Ease Of Engineering Research Articles

Fermentative production of 3-hydroxypropionic acid by using metabolically engineered Klebsiella pneumoniae strains

3-Hydroxypropionic acid (3-HP) is an industrially important platform chemical for super-absorbent or biodegradable polymers. Its production via biological methods is expected to be more competitive than chemical methods. Klebsiella pneumoniae is the most promising host due to its innate capabilities for 3-HP and vitamin-B12 production, ease of culture, and ease of engineering. In this study, step-by-step metabolic engineering and fermentation technologies were used to enhance the production of 3-HP. K. pneumoniae-derived ydcW gene was overexpressed using a plasmid after screening candidate genes. Major competing pathways encoded by dhaT, yqhD, ldhA, glpK, poxB, and pta-ackA were blocked. Additionally, it was demonstrated that simultaneous reinforcement of two native aldehyde dehydrogenase encoded by the ydcW gene preferring NADPH and the puuC gene preferring NADH, synergistically improved 3-HP production. Additional reinforcement of the acs gene to reduce acetate accumulation resulted in 93.7 g/L of 3-HP with a yield of 0.42 g/g·glycerol over a 72-h fed-batch fermentation. This performance is deemed sufficient for industrial applications.

Radiolabeled Affibody Molecules for PET Imaging.

Owing to their ease of engineering and production, chemical stability, size, and high target affinity and specificity, radiolabeled affibody molecules have been recognized as very promising molecular imaging probes in both preclinical and clinical settings. Herein we describe the methods for the preparation of affibody-chelator conjugates and their subsequent radiolabeling with 18F-AlF, 68Ga, 89Zr.

Immunogenicity and Pre-Clinical Efficacy of an OMV-Based SARS-CoV-2 Vaccine.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

Selection, identification and crystal structure of shark-derived single-domain antibodies against a green fluorescent protein

Shark variable domain of new antigen receptors (VNARs) are the smallest naturally occurring binding domains with properties of low complexity, small size, cytoplasmic expression, and ease of engineering. Green fluorescent protein (GFP) molecules have been analyzed in conventional microscopy, but their spectral characteristics preclude their use in techniques offering substantially higher resolution. Besides, the GFP molecules can be quenched in acidic environment, which makes it necessary to develop anti-GFP antibody to solve these problems. In view of the diverse applications of GFP and unique physicochemical features of VNAR, the present study aims to generate VNARs against GFP. Here, we identified 36 VNARs targeting eCGP123, an extremely stable GFP, by phage display from three immunized sharks. These VNARs bound to eCGP123 with affinity constant KD values ranging from 6.76 to 605 nM. Among them, two lead VNARs named aGFP-14 and aGFP-15 with nanomolar eCGP123-binding affinity were selected for in-depth characterization. aGFP-14 and aGFP-15 recognized similar epitopes on eCGP123. X-ray crystallography studies clarified the mechanism by which aGFP14 interacts with eCGP123. aGFP-14 also showed cross-reaction with EGFP, with KD values of 47.2 nM. Finally, immunostaining analyses demonstrated that aGFP-14 was able to bind effectively to the EGFP expressed in both cultured cells and mouse brain tissues, and can be used as a fluorescence amplifier for EGFP. Our research demonstrates a feasible idea for the screening and production of shark-derived VNARs. The two high-affinity VNARs developed in the study contribute to the diversity of GFP sdAbs and may enhance the applications of GFP.

Application of Metal-Organic Framework in Diagnosis and Treatment of Diabetes.

Diabetes-related chronic wounds are often accompanied by a poor wound-healing environment such as high glucose, recurrent infections, and inflammation, and standard wound treatments are fairly limited in their ability to heal these wounds. Metal–organic frameworks (MOFs) have been developed to improve therapeutic outcomes due to their ease of engineering, surface functionalization, and therapeutic properties. In this review, we summarize the different synthesis methods of MOFs and conduct a comprehensive review of the latest research progress of MOFs in the treatment of diabetes and its wounds. State-of-the-art in vivo oral hypoglycemic strategies and the in vitro diagnosis of diabetes are enumerated and different antimicrobial strategies (including physical contact, oxidative stress, photothermal, and related ions or ligands) and provascular strategies for the treatment of diabetic wounds are compared. It focuses on the connections and differences between different applications of MOFs as well as possible directions for improvement. Finally, the potential toxicity of MOFs is also an issue that we cannot ignore.

Saccharomyces cerevisiae as a Heterologous Host for Natural Products.

Cell factories can provide a sustainable supply of natural products with applications as pharmaceuticals, food-additives or biofuels. Besides being an important model organism for eukaryotic systems, Saccharomyces cerevisiae is used as a chassis for the heterologous production of natural products. Its success as a cell factory can be attributed to the vast knowledge accumulated over decades of research, its overall ease of engineering and its robustness. Many methods and toolkits have been developed by the yeast metabolic engineering community with the aim of simplifying and accelerating the engineering process.In this chapter, a range of methodologies are highlighted, which can be used to develop novel natural product cell factories or to improve titer, rate and yields of an existing cell factory with the goal of developing an industrially relevant strain. The addressed topics are applicable for different stages of a cell factory engineering project and include the choice of a natural product platform strain, expression cassette design for heterologous or native genes, basic and advanced genetic engineering strategies, and library screening methods using biosensors. The many engineering methods available and the examples of yeast cell factories underline the importance and future potential of this host for industrial production of natural products.

Molecularly Imprinted Polymer-Based Sensors for Priority Pollutants.

Globally, there is growing concern about the health risks of water and air pollution. The U.S. Environmental Protection Agency (EPA) has developed a list of priority pollutants containing 129 different chemical compounds. All of these chemicals are of significant interest due to their serious health and safety issues. Permanent exposure to some concentrations of these chemicals can cause severe and irrecoverable health effects, which can be easily prevented by their early identification. Molecularly imprinted polymers (MIPs) offer great potential for selective adsorption of chemicals from water and air samples. These selective artificial bio(mimetic) receptors are promising candidates for modification of sensors, especially disposable sensors, due to their low-cost, long-term stability, ease of engineering, simplicity of production and their applicability for a wide range of targets. Herein, innovative strategies used to develop MIP-based sensors for EPA priority pollutants will be reviewed.

Topological Nanophotonics: Toward Robust Quantum Circuits

In the last five years, the combination of advances in nanofabrication and a series of ingenious design ideas has enabled the implementation of topological concepts into nanophotonic platforms. In parallel, an unprecedented amount of research effort is being devoted to quantum technologies, with quantum information processing promising exponentially better performance than its classic counterpart for certain tasks. Nanophotonics is bound to play a key role in quantum information technologies given its compactness, low-power requirements, room-temperature operation, and the inherent high speed and low noise of photons. However, the scalability of quantum information systems in integrated photonic platforms is limited by scattering loss and other errors from random fabrication imperfections. The remarkable robustness to disorder and imperfections manifested by photonic states in topological systems emerges as a fascinating research avenue to tackle this issue. Furthermore, the ease of engineering and fabricating nanophotonic platforms with distinctive geometries and symmetries that can effectively generate and transport complex quantum states makes nanophotonics an ideal platform in which to realize topological protection of quantum photonic states. This article highlights the pioneering experimental efforts to understand the potential of topological protection on quantum photonic states and to outline a path toward robust quantum circuits.

Duplexed aptamers: history, design, theory, and application to biosensing.

Nucleic acid aptamers are single stranded DNA or RNA sequences that specifically bind a cognate ligand. In addition to their widespread use as stand-alone affinity binding reagents in analytical chemistry, aptamers have been engineered into a variety of ligand-specific biosensors, termed aptasensors. One of the most common aptasensor formats is the duplexed aptamer (DA). As defined herein, DAs are aptasensors containing two nucleic acid elements coupled via Watson-Crick base pairing: (i) an aptamer sequence, which serves as a ligand-specific receptor, and (ii) an aptamer-complementary element (ACE), such as a short DNA oligonucleotide, which is designed to hybridize to the aptamer. The ACE competes with ligand binding, such that DAs generate a signal upon ligand-dependent ACE-aptamer dehybridization. DAs possess intrinsic advantages over other aptasensor designs. For example, DA biosensing designs generalize across DNA and RNA aptamers, DAs are compatible with many readout methods, and DAs are inherently tunable on the basis of nucleic acid hybridization. However, despite their utility and popularity, DAs have not been well defined in the literature, leading to confusion over the differences between DAs and other aptasensor formats. In this review, we introduce a framework for DAs based on ACEs, and use this framework to distinguish DAs from other aptasensor formats and to categorize cis- and trans-DA designs. We then explore the ligand binding dynamics and chemical properties that underpin DA systems, which fall under conformational selection and induced fit models, and which mirror classical SN1 and SN2 models of nucleophilic substitution reactions. We further review a variety of in vitro and in vivo applications of DAs in the chemical and biological sciences, including riboswitches and riboregulators. Finally, we present future directions of DAs as ligand-responsive nucleic acids. Owing to their tractability, versatility and ease of engineering, DA biosensors bear a great potential for the development of new applications and technologies in fields ranging from analytical chemistry and mechanistic modeling to medicine and synthetic biology.

Blueprints for Biosensors: Design, Limitations, and Applications.

Biosensors are enabling major advances in the field of analytics that are both facilitating and being facilitated by advances in synthetic biology. The ability of biosensors to rapidly and specifically detect a wide range of molecules makes them highly relevant to a range of industrial, medical, ecological, and scientific applications. Approaches to biosensor design are as diverse as their applications, with major biosensor classes including nucleic acids, proteins, and transcription factors. Each of these biosensor types has advantages and limitations based on the intended application, and the parameters that are required for optimal performance. Specifically, the choice of biosensor design must consider factors such as the ligand specificity, sensitivity, dynamic range, functional range, mode of output, time of activation, ease of use, and ease of engineering. This review discusses the rationale for designing the major classes of biosensor in the context of their limitations and assesses their suitability to different areas of biotechnological application.

Comparing proteins and nucleic acids for next-generation biomolecular engineering

Nanostructures built from biomolecules such as proteins, DNA and RNA are attracting attention in many areas of biological and materials sciences. Such nanoscale engineering was pioneered with proteins, yet the use of DNA is rapidly gaining traction. What are the advantages of the different biopolymers and which is best suited for a given molecular structure, function or application? In this Review, we evaluate the different structural properties of proteins and nucleic acids, as well as possible designs and synthetic routes for functional nanostructures. By comparing protein engineering and DNA nanotechnology, we highlight molecular architectures that are relevant in biotechnology, biomedicine and synthetic biology research, and identify emerging areas for research such as hybrid materials composed of protein and DNA/RNA. Proteins, DNA and RNA can be used to build functional nanostructures. This Review compares protein and DNA/RNA in terms of biochemical properties and ease of engineering in three major areas of application: biomolecular recognition, biocatalysis and structural support.

Two-dimensional photonic crystal slabs as a tunable polarization mode converter

Two dimensional photonic crystal slabs with elliptical holes exposed to the normal incident laser light are proposed and designed as a tunable broadband polarization rotator. The photonic crystal electric field mode due to the eccentricity and orientation of the holes will provide a mean to exchange power between two orthogonal polarization states. Around guided resonance wavelengths of photonic crystals with 45° oriented elliptical holes, 20% polarization mode conversion is achieved. By tuning of the difference of the two radii of the elliptical holes we are able to span a bandwidth of 130nm and 70nm around wavelengths of 1445nm and 1650nm. The unique properties of these devices such as ease of engineering and simplified light coupling scheme will make them a promising polarization mode converter in high speed polarization-multiplexed optical transmission systems.

Artificial affinity proteins as ligands of immunoglobulins.

A number of natural proteins are known to have affinity and specificity for immunoglobulins. Some of them are widely used as reagents for detection or capture applications, such as Protein G and Protein A. However, these natural proteins have a defined spectrum of recognition that may not fit specific needs. With the development of combinatorial protein engineering and selection techniques, it has become possible to design artificial affinity proteins with the desired properties. These proteins, termed alternative scaffold proteins, are most often chosen for their stability, ease of engineering and cost-efficient recombinant production in bacteria. In this review, we focus on alternative scaffold proteins for which immunoglobulin binders have been identified and characterized.

An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas.

The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains.

DNA-based molecular architecture with spatially localized components

Performing computation inside living cells offers life-changing applications, from improved medical diagnostics to better cancer therapy to intelligent drugs. Due to its bio-compatibility and ease of engineering, one promising approach for performing in-vivo computation is DNA strand displacement. This paper introduces computer architects to DNA strand displacement "circuits", discusses associated architectural challenges, and proposes a new organization that provides practical composability. In particular, prior approaches rely mostly on stochastic interaction of freely diffusing components. This paper proposes practical spatial isolation of components, leading to more easily designed DNA-based circuits. DNA nanotechnology is currently at a turning point, with many proposed applications being realized [20, 9]. We believe that it is time for the computer architecture community to take notice and contribute.

Live recombinant vectors for AIDS vaccine development.

Live recombinant vectors entered the AIDS vaccine field with the realization that live attenuated HIV vaccines posed too great a safety risk, and that subunit vaccines elicited antibodies which lacked the breadth or potency needed to induce sterilizing immunity. Vectored vaccines provided a means to bring the cellular arm of the immune system into play by mimicking natural viral infection. By delivering antigens within host cells, processing and presentation could occur for induction of cellular immune responses. This recombinant vector approach, either alone or combined with other strategies, has produced impressive results. Recombinants have been generated from DNA and RNA viruses and bacteria. With few exceptions, each vector poses some risk, yet each possesses unique features that make it attractive. In addition to safety, key considerations in vector selection have included previous success as a vaccine against the wild-type agent or other pathogens; ability to induce potent, persistent immune responses; ability to target mucosal inductive sites and antigen presenting cells; lack of integration into the host genome; presence of pre-existing immunity in people; ease of mucosal administration; cloning capacity; ease of engineering and production; and stability of the final product. Here we up-date the status of several live recombinant vectors that have shown good potential in pre-clinical studies. Some have progressed to human clinical trials, and others will shortly. The abundance of vectors, coupled with the complexity arising from use of combination regimens with other vaccine types and heterologous vectors, will necessitate selection of the most promising candidates for large-scale efficacy trials in people. The sooner comparative studies can be designed and implemented in which live recombinant vectors containing the same inserted genes are evaluated head-to-head, the closer we will be to an eventual vaccine.