Properties of a "Split-and-Stuttering" Module of an Assembly Line Polyketide Synthase.

Notwithstanding the "one-module-one-elongation-cycle" paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as "stuttering", have been derived through and analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a -AT subfamily PKS that is also naturally split across two polypeptides.

Challenges and opportunities for engineering assembly-line polyketide biosynthesis in .

Assembly-line polyketide synthases generate natural products that have led to many live-saving drugs. The use of as a heterologous host for reconstituting these enormous and complex enzymatic machines has and will continue to be a critical strategy for understanding them. Here, we concisely summarize successful examples in exploiting for assembly-line polyketide biosynthesis as well as offer examples of new challenges in which this approach is primed to tackle.

Evolution and Diversity of Assembly-Line Polyketide Synthases.

Assembly-line polyketide synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in the clinic. Their present-day diversity is the result of an evolutionary path that has involved the emergence of a multimodular architecture and further diversification of assembly-line PKSs. In this review, we provide an overview of previous studies that investigated PKS evolution and propose a model that challenges the currently prevailing view that gene duplication has played a major role in the emergence of multimodularity.

Split cGAL, an intersectional strategy using a split intein for refined spatiotemporal transgene control in .

Bipartite expression systems, such as the GAL4-UAS system, allow fine manipulation of gene expression and are powerful tools for interrogating gene function. Recently, we established cGAL, a GAL4-based bipartite expression system for transgene control in , where a single promoter dictates the expression pattern of a cGAL driver, which then binds target upstream activation sequences to drive expression of a downstream effector gene. Here, we report a split strategy for cGAL using the split intein gp41-1 for intersectional control of transgene expression.

Cell-specific proteomic analysis in Caenorhabditis elegans.

Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine.

Cell surface display yields evolvable, clickable antibody fragments.

Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments.

Chemical tools for temporally and spatially resolved mass spectrometry-based proteomics.

Accurate measurements of the abundances, synthesis rates and degradation rates of cellular proteins are critical for understanding how cells and organisms respond to changes in their environments. Over the past two decades, there has been increasing interest in the use of mass spectrometry for proteomic analysis. In many systems, however, protein diversity as well as cell and tissue heterogeneity limit the usefulness of mass spectrometry-based proteomics.

Microfluidic fabrication of hydrogel microparticles containing functionalized viral nanotemplates.

We demonstrate rapid microfluidic fabrication of hybrid microparticles composed of functionalized viral nanotemplates directly embedded in polymeric hydrogels. Specifically, genetically modified tobacco mosaic virus (TMV) templates were covalently labeled with fluorescent markers or metalized with palladium (Pd) nanoparticles (Pd-TMV) and then suspended in a poly(ethylene glycol)-based solution. Upon formation in a flow-focusing device, droplets were photopolymerized with UV light to form microparticles.

Spatiotemporal controlled delivery of nanoparticles to injured vasculature.

There are a number of challenges associated with designing nanoparticles for medical applications. We define two challenges here: (i) conventional targeting against up-regulated cell surface antigens is limited by heterogeneity in expression, and (ii) previous studies suggest that the optimal size of nanoparticles designed for systemic delivery is approximately 50-150 nm, yet this size range confers a high surface area-to-volume ratio, which results in fast diffusive drug release. Here, we achieve spatial control by biopanning a phage library to discover materials that target abundant vascular antigens exposed in disease.

Multifunctional superparamagnetic Janus particles.

In this study, we report the microfluidic-based synthesis of a multifunctional Janus hydrogel particle with anisotropic superparamagnetic properties and chemical composition for the bottom-up assembly of hydrogel superstructures. In a uniform magnetic field, the resulting Janus magnetic particles fabricated in the present method exhibit chainlike or meshlike superstructure forms, the complexity of which can be simply modulated by particle density and composition. This controllable field-driven assembly of the particles can be potentially used as building blocks to construct targeted superstructures for tissue engineering.

Tuning curvature in flow lithography: a new class of concave/convex particles.

Polymeric particles of complex shapes and chemistry have been used for a wide variety of applications in the materials and bioengineering fields. An interesting means of introducing complexity is through curvature. In this work, stop-flow lithography is used to generate concave/convex particles at high throughputs of 3x10(4) particles/h.

PLGA-lecithin-PEG core-shell nanoparticles for controlled drug delivery.

Current approaches to encapsulate and deliver therapeutic compounds have focused on developing liposomal and biodegradable polymeric nanoparticles (NPs), resulting in clinically approved therapeutics such as Doxil/Caelyx and Genexol-PM, respectively. Our group recently reported the development of biodegradable core-shell NP systems that combined the beneficial properties of liposomal and polymeric NPs for controlled drug delivery. Herein we report the parameters that alter the biological and physicochemical characteristics, stability, drug release properties and cytotoxicity of these core-shell NPs.