Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.     

Design and characterization of modular scaffolds for tubulin assembly

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.     

Self-assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation

The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.     

Flexible and reconfigurable manufacturing systems paradigms

Reconfigurable Manufacturing System (RMS) is a new manufacturing systems paradigm that aims at achieving cost-effective and rapid system changes, as needed and when needed, by incorporating principles of modularity, integrability, flexibility, scalability, convertibility, and diagnosability. RMS promises customized flexibility on demand in a short time, while Flexible Manufacturing Systems (FMSs) provides generalized flexibility designed for the anticipated variations and built-in a priori. The characteristics of the two paradigms are outlined and compared. The concept of manufacturing system life cycle is presented. The main types of flexibility in manufacturing systems are discussed and contrasted with the various reconfiguration aspects including hard (physical) and soft (logical) reconfiguration. The types of changeability and transformability of manufacturing systems, their components as well as factories, are presented along with their enablers and compared with flexibility and reconfigurability. The importance of having harmonized human-machine manufacturing systems is highlighted and the role of people in the various manufacturing paradigms and how this varies in pursuit of productivity are illustrated. Finally, the industrial and research challenges presented by these manufacturing paradigms are discussed.     

TectoRNA: modular assembly units for the construction of RNA nano-objects

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.     

Reconfigurable modular automation systems for automotive power-train manufacture

This paper describes research towards the realization of reconfigurable modular automated machines and the associated engineering methods and tools necessary to support their lifecycle needs. UK-based research, in collaboration with the Ford Motor Company and several machine builders, has resulted in the development of full-scale prototype reconfigurable modular automation systems for both engine assembly and machining applications. The implementation of an assembly system is featured in this paper. An engineering environment and associated reconfigurable component-based control system architecture have been created aimed at supporting the lifecycle needs of a new generation of agile automated systems, i.e., providing reconfigurable, easily scalable automated machinery. This approach has the potential to fit within a wider collaborative automation strategy where manufacturing systems are implemented as a conglomerate of distributed, autonomous, and reusable units.     

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.     

Hydrophobic condensation and modular assembly model of protein folding

Despite several decades of intense study, protein folding problem remains elusive. In this paper, we review current knowledge and the prevailing thinking in the field, and summarize our work on the in vitro folding of a typical small globular protein, staphylococcal nuclease (SNase). Various thermodynamic and kinetic methods have been employed to determine the energetic and construct the energy landscape of folding. Data presented include, but not limit to, the identification of intermediate states, time courses of their spread and convergence on the landscape, and finally the often ignored step, the refinement of the overall conformation and hence the activation of the enzyme. Our goal is to have a complete perspective of the folding process starting from its initial unfolded state to the fully active native state. Analysis leads to these findings: the folding starts with the condensation of the hydrophobic side chains in different locales of the peptide chain. The newly forged hydrophobic environment facilitates formation of helix- and sheet-like frameworks at different domains. Consolidation and inter-docking of these frameworks or domains then stabilizes the overall conformation and refines the structure to activate the enzyme. Based on these observations we favor folding-by-parts and propose a modular assembly model for the in vitro folding of SNase.     

Functional assembly and characterization of a modular xylanosome for hemicellulose hydrolysis in yeast

Five trimeric xylanosomes were successfully assembled on the cell surface of Saccharomyces cerevisiae. Three dockerin‐tagged fungal enzymes, an endoxylanase (XynAc) from Thermomyces lanuginosus, a β‐xylosidase (XlnDt) from Aspergillus niger and an acetylxylan esterase (AwAXEf) from Aspergillus awamori, were displayed for the synergistic saccharification of birchwood xylan. The surface‐expression scaffoldins were modular constructs with or without carbohydrate binding modules from Thermotoga maritima (family 22) or Clostridium thermocellum (family 3). The synergy due to enzyme–enzyme and enzyme–substrate proximity, and the effects of binding domain choice and position on xylan hydrolysis were determined. The scaffoldin‐based enzymes (with no binding domain) showed a 1.6‐fold increase in hydrolytic activity over free enzymes; this can be attributed to enzyme–enzyme proximity within the scaffoldin. The addition of a xylan binding domain from T. maritima improved hydrolysis by 2.1‐fold relative to the scaffoldin without a binding domain (signifying enzyme–substrate synergy), and 3.3‐fold over free enzymes, with a xylose productivity of 105 mg g^?1 substrate after 72 h hydrolysis. This system was also superior to the xylanosome carrying the cellulose binding module from C. thermocellum by 1.4‐fold. Furthermore, swapping the xylan binding module position within the scaffoldin resulted in 1.5‐fold more hydrolysis when the binding domain was adjacent to the endoxylanase. These results demonstrate the applicability of designer xylanosomes toward hemicellulose saccharification in yeast, and the importance of the choice and position of the carbohydrate binding module for enhanced synergy. Biotechnol. Bioeng. 2013; 110: 275–285. © 2012 Wiley Periodicals, Inc.     

Biosynthetic systems for nonribosomal peptide antibiotic assembly

Modular peptide synthetases, which act as the protein templates for the synthesis of a large number of peptide antibiotics and siderophores, hold great potential for the development of novel compounds. Recently, significant progress has been made towards understanding their molecular architecture and substrate specificity. The first crystal structure of a peptide synthetase has been solved, and the enzymes responsible for post-translational modification of peptide synthetases have recently been discovered. These will allow addressing important yet poorly understood mechanistic aspects.     

Equipment selection and task assignment for multiproduct assembly system design

A multiproduct assembly system produces a family of similar products, where the assembly of each product entails an ordered set of tasks. An assembly system consists of a sequence of workstations. For each workstation, we assign a subset of the assembly tasks to be performed at the workstation and select the type of assembly equipment or resource to be used by the workstation. The assembly of each product requires a visit to each workstation in the fixed sequence. The problem of system design is to find the system that is capable of producing all the products in the desired volumes at minimum cost. The system cost includes the fixed capital costs for the assembly equipment and tools and the variable operating costs for the various workstations. We present and illustrate an optimization procedure that assigns tasks to workstations and selects assembly equipment for each workstation.     

A line-balancing strategy for designing flexible assembly systems

We present a rough-cut analysis tool that quickly determines a few potential cost-effective designs at the initial design stage of flexible assembly systems (FASs) prior to a detailed analysis such as simulation. It uses quantitative methods for selecting and configuring the components of an FAS suitable for medium to high volumes of several similar products. The system is organized as a series of assembly stations linked with an automated material-handling system moving parts in a unidirectional flow. Each station consists of a single machine or of identical parallel machines. The methods exploit the ability of flexible hardware to switch almost instantaneously from product to product. Our approach is particularly suitable where the product mix is expected to be stable, since we combine the hardware-configuration phase with the task-allocation phase. For the required volume of products, we use integer programming to select the number of stations and the number of machines at each station and to allocate tasks to stations. We use queueing network analysis, which takes into account the mean and variance of processing times among different products to determine the necessary capacity of the material-handling system. We iterate between the two analyses to find the combined solution with the lowest costs. Work-in-process costs are also included in the analysis. Computational results are presented.     

Analytical performance models for closed-loop flexible assembly systems

Flexible Assembly Systems (FASs), which form an important subset of modern manufacturing systems, are finding increasing use in today's industry. In the planning and design phase of these systems, it is useful to have tools that predict system performance for various operating conditions. In this article, we present such a performance analysis tool based on queueing approximation for a class of FASs, namely, closed-loop flexible assembly systems (CL-FASs). For CL-FASs, we describe iterative algorithms for computing steady-state performance measures, including production rate and station utilizations. These algorithms are computationally simple and have a fast convergence rate. We derive a new approximation to correct the mean delay at each queue. This improves the accuracy of performance prediction, especially in the case of small CL-FASs. Comparisons with simulation results indicate that the approximation technique is reasonably accurate for a broad range of parameter values and system sizes. This makes possible efficient (fast and computationally inexpensive) analysis of CL-FASs under various conditions.     

A concurrent development tool for flexible assembly systems

Global competition and the rapid pace of technological change now require the almost continual introduction of product upgrades by any manufacturer. Thus, such a manufacturer is likely to market older and newer versions of a product simultaneously, not to mention niche-specific editions of any product upgrade. An increasingly successful response to this product proliferation is the implementation of flexible assembly systems. In the context of a flexible assembly system (FAS), the ability to estimate the impact of various product and process options on the maximal level of system output becomes crucial to managing the ever-changing product mix. This paper presents a tool for such impact estimation that can facilitate concurrent development and engineering. Experience with an actual FAS is the basis for the reported results. The tool is a specialized combination of discreteevent computer simulation, experimental design, and regression analysis. Application of the tool assumes FAS use with a cellular manufacturing philosophy. Thus, uncluttered process flow for a family of products in the sense of group technology places the focus on potential bottlenecks. The new tool here models the impact of process and product options on bottleneck and, hence, FAS behavior.     

Effect of reconfiguration costs on planning for capacity scalability in reconfigurable manufacturing systems

In a globally competitive market for products, manufacturers are faced with an increasing need to improve their flexibility, reliability, and responsiveness to meet the demands of their customers. Reconfigurable manufacturing systems (RMS) have become an important manufacturing paradigm, because they broadly encompass the ability to react efficiently to this environment by providing the exact capacity and functionality needed when needed. This paper studies how such new systems can manage their capacity scalability planning in a cost effective manner. An approach for modeling capacity scalability planning is proposed. The development of the model is based on set theory and the regeneration point theorem which is mapped to the reconfigurable manufacturing paradigm as the capacity scalability points of that system. The cost function of the model incorporates both the physical capacity cost based on capacity size and costs associated with the reconfiguration process which referred to as the scalability penalty cost and scalability effort cost. A dynamic programming (DP) approach is manipulated for the development of optimal capacity scalability plans. The effect of the reconfiguration costs on the capacity scalability planning horizon and overall cost is investigated. The results showed the relation between deciding on the optimal capacity scalability planning horizon and the different reconfiguration costs. Results also highlighted the fact that decreasing costs of reconfiguration will lead to cost effective implementation of reconfigurable manufacturing systems.