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Genetically engineered (GE) algae offer the promise of producing food, fuel, and other valuable products with reduced requirements for land and fresh water. While the gains in productivity measured in GE terrestrial crops are predicted to be mirrored in GE algae, the stability of phenotypes and ecological risks posed by GE algae in large-scale outdoor cultivation remain unknown. Here, we describe the first US Environmental Protection Agency (EPA)-sanctioned experiment aimed at understanding how GE algae perform in outdoor cultivation. Acutodesmus dimorphus was genetically engineered by the addition of two genes, one for enhanced fatty acid biosynthesis, and one for recombinant green fluorescence protein (GFP) expression; both the genes and their associated phenotypes were maintained during fifty days of outdoor cultivation. We also observed that while the GE algae dispersed from the cultivation ponds, colonization of the trap ponds by the GE strain declined rapidly with increasing distance from the source cultivation ponds. In contrast, many species of indigenous algae were found in every trap pond within a few days of starting the experiment. When inoculated in water from five local lakes, the GE algae's effect on biodiversity, species composition, and biomass of native algae was indiscernible from those of the wild-type (wt) progenitor algae, and neither the GE nor wt algae were able to outcompete native strains. We conclude that GE algae can be successfully cultivated outdoors while maintaining GE traits, and that for the specific GE algal strain tested here they did not outcompete or adversely impact native algae populations when grown in water taken from local lakes. This study provides an initial evaluation of GE algae in outdoor cultivation and a framework to evaluate GE algae risks associated with outdoor GE algae production. [ABSTRACT FROM AUTHOR]

As the industry surrounding microalgae continues to develop, there is a growing need for reliable, ready-to-use technologies for measuring the growth and composition of algal cultures. These can be new technologies or adapted existing technologies presently used for similar applications in other systems. Here we demonstrate the use of an LED fluorometer for the rapid estimation of dry weight, protein, and lipid content from two strains of microalgae. The instrument was successfully used to determine the dry weight of Chlamydomonas reinhardtii (CC-3491) and Scenedesmus dimorphus (UTEX 1237) cultures, at densities up to 1.58 g/L. Soluble protein was also measured using the instrument, and was highly comparable (average within 3%) to results obtained using both the Bradford and Lowry methods. Lastly, neutral lipid accumulation induced by nitrogen starvation was estimated via BODIPY 495/505 fluorescence. The basic methods developed here can easily be applied to any strain of microalgae or cyanobacteria, and demonstrate reliable, cost-effective, single-instrument methods for the determination of several key parameters in the cultivation of photosynthetic microorganisms. [ABSTRACT FROM AUTHOR]

Polyketides and non-ribosomal peptides are in a class of natural products important both as drug sources and as dangerous toxins and virulence factors. While studies over the last two decades have provided substantial characterization of the modular synthases that produce these compounds at the genetic level, their understanding at the protein level is much less understood. New proteomic platforms called an orthogonal active site identification system (OASIS) and proteomic interrogation of secondary metabolism (PrISM) have been developed to identify and quantify natural product synthase enzymes. Reviewed here, these tools offer the means to discover and analyze modular synthetic pathways that are limited by genetic techniques, opening the tools of contemporary proteomics to natural product sciences. [Copyright &y& Elsevier]

Abstract: Polyketides and nonribosomal peptides constitute two classes of small molecule natural products that are well‐known for their ability to impact important biological processes in a multitude of ways. The modular biosynthetic enzymes responsible for production of these compounds (PKS and NRPS enzymes) have been the subject of extensive genetic, biochemical, and structural characterization, in part due to the potential utility their successful reengineering may have for the production of new therapeutics. In this chapter, we provide background as well as specific techniques in which synthetically produced small molecule probes have been applied to help better understand the mechanism and structure of PKS and NRPS biosynthetic pathways, as well as to help streamline their discovery process. The continued development and application of these methods has the potential to greatly complement our current approaches to the study of natural product biosynthesis. [Copyright &y& Elsevier]

Abstract: Surface-based assays have been used extensively for the functional and structural analysis of biomolecules such as DNA or proteins. These experiments are established by the analysis of binding between acceptor molecules and immobilized receptors on a platform. Site-specific printing of receptor molecules on gold, glass, or polycarbonate (PC) surfaces is conventionally performed by the chemical derivatization of a surface, priming it to covalently bind to subsequently deposited receptor molecules. Unlike conventional methods, we have developed a new fabrication method for bioactive PC surfaces by directly molding PC granules doped with receptor molecules. PC-based receptor molecules were synthesized and commercially available PC granules were doped with these synthesized molecules. In our proof-of-concept study, PC doped with dye 1 (Fig. 1) was used as the receptor molecule. Using an aluminum mold and a hot press machine, PC-based objects were manufactured through compression molding using doped PC granules. Affinity analysis was evaluated by monitoring the localization of a monoclonal antibody elicited against dye 1 to the surface of the molded platforms by fluorescence microscopy. The results illustrated effective binding of an anti-dye 1 monoclonal antibody to the surface, substantiating successful display of assemblies of molecular receptors on the surface through compression molding. Although conventional surface functionalization methods impose limited applications and alter desired opto-mechanical properties of the polymer, our investigation provides a versatile means for the fabrication of bioactive PC-based platforms. It can also be used for engineering and imbedding receptor arrays within three-dimensional objects with applications to the production of opto-medical devices or biosensors. [Copyright &y& Elsevier]

Although chemical methods to modify proteins in a sequence-specific manner have yet to be developed, site-specific post-translational modification of proteins has recently emerged as a major focus in biological chemistry. Post-translational modification with functionalized substrate analogues opens up several unique avenues to induce selective reactivity into proteins in a sequence-specific manner, and can be applied to protein identification and manipulation in both in vitro and in vivo contexts. Further in vivo applications of this method will enable the imaging of cellular processes, avoiding nonspecific labeling and probe scattering, major complications observed in nonenzymatic methods. Additionally, new tools for in vitro protein modification have been developed that offer more versatile ways to study protein structure and function. [Copyright &y& Elsevier]

Plants are a sustainable resource for valuable natural chemicals best illustrated by large-scale farming centered on specific products. Here, we review recent discoveries of plant metabolic pathways producing natural products with unconventional biomolecular structures. Prenylation of polyketides by aromatic prenyltransferases (aPTases) ties together two of the major groups of plant specialized chemicals, terpenoids and polyketides, providing a core modification leading to new bioactivities and downstream metabolic processing. Moreover, PTases that biosynthesize Z -terpenoid precursors for small molecules such as lycosantalene have recently been found in the tomato family. Gaps in our understanding of how economically important compounds such as cannabinoids are produced are being identified using next-generation ‘omics’ to rapidly advance biochemical breakthroughs at an unprecedented rate. For instance, olivetolic acid cyclase, a polyketide synthase (PKS) co-factor from Cannabis sativa , directs the proper cyclization of a polyketide intermediate. Elucidations of spatial and temporal arrangements of biosynthetic enzymes into metabolons, such as those used to control the efficient production of natural polymers such as rubber and defensive small molecules such as linamarin and lotaustralin, provide blueprints for engineering streamlined production of plant products. [ABSTRACT FROM AUTHOR]

Type III plant polyketide synthases (PKSs) biosynthesize a dazzling array of polyphenolic products that serve important roles in both plant and human health. Recent advances in structural characterization of these enzymes and new tools from the field of chemical biology have facilitated exquisite probing of plant PKS iterative catalysis. These tools have also been used to exploit type III PKSs as biocatalysts to generate new chemicals. Going forward, chemical, structural and biochemical analyses will provide an atomic resolution understanding of plant PKSs and will serve as a springboard for bioengineering and scalable production of valuable molecules in vitro, by fermentation and in planta. [Copyright &y& Elsevier]

Highlights: [•] Engineering of the fatty acid synthase in algae and cyanobacteria is desirable to alter lipid production and composition. [•] Initial engineering efforts demonstrate that fatty acid biosynthesis is not easily perturbed. [•] Successful engineering will require a deeper understanding of FAS protein interactions and regulation. [ABSTRACT FROM AUTHOR]

Abstract: Recent advances in the structural study of fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic enzymes have illuminated our understanding of modular enzymes of the acetate pathway. However, one significant and persistent challenge in such analyses is resolution of the acyl carrier protein (ACP), a small (∼9kDa) protein to which biosynthetic intermediates are tethered throughout the biosynthetic cycle. Here we report a chemoenzymatic crosslinking strategy in which the installation of a historical suicide substrate scaffold upon the 4′-phosphopantetheine (PPant) arm of the ACP is used to capture the active site of acyl carrier protein dehydratase (DH) domains in FAS. Through the synthesis of a small panel of related probes we identify structural features essential for ACP–DH crosslinking, and apply gel-based assays to demonstrate the stability as well as purification strategies for isolation of the chemoenzymatically modified ACP. Applying these carrier protein crosslinking techniques to the structural analysis of FAS and PKS complexes has the potential to provide snapshots of these biosynthetic assembly lines at work. [ABSTRACT FROM AUTHOR]

The alpha/beta hydrolase (ABH) superfamily is a widespread and functionally malleable protein fold recognized for its diverse biochemical activities across all three domains of life. ABH enzymes possess unexpected catalytic activity in the green plant lineage through selective alterations in active site architecture and chemistry. Furthermore, the ABH fold serves as the core structure for phytohormone and ligand receptors in the gibberellin, strigolactone, and karrikin signaling pathways in plants. Despite recent discoveries, the ABH family is sparsely characterized in plants, a sessile kingdom known to evolve complex and specialized chemical adaptations as survival responses to widely varying biotic and abiotic ecologies. This review calls attention to the ABH superfamily in the plant kingdom to highlight the functional adaptability of the ABH fold. [ABSTRACT FROM AUTHOR]

Polyketide biosynthesis engages a series of well-timed biosynthetic operations to generate elaborate natural products from simple building blocks. Mimicry of these processes has offered practical means for total synthesis and provided a foundation for reaction discovery. We now report an unusual intramolecular trans -amidation reaction discovered while preparing stabilized probes for the study of actinorhodin biosynthesis. This rapid cyclization event offers insight into the natural cyclization process inherent to the biosynthesis of type II polyketide antibiotics. [ABSTRACT FROM AUTHOR]

Enoyl-acyl carrier protein reductase (ENR), a critical enzyme in type II fatty acid biosynthesis, is a promising target for drug discovery against hepatocyte-stage Plasmodium falciparum . In order to identify Pf ENR-specific inhibitors, we docked 70 FDA-approved, bioactive, and/or natural product small molecules known to inhibit the growth of whole-cell blood-stage P. falciparum into several Pf ENR crystallographic structures. Subsequent in vitro activity assays identified a noncompetitive low-micromolar Pf ENR inhibitor, celastrol, from this set of compounds. [ABSTRACT FROM AUTHOR]

Eukaryotic microalgae and cyanobacteria have recently reemerged as promising organisms in the effort to develop sustainable options for production of food and fuel. However, substantial discrepancies consistently arise between laboratory and outdoor cultivation, and gains demonstrated using laboratory technologies have not paralleled gains observed in field demonstrations. For these reasons, a low-maintenance system and process for research-scale outdoor cultivation of a variety of both freshwater and marine microalgae and cyanobacteria was developed. Nine genera were evaluated in the system, demonstrating cultivation of both laboratory model and commercial-production organisms. Hundreds to thousands of grams of dry biomass could be produced in a single growth cycle, suitable for a variety of uses including inoculum generation, protein production, and biofuel applications. Following testing in outdoor stock-ponds, Scenedesmus and Nannochloropsis were grown semi-continuously in an 8000 L airlift-driven raceway, yielding in total over 8 kg of dry biomass for each strain. [ABSTRACT FROM AUTHOR]

Abstract: Protein–protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target. [Copyright &y& Elsevier]

Abstract: Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory properties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that a secondary structure–activity relationship is an important factor in the antibiotic activity of these compounds. [Copyright &y& Elsevier]

Abstract: Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP–KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex. [Copyright &y& Elsevier]

Abstract: The critical role of protein–protein interactions in the chemistry of polyketide synthases is well established. However, the transient and weak nature of these interactions, in particular those involving the acyl carrier protein (ACP), has hindered efforts to structurally characterize these interactions. We describe a chemo-enzymatic approach that crosslinks the active sites of ACP and their cognate ketosynthase (KS) domains, resulting in the formation of a stable covalent adduct. This process is driven by specific protein–protein interactions between KS and ACP domains. Suitable manipulation of the reaction conditions enabled complete crosslinking of a representative KS and ACP, allowing isolation of a stable, conformationally constrained adduct suitable for high-resolution structural analysis. [Copyright &y& Elsevier]

Abstract: Presented within are syntheses of the pladienolide B and FD-895 side-chains, as well as models of the essential ring-closing metathesis and Stille coupling that will be used to complete their total syntheses. Several analogs of the pladienolide B side-chain were also prepared in order to evaluate the scope of the methodology and to create a library of structures that could be used for stereochemical and SAR analyses. [Copyright &y& Elsevier]

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 Å structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction (double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD. [ABSTRACT FROM AUTHOR]

Modular synthases, such as fatty acid, polyketide, and non-ribosomal peptide synthases (NRPSs), are sophisticated machineries essential in both primary and secondary metabolism. Various techniques have been developed to understand their genetic background and enzymatic abilities. However, uncovering the actual biosynthetic pathways remains challenging. Herein, we demonstrate a pipeline to study an assembly line synthase by interrogating the enzymatic function of each individual enzymatic domain of BpsA, a NRPS that produces the blue 3,3′-bipyridyl pigment indigoidine. Specific inhibitors for each biosynthetic domain of BpsA were obtained or synthesized, and the enzymatic performance of BpsA upon addition of each inhibitor was monitored by pigment development in vitro and in living bacteria. The results were verified using genetic mutants to inactivate each domain. Finally, the results complemented the currently proposed biosynthetic pathway of BpsA. [ABSTRACT FROM AUTHOR]