Molecular Assembly Lines Research Articles

High-throughput reprogramming of an NRPS condensation domain

Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.

Continuous Transport of a Nanoparticle on a Solid Surface

Abstract Long-distance transport of a nanoparticle on a solid surface remains a challenge in nanotechnology. Here, we design a nanoscale motor device for continuously transporting a nanoparticle on a beam surface. The device is composed of repeated units of clamped beams on which a harmonic excitation is applied to induce a gradient in atomic density on their surface, and such atomic density consequently creates a driving force on the nanoparticle attached to the device surface. The design requirements that should be satisfied by the device attributes are analytically derived, and the effect of the device attributes on the device transport performance is discussed. In addition, molecular dynamics simulations for a typical device of a graphene sheet transported on a silver beam are conducted to verify the analytical results. The proposed design provides a starting point for continuously transporting a nanoobject on a solid surface and has great potential in various applications such as nanomotors and molecular assembly lines.

Multiple Functions of the Type II Thioesterase Associated with the Phoslactomycin Polyketide Synthase.

Polyketide synthases (PKSs) are molecular assembly lines that condense basic chemical building blocks for the production of structurally diverse polyketides. Many PKS biosynthetic gene clusters contain a gene encoding for a type II thioesterase (TEII). It is believed that TEIIs exert a proofreading function and restore or increase the productivity of PKSs by removing aberrant modifications on the acyl-carrier proteins (ACPs) of the PKS assembly line. Yet biochemical evidence is still sparse. Here, we investigated the function of PnG, the TEII of the phoslactomycin PKS (Pn PKS), in the context of its cognate assembly line in vitro. Biochemical analysis revealed that PnG preferentially hydrolyzes alkyl-ACPs over (alkyl)malonyl-ACPs by up to three orders of magnitude, supporting a proofreading role of the enzyme. We further demonstrate that PnG increases the in vitro production of different native and non-native tetra-, penta-, and hexaketide derivatives of phoslactomycin by more than one order of magnitude and show that these effects are caused by the initial clearing of the Pn PKS, as well as proofreading of the active assembly line. Finally, we demonstrate that PnG is able to release intermediate but notably also terminal polyketides from the Pn PKS. This allows PnG to functionally replace and overcome the terminal TEI activity of chimeric in vitro Pn PKS systems, as showcased with a phoslactomycin hexaketide system. Altogether, our experiments provide detailed insights into the molecular mechanisms and the multiple functions of PnG in its native context, as well as their potential use in future applications.

Molecular Assembly Lines in Active Droplets.

Large protein complexes are assembled from protein subunits to form a specific structure. In our theoretic work, we propose that assembly into the correct structure could be reliably achieved through an assembly line with a specific sequence of assembly steps. Using droplet interfaces to position compartment boundaries, we show that an assembly line can be self-organized by active droplets. As a consequence, assembly steps can be arranged spatially so that a specific order of assembly is achieved and incorrect assembly is strongly suppressed.

Lithiation–borylation methodology in the total synthesis of natural products

Robust synthetic methods that show a broad substrate scope are of great utility in the synthesis of complex organic molecules. Within this arena, synthetic methods that employ boronic esters are especially useful because they undergo a wide variety of transformations with very high levels of stereoselectivity. In particular, boronic esters can undergo single or multiple homologations using enantioenriched metal carbenoids. The addition of a suitable enantioenriched lithium or magnesium carbenoid to a boronic ester, with subsequent 1,2-migration, gives a homologated boronic ester with high stereocontrol. This process, termed lithiation–borylation, can be iterative, which allows a carbon chain to be extended one atom at a time with remarkable precision. The iterative homologation has been likened to a molecular assembly line and resembles the way nature assembles natural products, for example, in polyketide synthase machinery. The application of lithiation–borylation chemistry to the synthesis of a broad variety of natural products is discussed in this Review.

Seeing gene expression in cells: the future of structural biology.

Although much is known about the machinery that executes fundamental processes of gene expression in cells, much also remains to be learned about how that machinery works. A recent paper by O'Reilly et al. reports a major step forward in the direct visualization of central dogma processes at submolecular resolution inside bacterial cells frozen in a native state. The essential methodologies involved are cross-linking mass spectrometry (CLMS) and cryo-electron tomography (cryo-ET). In-cell CLMS provides in vivo protein interaction maps. Cryo-ET allows visualization of macromolecular complexes in their native environment. These methods have been integrated by O'Reilly et al. to describe a dynamic assembly in situ between a transcribing RNA polymerase (RNAP) and a translating ribosome - a complex known as the expressome - in the model bacterium Mycoplasma pneumoniae 1. With the application of improved data processing and classification capabilities, this approach has allowed unprecedented insights into the architecture of this molecular assembly line, confirming the existence of a physical link between RNAP and the ribosome and identifying the transcription factor NusA as the linking molecule, as well as making it possible to see the structural effects of drugs that inhibit either transcription or translation. The work provides a glimpse into the future of integrative structural cell biology and can serve as a roadmap for the study of other molecular machineries in their native context.

Molecular Chaperones: Molecular Assembly Line Brings Metabolism and Immunity in Shape.

Molecular chaperones are a set of conserved proteins that have evolved to assist the folding of many newly synthesized proteins by preventing their misfolding under conditions such as elevated temperatures, hypoxia, acidosis and nutrient deprivation. Molecular chaperones belong to the heat shock protein (HSP) family. They have been identified as important participants in immune functions including antigen presentation, immunostimulation and immunomodulation, and play crucial roles in metabolic rewiring and epigenetic circuits. Growing evidence has accumulated to indicate that metabolic pathways and their metabolites influence the function of immune cells and can alter transcriptional activity through epigenetic modification of (de)methylation and (de)acetylation. However, whether molecular chaperones can regulate metabolic programs to influence immune activity is still largely unclear. In this review, we discuss the available data on the biological function of molecular chaperones to immune responses during inflammation, with a specific focus on the interplay between molecular chaperones and metabolic pathways that drive immune cell fate and function.

Structural basis for chain release from the enacyloxin polyketide synthase.

SummaryModular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N- and C-terminal docking domains are usually responsible for mediating interactions between subunits. Here we show that communication between two nonribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a β-hairpin docking domain. The structures, interactions and dynamics of these subunits are characterised using several complementary biophysical techniques, providing extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/β-hairpin docking domain pairs mediate subunit interactions in numerous nonribosomal peptide and hybrid polyketide-nonribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and β-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Using Ketoreductases for the Chemoenzymatic Synthesis of a Stereotriad

Polyketides are diverse natural products of high biological activity produced from complex molecular assembly lines called polyketide synthases (PKSs). Some of the most important bioactive molecules are polyketides, including geldanamycin, doxycycline, tetracycline, and erythromycin. Other polyketides have been shown to have antifungal, antiparasitic, immunosuppressive, and even anticancer properties. Of particular focus here is the ketoreductase (KR), one of the many possible domains within PKSs. The KR reduces carbonyl groups stereoselectively and controls the majority of stereocenters in the polyketide product. KRs have been used in a bottom‐up approach here, harnessing their stereoselective reductions to produce products that would traditionally be difficult and time‐consuming to synthesize. A diketide was first synthesized chemically and reduced with a tylosin KR from Streptomyces fradiae (TylKR2). This reduced diketide was then extended into a triketide through the Masamune C‐acylation reaction. Finally, TylKR2 and a mycolactone KR from a bacterial artificial chromosome (MycKR6) were found to reduce S‐(2‐acetamidoethyl)(4R,5R)‐5‐methoxy‐4‐methyl‐3‐oxoheptanethioate to S‐(2‐acetamidoethyl) (3S,4S,5R)‐3‐hydroxy‐5‐methoxy‐4‐methylheptanethioate in the case of TylKR2 and to S‐(2‐acetamidoethyl) (3R,4S,5R)‐3‐hydroxy‐5‐methoxy‐4‐methylheptanethioate in the case of MycKR6. The reductions were performed in vitro using the glucose dehydrogenase regeneration system with NADP+. In this way, each of the 3 stereocenters was set chemoenzymatically using KRs to form a stereotriad. With similar KRs, it is likely that all 8 possible stereoisomers could be synthesized. Ultimately, these di‐ and triketide building blocks could be extremely useful functional groups to experiment with in drug synthesis and may result in more potent or effective medicines. Additionally, this work could be extended into a methodology for setting as many stereocenters as desired ‐ possibly one day allowing for customizable polyketide synthesis in the same way oligonucleotides and polypeptides can be made today.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Real Time Actuation of a DNA Based Robotic Arm

Since the early days of nanotechnology, scientists have dreamt of nanoscale mechanical systems that resemble macroscale industrial assembly lines. Molecular self-assembly with DNA molecules, used for the bottom-up creation of complex molecular constructs, has been shown to hold large potential to push towards a realization of this vision. Starting out from simple mechanical mechanisms, increasingly intricate systems have been demonstrated in the past decade. A wide collection of mechanical elements, potentially used as components for even more elaborate systems have been developed over the past years. These elements were based on a variety of different concepts to facilitate conformational changes, sliding motion and rotation. We here demonstrate a DNA-based molecular robotic arm integrated on a square base plate created with the DNA origami technique, expanding the repertoire of available building components for DNA assembled nanomachines. A flexible connection between base plate and arm allows rotation of the arm with respect to the specifically addressable base plate. Based on a one pot folding approach, the structure efficiently self-assembles. With the help of single-molecule fluorescence techniques, diffusive arm rotation and temporary fixation at different docking positions on the base plate is observed on a millisecond time scale. Utilizing the high intrinsic charge of our DNA nanoconstruct we also demonstrate the use of electrical fields to actively control the arm's angle relative to the base plate. Actuation of the robot arm only requires milliseconds, which is many orders of magnitude faster than established methods for the operation of synthetic DNA nanomachines. For future work, the system promises to act a as versatile rapid prototyping platform for the development of components for molecular assembly lines and highly parallelizable nanoscale force spectroscopy.

Context-Specific Regulation of Coupled Transcription-Translation Modules Predicts Pervasive Ribosome Specialization

The ribosome is a defining structure of all cellular organisms and represents the uniquely biological process of translation. Although ribosomes are typically represented as molecular assembly lines that constitutively perform coded protein synthesis, translation is a system property that emerges from interactions among diverse types of cellular components, including different classes of RNAs, proteins, and amino acids. The prevailing view that all ribosomes within a cell are structurally identical and functionally equivalent was challenged long ago, and is becoming increasingly less tenable with modern experimental tools. Through analysis of environmental and gene regulatory influence networks of H. salinarum, E. coli, and S. cerevisiae, we have discovered a generalized pattern of extensive, environment-specific modularity and heterogeneity in translational subsystems across these representative organisms from each of the three domains of life (Archaea, Bacteria and Eukarya). Systems analysis of H. salinarum growth-associated changes in total mRNA abundance (RNA-seq), ribosome associated transcripts (ribosome profiling), and protein abundance (SWATH-MS proteomics) further support the hypothesis that ribosomes are less of a singular entity, and more of a mosaic patchwork. These data provide evidence that variability in translational subsystems might confer intrinsic regulation of protein synthesis by selectively translating subsets of the transcriptome. We discuss the implication of linked translation-transcription modules as a minimal set of molecules for steering physiological state transitions, as well as acting as a fundamental constraint on biological evolution.

Molecular Assembly Line: Stepwise Hydrogenation of Multifunctional Substrates over Catalyst Mixtures

AbstractThe synthesis of many chemical products requires multiple steps. To simplify the overall synthesis by combining consecutive reaction steps, the concept of a “molecular assembly line” promises to yield high reaction rates and selectivities. In this study, mixtures of carbon nanotube (CNT)‐supported catalysts were used to demonstrate the potential of this concept on hand of the hydrogenation of nitrobenzene via aniline to cyclohexylamine. Mixtures of Pt/CNT, having a high activity in nitrobenzene hydrogenation, and of Ru/CNT, highly selective for the hydrogenation of aniline to cyclohexylamine, provided high activity at constant high selectivity. Varying the Pt:Ru ratio to 5:95 was found to be the best case scenario, for which the rates of the two consecutive reaction steps are balanced. The use of catalyst mixtures rather than bimetallic catalysts avoids complex phenomena such as phase separation or migration of one of the metals to the surface of the bimetallic particles.

Rapid Vortex Fluidics: Continuous Flow Synthesis of Amides and Local Anesthetic Lidocaine.

Thin film flow chemistry using a vortex fluidic device (VFD) is effective in the scalable acylation of amines under shear, with the yields of the amides dramatically enhanced relative to traditional batch techniques. The optimized monophasic flow conditions are effective in ≤80 seconds at room temperature, enabling access to structurally diverse amides, functionalized amino acids and substituted ureas on multigram scales. Amide synthesis under flow was also extended to a total synthesis of local anesthetic lidocaine, with sequential reactions carried out in two serially linked VFD units. The synthesis could also be executed in a single VFD, in which the tandem reactions involve reagent delivery at different positions along the rapidly rotating tube with in situ solvent replacement, as a molecular assembly line process. This further highlights the versatility of the VFD in organic synthesis, as does the finding of a remarkably efficient debenzylation of p-methoxybenzyl amines.

Function of polyketide synthases from the SAXS structure of a complete module

The modular polyketide synthases (PKS), common to many bacterial antibiotic-producers, are huge multienzyme, multidomain systems which can be likened to molecular assembly lines.1 An understanding of the structural and mechanistic aspects which underpin interdomain cooperation during biosynthesis by modular PKS is crucial to ongoing efforts to re-engineer these systems for the production of novel compounds of medicinal value.2 Previous attempts to solve the structures of PKS modules by X-ray crystallography have been unsuccessful, likely due to the high, inherent flexibility of the multienzymes. Here, we have employed an alternative technique, small-angle X-ray scattering (SAXS), in combination with NMR structure elucidation and homology modelling of individual domains, to obtain the first low-resolution structural data on an entire apo PKS module. The model system comprises a multidomain region of the virginiamycin M1 PKS from Streptomyces virginiae, a target of relevance for its involvement in production of the commercial antibiotic dalfopristin. The investigated region, which comprises a ketosynthase (KS) and two consecutive acyl carrier protein (ACP) domains, interacts with a wide range of protein partners, including notably, a complex of discrete enzymes responsible for β-methylation of the polyketide, and the N-terminus of the downstream PKS protein, VirFG.3 In our solved structure (Fig. 1), the homodimeric KS, which is flanked by well-folded linker regions, occupies the center of the module. While the first ACP is located close to the KS, the second is situated at the end of a flexible linker, and mobile. Taken together, these data provide a physical explanation for the functional non-equivalence previously observed for certain tandem ACPs of this class of PKS. Furthermore, the overall open shape of the module renders the second ACP highly accessible, which may be critical for its interaction with its multiple in trans catalytic partners. Finally, our analysis redefines the function of a putative dimerization motif of tandem ACPs as a docking domain, suggesting that the module likely adopts a more closed form in order to affect transfer of the chain extension intermediate to the subsequent module.

Nanoshuttles propelled by motor proteins sequentially assemble molecular cargo in a microfluidic device.

Nanoshuttles powered by the molecular motor kinesin have the potential to capture and concentrate rare molecules from solution as well as to transport, sort and assemble them in a high-throughput manner. One long-thought-of goal has been the realisation of a molecular assembly line with nanoshuttles as workhorses. To harness them for this purpose might allow the community to engineer novel materials and nanodevices. The central milestone towards this goal is to expose nanoshuttles to a series of different molecules or building blocks and load them sequentially to build hierarchical structures, macromolecules or materials. Here, we addressed this challenge by exploiting the synergy of two so far mostly complementary techniques, nanoshuttle-mediated active transport and pressure-driven passive transport, integrated into a single microfluidic device to demonstrate the realisation of a molecular assembly line. Multiple step protocols can thus be miniaturised to a highly parallelised and autonomous working lab-on-a-chip: in each reaction chamber, analytes or building blocks are captured from solution and are then transported by nanoshuttles across fluid flow boundaries in the next chamber. Cargo can thus be assembled, modified, analysed and eventually unloaded in a procedure that requires only one step by its operator.

Flexing and Stretching in Nonribosomal Peptide Synthetases

Re-engineering of nonribosomal peptide synthetase molecular assembly lines has been hampered by a lack of detailed knowledge concerning inter-domain substrate transfer. Recent structural studies of catalytically relevant domain-domain interactions provide valuable insights into this problem (Liu etal., 2011; Sundlov etal., 2012 [in this issue of Chemistry & Biology]).

Direct observation of stepwise movement of a synthetic molecular transporter

Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor and fuel, all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold, and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.

Crystal Structure of a Molecular Assembly Line

Crystal Structure of a Molecular Assembly Line

Enzymes line up for assembly

Many enzymes have a series of catalytic sites, lined up like beads on a string. A previously unknown link in one of these molecular assembly lines involves an unexpected approach to a common biochemical reaction.

Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with approximately 10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (10(3) to 10(4) clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts.