Comparison of two codon optimization strategies to enhance recombinant protein production in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes. The use of codon-optimized genes may overcome this problem. However, designing a gene for optimal expression requires choosing from a vast number of possible DNA sequences and different codon optimization methods have been used in the past decade. Here, a comparative study of the two most common methods is presented using calf prochymosin as a model.</p> <p>Results</p> <p>Seven sequences encoding calf prochymosin have been designed, two using the "one amino acid-one codon" method and five using a "codon randomization" strategy. When expressed in <it>Escherichia coli</it>, the variants optimized by the codon randomization approach produced significantly more proteins than the native sequence including one gene that produced an increase of 70% in the amount of prochymosin accumulated. On the other hand, no significant improvement in protein expression was observed for the variants designed with the one amino acid-one codon method. The use of codon-optimized sequences did not affect the quality of the recovered inclusion bodies.</p> <p>Conclusions</p> <p>The results obtained in this study indicate that the codon randomization method is a superior strategy for codon optimization. A significant improvement in protein expression was obtained for the largely established process of chymosin production, showing the power of this strategy to reduce production costs of industrial enzymes in microbial hosts.</p

An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background: Biodiesels produced from transesterification of vegetable oils have a major quality problem due to thepresence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitateshave been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods toremove these compounds are not available. Here we describe a novel method for the efficient removal of SGs frombiodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.Results: A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli culturesexpressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for theselection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs fromcrude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to thetotal contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blockingtendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability toindustrial settings.Conclusions: The results presented in this work provide a novel path for the removal of steryl glucosides from biodieselusing a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption ofthis renewable fuel.Fil: Peirú, Salvador. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Keclon; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aguirre, Andres. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Keclon; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Eberhardt, Maria Florencia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Braia, Mauricio Javier. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cabrera, Rodolfo Ariel. Unitec Bio; ArgentinaFil: Menzella, Hugo Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Keclon; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Targeting eosinophils: severe asthma and beyond

Recent research in the field of bronchial asthma has mainly focused on eosinophilic disease phenotype. Several trials proved the efficacy and safety profile of eosinophils and interleukin (IL)-5 targeting molecules, currently approved for severe asthma and available on the market. They include mepolizumab and reslizumab, IL-5 blocking molecules, and benralizumab, targeting the IL-5 receptor and eliciting a NK cell-mediated antibody-dependent cellular cytotoxicity against eosinophils. Eosinophilic inflammation represents the common pathophysiological background of several conditions, providing the rationale for the use of the same biologics beyond asthma. Although with different evidence grade, from clinical trials to case reports, anti-IL-5 biologics have been investigated in eosinophilic granulomatosis with polyangitis, allergic bronchopulmonary aspergillosis, chronic eosinophilic pneumonia, nasal polyposis, hypereosinophilic syndrome, and eosinophilic esophagitis. However, non-negligible differences between asthma and other eosinophilic diseases, particularly in eosinophils homing (blood and/or tissues), target organs and thus clinical features, probably account for the different response to the same drug in different clinical conditions and highlights the need for tailoring the therapeutic approach by modulating the drug dose and/or by combination therapy with multiple drugs. The optimal safety and tolerability profile of anti-IL-5 drugs warrants further and larger experimental and real-life investigations, which are needed especially in the field of non-asthma eosinophilic diseases. This review aims at summarizing the rationale for the use of biologics in eosinophilic diseases and their mechanisms of action. The current efficacy and safety evidence about eosinophils and IL-5 targeting molecules in asthma and in eosinophilic conditions beyond bronchi is also discussed

Rational Design and Assembly of Synthetic Trimodular Polyketide Synthases

SummaryType I polyketide synthases (PKSs) consist of modules that add two-carbon units in polyketide backbones. Rearranging modules from different sources can yield novel enzymes that produce unnatural products, but the rules that govern module-module communication are still not well known. The construction and assay of hybrid bimodular units with synthetic PKS genes were recently reported. Here, we describe the rational design of trimodular PKSs by combining bimodular units. A cloning-expression system was developed to assemble and test 54 unnatural trimodular PKSs flanked by the loading module and the thioesterase from the erythromycin synthase. Remarkably, 96% of them produced the expected polyketide. The obtained results represent an important milestone toward the ultimate goal of making new bioactive polyketides by rational design. Additionally, these results show a path for the production of customized tetraketides by fermentation, which can be an important source of advanced intermediates to facilitate the synthesis of complex products

Severe refractory asthma: Current treatment options and ongoing research

Patients with severe asthma have a greater risk of asthma-related symptoms, morbidities, and exacerbations. Moreover, healthcare costs of patients with severe refractory asthma are at least 80% higher than those with stable asthma, mainly because of a higher use of healthcare resources and chronic side effects of oral corticosteroids (OCS). The advent of new promising biologicals provides a unique therapeutic option that could achieve asthma control without OCS. However, the increasing number of available molecules poses a new challenge: the identification and selection of the most appropriate treatment. Thanks to a better understanding of the basic mechanisms of the disease and the use of predictive biomarkers, especially regarding the Th2-high endotype, it is now easier than before to tailor therapy and guide clinicians toward the most suitable therapeutic choice, thus reducing the number of uncontrolled patients and therapeutic failures. In this review, we will discuss the different biological options available for the treatment of severe refractory asthma, their mechanism of action, and the overlapping aspects of their usage in clinical practice. The availability of new molecules, specific for different molecular targets, is a key topic, especially when considering that the same targets are sometimes part of the same phenotype. The aim of this review is to help clarify these doubts, which may facilitate the clinical decision-making process and the achievement of the best possible outcomes

Precision Medicine in Targeted Therapies for Severe Asthma: Is There Any Place for "omics" Technology?

According to the current guidelines, severe asthma still represents a controversial topic in terms of definition and management. The introduction of novel biological therapies as a treatment option for severe asthmatic patients paved the way to a personalized approach, which aims at matching the appropriate therapy with the different asthma phenotypes. Traditional asthma phenotypes have been decomposing by an increasing number of asthma subclasses based on functional and physiopathological mechanisms. This is possible thanks to the development and application of different omics technologies. The new asthma classification patterns, particularly concerning severe asthma, include an increasing number of endotypes that have been identified using new omics technologies. The identification of endotypes provides new opportunities for the management of asthma symptoms, but this implies that biological therapies which target inflammatory mediators in the frame of specific patterns of inflammation should be developed. However, the pathway leading to a precision approach in asthma treatment is still at its beginning. The aim of this review is providing a synthetic overview of the current asthma management, with a particular focus on severe asthma, in the light of phenotype and endotype approach, and summarizing the current knowledge about "omics" science and their therapeutic relevance in the field of bronchial asthma

Precision Medicine in Targeted Therapies for Severe Asthma:Is There Any Place for "omics" Technology?

According to the current guidelines, severe asthma still represents a controversial topic in terms of definition and management. The introduction of novel biological therapies as a treatment option for severe asthmatic patients paved the way to a personalized approach, which aims at matching the appropriate therapy with the different asthma phenotypes. Traditional asthma phenotypes have been decomposing by an increasing number of asthma subclasses based on functional and physiopathological mechanisms. This is possible thanks to the development and application of different omics technologies. The new asthma classification patterns, particularly concerning severe asthma, include an increasing number of endotypes that have been identified using new omics technologies. The identification of endotypes provides new opportunities for the management of asthma symptoms, but this implies that biological therapies which target inflammatory mediators in the frame of specific patterns of inflammation should be developed. However, the pathway leading to a precision approach in asthma treatment is still at its beginning. The aim of this review is providing a synthetic overview of the current asthma management, with a particular focus on severe asthma, in the light of phenotype and endotype approach, and summarizing the current knowledge about “omics” science and their therapeutic relevance in the field of bronchial asthma

Towards precision medicine: The application of omics technologies in asthma management

Asthma is a chronic obstructive respiratory disease characterised by bronchial inflammation. Its biological and clinical features have been widely explored and a number of pharmacological treatments are currently available. Currently several aspects of asthma pathophysiological background remain unclear, and this is represent a limitation for the traditional asthma phenotype approach. In this scenario, the identification of new molecular and clinical biomarkers may be helpful in order to better understand the disease, define specific diagnostic tools and highlight relevant novel targets for pharmacological treatments. Omics technologies offer innovative research tools for addressing the above mentioned goals. However, there is still a lot to do both in the fields of basic research and in the clinical application. Recently, genome-wide association studies, microRNAs and proteomics are contributing to enrich the available data for the identification of new asthma biomarkers. A precise approach to the patient with asthma, particularly with severe uncontrolled asthma, requires new and specific therapeutic targets, but also proper tools able to drive the clinician in tailoring the treatment. On the other hand, there is a need of predictors to treatment's response, particularly in the field of biological drugs, whose sustainability implies a correct and precise selection of the patients. Translating acquired omics knowledge in clinical practice may address the unmet needs described above, but large-scale studies are required in order to confirm their relevance and effectiveness in daily practice. Thus in our opinion the application of omics is still lagging in the real-life setting

Self-administration of omalizumab: why not? A literature review and expert opinion

Introduction: Omalizumab is used to treat severe uncontrolled allergic asthma and chronic spontaneous urticaria (CSU), and is approved for self-administration in prefilled syringes. It is thus important to understand the advantages, critical issues, and indications for home administration.Areas covered: The present review summarizes the available evidence on home administration of omalizumab in asthma and CSU to illustrate the advantages derived from self-administration of patients in this setting.Expert opinion: The available data suggest that patients can safely administer biologics at home with suitable training, and that home administration is time saving and cost-effective. The majority of patients with severe asthma or CSU treated with omalizumab are likely to be suitable candidates for self-administration, which can be proposed to anyone that the clinician deems suitable. In addition to clinicians, pharmacists can also play a key role in managing patients who are prescribed home administration. A practical flow chart is proposed on selection of patients and their management during home administration. Self-administration of biologics can be considered as a valid alternative to traditional injections in a clinical setting, and the evidence has shown that no major issues need to be overcome in terms of safety or efficacy