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

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

Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR

Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.Heckel S, Pacini A, Paredes F, Petreli M.V, Perez M, Adriani N, et al. (2022) Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR. PLoS ONE 17(11): e0271860. https://doi.org/ 10.1371/journal.pone.0271860Fil: Heckel, Sofía. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Pacini, Antonella. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Paredes, Franco. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Petreli, María Victoria. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Perez, Marilina. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Adriani, Natalia. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Ibarra, Guadalupe. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Sesma, Juliana. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Heckel, Sofía. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET); Argentina.Fil: Pacini, Antonella. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET); Argentina.Fil:. Sesma, Juliana. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET); Argentina.Fil: Heckel, Sofía. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Paredes, Franco. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Petreli, María Victoria. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Ibarra, Guadalupe. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Menzella, Hugo G. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Menzella, Hugo G. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Colaneri, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Sesma, Juliana. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina

Atypical pathogens in hospitalized patients with community-acquired pneumonia: A worldwide perspective

Background: Empirical antibiotic coverage for atypical pathogens in community-acquired pneumonia (CAP) has long been debated, mainly because of a lack of epidemiological data. We aimed to assess both testing for atypical pathogens and their prevalence in hospitalized patients with CAP worldwide, especially in relation with disease severity. Methods: A secondary analysis of the GLIMP database, an international, multicentre, point-prevalence study of adult patients admitted for CAP in 222 hospitals across 6 continents in 2015, was performed. The study evaluated frequency of testing for atypical pathogens, including L. pneumophila, M. pneumoniae, C. pneumoniae, and their prevalence. Risk factors for testing and prevalence for atypical pathogens were assessed through univariate analysis. Results: Among 3702 CAP patients 1250 (33.8%) underwent at least one test for atypical pathogens. Testing varies greatly among countries and its frequency was higher in Europe than elsewhere (46.0% vs. 12.7%, respectively, p &lt; 0.0001). Detection of L. pneumophila urinary antigen was the most common test performed worldwide (32.0%). Patients with severe CAP were less likely to be tested for both atypical pathogens considered together (30.5% vs. 35.0%, p = 0.009) and specifically for legionellosis (28.3% vs. 33.5%, p = 0.003) than the rest of the population. Similarly, L. pneumophila testing was lower in ICU patients. At least one atypical pathogen was isolated in 62 patients (4.7%), including M. pneumoniae (26/251 patients, 10.3%), L. pneumophila (30/1186 patients, 2.5%), and C. pneumoniae (8/228 patients, 3.5%). Patients with CAP due to atypical pathogens were significantly younger, showed less cardiovascular, renal, and metabolic comorbidities in comparison to adult patients hospitalized due to non-atypical pathogen CAP. Conclusions: Testing for atypical pathogens in patients admitted for CAP in poorly standardized in real life and does not mirror atypical prevalence in different settings. Further evidence on the impact of atypical pathogens, expecially in the low-income countries, is needed to guidelines implementation

Production of the Potent Antibacterial Polyketide Erythromycin C in Escherichia coli

An Escherichia coli strain capable of producing the potent antibiotic erythromycin C (Ery C) was developed by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. The megalomicin gene cluster was used as the source for the construction of two artificial operons that contained the genes encoding the deoxysugar biosynthetic and tailoring enzymes necessary to convert 6dEB to Ery C. The reconstructed mycarose operon contained the seven genes coding for the enzymes that convert glucose-1-phosphate (G-1-P) to TDP-l-mycarose, a 6dEB mycarosyl transferase, and a 6dEB 6-hydroxylase. The activity of the pathway was confirmed by demonstrating conversion of exogenous 6dEB to 3-O-α-mycarosylerythronolide B (MEB). The reconstructed desosamine operon contained the six genes necessary to convert TDP-4-keto-6-deoxyglucose, an intermediate formed in the mycarose pathway, to TDP-d-desosamine, a desosamine transferase, a 6dEB 12-hydroxylase, and the rRNA methyltransferase ErmE; the last was required to confer resistance to the host cell upon production of mature macrolide antibiotics. The activity of this pathway was demonstrated by conversion of MEB to Ery C. When the mycarose and desosamine operons were expressed in an E. coli strain engineered to synthesize 6dEB, Ery C and Ery D were produced. The successful production of Ery C in E. coli shows the potentiality of this model microorganism to synthesize novel 6-deoxysugars and to produce bioactive glycosylated compounds and also establishes the basis for the future use of E. coli both in the production of new glycosylated polyketides and for the generation of novel bioactive compounds through combinatorial biosynthesis

Correction to: Low cost and sustainable hyaluronic acid production in a manufacturing platform based on Bacillus subtilis 3NA strain

Fe de erratas del artículo Low cost and sustainable hyaluronic acid production in a manufacturing platform based on Bacillus subtilis 3NA strain.Fil: Cerminati, Sebastián. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina.Fil: Leroux, Mélanie. Université Grenoble Alpes. Centre National de la Recherche Scientifique. Centre de Recherches sur les Macromolécules Végétales; France.Fil: Anselmi, Pablo Ariel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina.Fil: Peirú, Salvador. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina.Fil: Alonso, Juan C. Centro Nacional de Biotecnología. Department of Microbial Biotechnology; Spain.Fil: Priem, Bernard. Université Grenoble Alpes. Centre National de la Recherche Scientifique. Centre de Recherches sur les Macromolécules Végétales; France.Fil: Menzella, Hugo G. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina

The Antifungal Polyketide Ambruticin Targets the HOG Pathway▿

The polyketide ambruticin is an attractive candidate for drug development as an antifungal agent, but its mechanism of action has not yet been elucidated. Here we present evidence that ambruticin exerts its effect by targeting HOG, the osmotic stress control pathway, through Hik1, a group III histidine kinase

Low cost and sustainable hyaluronic acid production in a manufacturing platform based on Bacillus subtilis 3NA strain

Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genus Streptococcus grown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large-scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption. Bacillus subtilis 3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed. A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100%. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP free platform that could be adaptable for production of other biomolecules.Fil: Cerminati, Sebastián. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina.Fil: Leroux, Mélanie. Université Grenoble Alpes. Centre National de la Recherche Scientifique. Centre de Recherches sur les Macromolécules Végétales; France.Fil: Anselmi, Pablo Ariel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina.Fil: Peirú, Salvador. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina.Fil: Alonso, Juan C. Centro Nacional de Biotecnología. Department of Microbial Biotechnology; Spain.Fil: Priem, Bernard. Université Grenoble Alpes. Centre National de la Recherche Scientifique. Centre de Recherches sur les Macromolécules Végétales; France.Fil: Menzella, Hugo G. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ-CONICET); Argentina

Using Chemobiosynthesis and Synthetic Mini-Polyketide Synthases To Produce Pharmaceutical Intermediates in Escherichia coli▿ †

Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure intermediates for the synthesis of complex molecules. Here, we describe a method to produce polyketide intermediates possessing multiple stereogenic centers by combining chemobiosynthesis and engineered mini-polyketide synthases (PKSs). Chemobiosynthesis allows the introduction of unnatural moieties, while a library of synthetic bimodular PKSs expressed from codon-optimized genes permits the introduction of a variety of ketide units. To validate the approach, intermediates for the synthesis of trans-9,10-dehydroepothilone D were generated. The designer molecules obtained have the potential to greatly reduce the manufacturing cost of epothilone analogues, thus facilitating their commercial development as therapeutic agents