field owing to their high insecticidal activity and environmental compatibility. Milbemycin A3 and A4 with high bioactivities are the main components of milbemycin-derived products, which require a component ratio A4:A3 of 2.3- to 4.0-fold. Streptomyces bingchenggensis BC04 is a promising milbemycin producer, whereas the component ratio of its products (A4:A3 of 9.0-fold) could not meet the requirement for industrial production. To address this issue, we reconstructed the precursor biosynthetic pathways to fine tune the supply of different acyl-coenzyme A precursors required for milbemycin biosynthesis. Based on an analysis of the intracellular acyl-coenzyme A precursors, we reconstructed stepwise heterogeneous biosynthetic pathways of extender units for milbemycin biosynthesis. Then, we coordinated the supply of milbemycin biosynthetic starter units with temporal promoters. Thanks to these manipulations, we obtained an engineered strain with 39.5% milbemycin titer improvement to 3417.88 mg/L and a qualified component ratio A4:A3 of 3.3-fold. This work demonstrated that coordinating the precursor supply is a simple and effective approach to optimize the component ratio of A4:A3 in milbemycin fermentation products. Moreover, this strategy might also be useful to construct high-yield strains with optimized component ratios of fermentation products in other Streptomyces.

Enhancing armeniaspirols production through multi-level engineering of a native Streptomyces producer

BackgroundNature has provided unique molecular scaffolds for applications including therapeutics, agriculture, and food. Due to differences in ecological environments and laboratory conditions, engineering is often necessary to uncover and utilize the chemical diversity. Although we can efficiently activate and mine these often complex 3D molecules, sufficient production of target molecules for further engineering and application remain a considerable bottleneck. An example of these bioactive scaffolds is armeniaspirols, which are potent polyketide antibiotics against gram-positive pathogens and multi-resistance gram-negative Helicobacter pylori. Here, we examine the upregulation of armeniaspirols in an alternative Streptomyces producer, Streptomyces sp. A793.ResultsThrough an incidental observation of enhanced yields with the removal of a competing polyketide cluster, we observed seven-fold improvement in armeniaspirol production. To further investigate the improvement of armeniaspirol production, we examine upregulation of armeniaspirols through engineering of biosynthetic pathways and primary metabolism; including perturbation of genes in biosynthetic gene clusters and regulation of triacylglycerols pool.ConclusionWith either overexpression of extender unit pathway or late-stage N-methylation, or the deletion of a competing polyketide cluster, we can achieve seven-fold to forty nine-fold upregulation of armeniaspirol production. The most significant upregulation was achieved by expression of heterologous fatty acyl-CoA synthase, where we observed not only a ninety seven-fold increase in production yields compared to wild type, but also an increase in the diversity of observed armeniaspirol intermediates and analogs.

Biosynthesis-Guided Discovery and Engineering of α-Pyrone Natural Products from Type I Polyketide Synthases.

Natural products containing the α-pyrone moiety are produced by polyketide synthases (PKSs) in bacteria, fungi, and plants. The conserved biosynthetic logic for the production of the α-pyrone moiety involves the cyclization of a triketide intermediate which also off-loads the polyketide from the activating thioester. In this study, we show that truncating a tetraketide natural product producing PKS assembly line allows for a thioesterase-independent off-loading of an α-pyrone polyketide natural product, one which we find to be natively present in the extracts of the bacterium that otherwise furnishes the tetraketide natural product. By engineering the truncated PKS in vitro, we demonstrate that a ketosynthase (KS) domain with relaxed substrate selectivity when coupled with in trans acylation of polyketide extender units can expand the chemical space of α-pyrone polyketide natural products. Findings from this study point toward heterologous intermolecular protein-protein interactions being detrimental to the efficiency of engineered PKS assembly lines.

Control Parameter Analysis for an SRM Inverter for Range Extender Units with Integrated DC–DC Converter

In modern electrical traction drives, a variable dc-link voltage can be applied to increase efficiency in partial load. A dc-dc converter can supply a variable dc-link voltage and transfer energy between the battery and the dc-link. Hybrid cars combine the advantages of long driving ranges with the partial zero-emission operation. In series-hybrid drive trains, a switched reluctance machine can be used as a generator for the range extender unit. This paper presents at first the mechanism of energy transfer between the battery and the dc-link. Further, a control parameter analysis of an inverter topology for a switched reluctance machine, which combines the dc-dc converter for the power transfer between the battery and the dc-link with the inverter for the generator, is investigated. For this analysis, the control parameters turn-on, freewheeling and turn-off angle are varied to test the controllability of the energy transfer between these two components. The simulation results of the presented analysis are finally validated with measurements on the test bench.

Biosynthesis of Tetronates by a Nonribosomal Peptide Synthetase-Polyketide Synthase System.

A cryptic tetronate biosynthetic pathway was identified in Kitasatospora niigatensis DSM 44781 via heterologous expression. Distinct from the currently known biosynthetic pathways, this system utilizes a partially functional nonribosomal peptide synthetase and a broadly selective polyketide synthase to direct the assembly and lactonization of the tetronate scaffold. By employing a permissive crotonyl-CoA reductase/carboxylase to provide different extender units, seven new tetronates (kitaniitetronins A-G) were obtained via precursor-directed biosynthesis.

Acyltransferase Domain Exchange between Two Independent Type I Polyketide Synthases in the Same Producer Strain of Macrolide Antibiotics.

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activity. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without any supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon-elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.