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To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli., This work was supported by the Spanish MICINN, project number CTQ2011-28398-CO2-01, the research group 2009SGR281, and by the Bioprocess Engineering and Applied Biocatalisys Group, Department of Chemical, Biological and Environmental Engineering of the Universitat Autònoma de Barcelona, Cerdanyola del Valles (Spain).

Nanoparticle Tracking Analysis (NTA) has been employed to measure the particle concentration and size distribution of magnetosomes extracted and purified from Magnetospirillum gryphiswaldense MSR-1, and then exposed to probe ultrasonication for various times, or 1% (w/v) sodium dodecyl sulphate (SDS) for 1 h. Particle concentration increased 3.7-fold over the first 15 min of ultrasonication (from 2 × 108 to >7.3 × 108 particles mL−1), but fell steeply to ∼3.6 × 108 particles mL−1 after 20 min. NTA of untreated magnetosome preparation confirmed a wide particle distribution dominated by larger species (D[1,0] = 312 nm; Dn50 = 261 nm; mode = 243 nm) with no particles in the size range of isolated single magnetosomes. After 5 min of ultrasonication the whole particle size distribution shifted to smaller size (D[1,0] = 133 nm; Dn50 = 99 nm; mode = 36 nm, corresponding to individual magnetosomes), but longer treatment times (15 and 20 min) reversed the previous transition; all characteristic numbers of the particle size distributions increased and very few small particles were detected. Side-by-side comparison of NTA and TEM sizing data revealed remarkable similarity at low ultrasonication times, with both showing single magnetosomes accounted for ∼30% population after 5 min. Exposure of magnetosomes to SDS resulted in a ∼3-fold increase in particle concentration to 5.8 × 108 particles mL−1, narrowing of the size distribution and gross elimination of particles below 60 nm. We conclude that NTA is a rapid cost-effective technique for measuring particle concentration, size distribution and aggregation state of magnetosomes in solution.

Nanoparticle Tracking Analysis (NTA) has been employed to measure the particle concentration and size distribution of magnetosomes extracted and purified fromMagnetospirillum gryphiswaldenseMSR-1, and then exposed to probe ultrasonication for various times, or 1% (w/v) sodium dodecyl sulphate (SDS) for 1 h. Particle concentration increased 3.7-fold over the first 15 min of ultrasonication (from 2 × 108to >7.3 × 108particles mL−1), but fell steeply to ~3.6 × 108particles mL−1after 20 min. NTA of untreated magnetosome preparation confirmed a wide particle distribution dominated by larger species (D[1,0] = 312 nm; Dn50= 261 nm; mode = 243 nm) with no particles in the size range of isolated single magnetosomes. After 5 min of ultrasonication the whole particle size distribution shifted to smaller size (D[1,0] = 133 nm; Dn50= 99 nm; mode = 36 nm, corresponding to individual magnetosomes), but longer treatment times (15 and 20 min) reversed the previous transition; all characteristic numbers of the particle size distributions increased and very few small particles were detected. Side-by-side comparison of NTA and TEM sizing data revealed remarkable similarity at low ultrasonication times, with both showing single magnetosomes accounted for ~30% population after 5 min. Exposure of magnetosomes to SDS resulted in a ~3-fold increase in particle concentration to 5.8 × 108particles mL−1, narrowing of the size distribution and gross elimination of particles below 60 nm. We conclude that NTA is a rapid cost-effective technique for measuring particle number, size distribution and aggregation state of magnetosomes in solution, but requires further work to improve its resolving power.

Magnetosomes are nano-sized magnetic nanoparticles with exquisite properties that can be used in a wide range of healthcare and biotechnological applications. They are biosynthesised by magnetotactic bacteria (MTB) such as Magnetospirillum gryphiswaldense MSR-1 (Mgryph). However, magnetosome bioprocessing yields low quantities compared to chemical synthesis of magnetic nanoparticles. Therefore, the understanding of the intracellular metabolites and the metabolic networks related to Mgryph growth and magnetosome formation are vital to unlock the potential of this organism to develop improved bioprocesses. In this work, we investigated the metabolism of Mgryph using untargeted metabolomics. Liquid chromatography-mass spectrometry (LC-MS) was performed to profile spent medium samples of Mgryph cells grown under O2-limited (n=6) and O2-rich conditions (n=6) corresponding to magnetosome- and non-magnetosome producing cells, respectively. Cross-validated multivariate, univariate and pathway enrichment analyses were conducted to identify significantly altered metabolites and pathways. Rigorous metabolite identification was carried out using authentic standards, Mgryph-specific metabolite database and MS/MS mzCloud database. PCA and OPLS-DA showed clear separation and clustering of sample groups with cross-validation values of R2X=0.76, R2Y=0.99 and Q2=0.98 in OPLS-DA. As a result, 50 metabolites linked to 45 metabolic pathways were found significantly altered in the tested conditions including glycine, serine and threonine; butanoate; alanine, aspartate and glutamate metabolism; aminoacyl-tRNA biosynthesis and; pyruvate and citric acid cycle (TCA) metabolisms. Our findings demonstrate the potential of LC-MS to characterise key metabolites in Mgryph and will contribute to further understand the metabolic mechanisms that affect Mgryph growth and magnetosome formation.

Magnetosomes are nano-sized magnetic nanoparticles with exquisite properties that can be used in a wide range of healthcare and biotechnological applications. They are biosynthesised by magnetotactic bacteria (MTB), such as

BACKGROUND The extraction of biopharmaceuticals from plasma and serum often employs overly complicated antiquated procedures that can inflict serious damage on especially prone protein targets and which afford low purification power and overall yields. This paper describes systematic development of a high-gradient magnetic fishing process for recovery of immunoglobulins from unclarified antiserum. RESULTS Non-porous superparamagnetic particles were transformed into hydrophobic-charge induction adsorbents and then used to recover immunoglobulins from rabbit antiserum feedstocks. Comprehensive characterisation tests conducted with variously diluted clarified antiserum on a magnetic rack revealed that immunoglobulin binding was rapid (equilibrium reached in 72% of the immunoglobulin present in an unclarified antiserum feed was recovered in 0.5 h in >3-fold purified form. CONCLUSIONS Fast magnetic particle based capture of antibodies from an unclarified high-titre feed has been demonstrated. Efficient product recovery from ultra-high titre bioprocess liquors by high-gradient magnetic fishing requires that improved magnetic adsorbents displaying high selectivity, ultra-high capacity and operational robustness are used with 'state-of-the-art' rotor-stator magnetic separators. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ethyl lactate is a hydrophilic solvent produced from bio-renewable sources (bioethanol and lactic acid produced from corn fermentation) that is considered as a “green” solvent due to its extremely low toxicity, biodegradability and negligible eco-toxicity. This work focuses on the utilization of ethyl lactate to form aqueous two-phase systems (ATPS) in the presence of inorganic salts for the extraction of antibiotics from aqueous solutions. The performance of three thiosulfate salts (Na2S2O3, K2S2O3 and (NH4)2S2O3) as salting-out media for the extraction of chloramphenicol and tetracycline from their aqueous solutions was examined. In this respect, cloud points for the ternary solutions composed of ethyl lactate, water and salt were determined at atmospheric pressure (0.1 MPa) and 298.2 K. Partition coefficients of chloramphenicol and tetracycline between the two phases were determined by chemical analysis of phases in equilibrium for different initial compositions at 298.2 K. This paper is the first report to demonstrate the ability of the ATPS based on ethyl lactate to efficiently separate antibiotics. Thus, ATPS based on ethyl lactate represents a new and green platform for the extraction of antibiotics from aqueous solutions which can facilitate their detection, identification and quantification in surface waters as well as their extraction from fermentation broths.

Volatile fatty acids (VFAs) derived from organic wastes are being considered as low-cost feedstock for microbial lipid production as a valuable alternative to plant derived oils/biodiesel. In this study, VFAs were produced from anaerobic open culture fermentation of wastepaper and subsequently, used as a feedstock for lipid production by Cryptococcus curvatus. Total VFAs, yield and productivity achieved from waste office paper (WOP) and waste newspaper (WNP) were 17.3 and 10.2 g/L, 0.17 and 0.10 g/g TS, and 0.86 and 0.51 g/L/day, respectively. Biomass, lipid content and productivity achieved utilizing VFAs derived from WOP and WNP were 4.3 and 2.9 g/L, 41.2 and 27.7% DCW, and 0.037 and 0.033 g/L/h, respectively. The dominance of fatty acids such as oleic, palmitic, stearic and linoleic acid in the lipids suggests a high level of similarity with plant/vegetable oils used for biodiesel production. Therefore, VFAs derived from wastepaper could be potentially used as feedstock to produce microbial lipids towards cost-effective production of biodiesel.

Highlights • Magnetosomes are natural intracellular, membrane-bound, magnetic nanoparticles. • Magnetosomes have a variety of clinical and biotechnological applications. • Magnetosomes are currently difficult to produce at large scale. • We developed a simple, scalable, fermentation strategy for magnetosome production. • The methods developed will aid development of magnetosome technologies., The development of a simple pH-stat fed-batch fermentation strategy for the production of Magnetospirillum gryphiswaldense MSR-1 and magnetosomes (nanoscale magnetic organelles with biotechnological applications) is described. Flow cytometry was exploited as a powerful analytical tool for process development, enabling rapid monitoring of cell morphology, physiology and polyhydroxyalkanoate production. The pH-stat fed-batch growth strategy was developed by varying the concentrations of the carbon source (lactic acid) and the alternative electron acceptor (sodium nitrate) in the feed. Growth conditions were optimized on the basis of biomass concentration, cellular magnetism (indicative of magnetosome production), and intracellular iron concentration. The highest biomass concentration and cellular iron content achieved were an optical density at 565 nm of 15.5 (equivalent to 4.2 g DCW·L−1) and 33.1 mg iron·g−1 DCW, respectively. This study demonstrates the importance of analyzing bacterial physiology during fermentation development and will potentially aid the industrial production of magnetosomes, which can be used in a wide range of biotechnology and healthcare applications.

Magnetotactic bacteria (MTB) are a diverse group of bacteria that synthesise magnetosomes, magnetic membrane-bound nanoparticles that have a variety of diagnostic, clinical and biotechnological applications. We present the development of rapid methods using flow cytometry to characterize several aspects of the physiology of the commonly-used MTB Magnetospirillum gryphiswaldense MSR-1. Flow cytometry is an optical technique that rapidly measures characteristics of individual bacteria within a culture, thereby allowing determination of population heterogeneity and also permitting direct analysis of bacteria. Scatter measurements were used to measure and compare bacterial size, shape and morphology. Membrane permeability and polarization were measured using the dyes propidium iodide and bis-(1,3-dibutylbarbituric acid) trimethine oxonol to determine the viability and ‘health’ of bacteria. Dyes were also used to determine changes in concentration of intracellular free iron and polyhydroxylakanoate (PHA), a bacterial energy storage polymer. These tools were then used to characterize the responses of MTB to different O2 concentrations and iron-sufficient or iron-limited growth. Rapid analysis of MTB physiology will allow development of bioprocesses for the production of magnetosomes, and will increase understanding of this fascinating and useful group of bacteria.

The development and application of biotechnology-based strategies has had a great socio-economical impact and is likely to play a crucial role in the foundation of more sustainable and efficient industrial processes. Within biotechnology, metabolic engineering aims at the directed improvement of cellular properties, often with the goal of synthesizing a target chemical compound. The use of computer-aided design (CAD) tools, along with the continuously emerging advanced genetic engineering techniques have allowed metabolic engineering to broaden and streamline the process of heterologous compound-production. In this work, we review the CAD tools available for metabolic engineering with an emphasis, on retrosynthesis methodologies. Recent advances in genetic engineering strategies for pathway implementation and optimization are also reviewed as well as a range of bionalytical tools to validate in silico predictions. A case study applying retrosynthesis is presented as an experimental verification of the output from Retropath, the first complete automated computational pipeline applicable to metabolic engineering. Applying this CAD pipeline, together with genetic reassembly and optimization of culture conditions led to improved production of the plant flavonoid pinocembrin. Coupling CAD tools with advanced genetic engineering strategies and bioprocess optimization is crucial for enhanced product yields and will be of great value for the development of non-natural products through sustainable biotechnological processes.

An unsteady, unstructured, unsegregated and based on first principles mathematical model has been proposed to describe IPTG (isopropyl-β- d -tiogalactopiranoside) transport in induced fed-batch cultures of E. coli M15 Δ glyA [pQEαβrham] [pREP4] producing rhamnulose 1-phosphate aldolase (RhuA). The model predicts extracellular and intracellular IPTG concentration. Experimental extracellular IPTG concentrations under different operational conditions were obtained by HPLC–MS analysis. These experimental data were used to fit the parameters of the model. The model was also able to predict the experimental behavior of two different E. coli strains producing fuculose 1-phosphate aldolase (FucA). IPTG transport to cells was the contribution of three processes: a diffusion process, and two active processes (one non-specific and another specific).

For recombinant protein production in E. coli fed-batch cultures, post-induction conditions have great influence in the quantity and quality of the product. The present paper covers the effect of different factors affecting the cellular environment in recombinant aldolase (rhamnulose-1-phosphate aldolase, RhuA) production. An operational mode employing an exponential addition profile for constant specific growth rate has been analyzed, in order to understand and define possible modifications with influence on post-induction cellular behavior. A constant addition profile has been demonstrated to render higher specific aldolase production than the exponential addition profile, probably due to a more constant environment for the cells. On the other hand, amino acid (leucine) supplementation has proven to increase protein quality in terms of activity units (U) per unit mass of RhuA (U mg−1 RhuA), alleviating metabolic overload. Based on the above, a production process was set up and scaled up to pilot plant. Resulting production was double that of a standard laboratory operation, 45,000 U L−1, and almost all the protein retained the 6xHis-tag with the highest quality, 11.3 U mg−1 RhuA.

The over-expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition or activation of secondary metabolism pathways. Moreover, the expression of other plasmid-encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth kinetics.In this work, we have developed a second-generation system suitable for Escherichia coli expression with an antibiotic-free plasmid maintenance mechanism based on a glycine auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression was minimized by tuning the expression levels of the repressor protein (LacI) and glyA using a library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. The engineered antibiotic-free expression system was applied to the l-fuculose phosphate aldolase (FucA) over-production, showing an increase in production up to 3.8-fold in terms of FucA yield (mgg-1DCW) and 4.5-fold in terms of FucA activity (AUg-1DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAcgDCW-1.Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity. © 2015 Elsevier B.V., This work was supported by the Spanish MICINN, project number CTQ2011-28398-CO2-01 and the research group 2009SGR281 and by the Bioprocess Engineering and Applied Biocatalisys Group, department of Chemical Engineering of the Universitat Autonoma de Barcelona, Cerdanyola del Valles (Spain). M.P. acknowledges the Universitat Autònoma de Barcelona for the pre-doctoral fellowship. Appendix A

International audience; Metabolic engineering has succeeded in biosynthesis of numerous commodity or high value compounds. However, the choice of pathways and enzymes used for production was many times made ad hoc, or required expert knowledge of the specific biochemical reactions. In order to rationalize the process of engineering producer strains, we developed the computer-aided design (CAD) tool RetroPath that explores and enumerates metabolic pathways connecting the endogenous metabolites of a chassis cell to the target compound. To experimentally validate our tool, we constructed 12 top-ranked enzyme combinations producing the flavonoid pinocembrin, four of which displayed significant yields. Namely, our tool queried the enzymes found in metabolic databases based on their annotated and predicted activities. Next, it ranked pathways based on the predicted efficiency of the available enzymes, the toxicity of the intermediate metabolites and the calculated maximum product flux. To implement the top-ranking pathway, our procedure narrowed down a list of nine million possible enzyme combinations to 12, a number easily assembled and tested. One round of metabolic network optimization based on RetroPath output further increased pinocembrin titers 17-fold. In total, 12 out of the 13 enzymes tested in this work displayed a relative performance that was in accordance with its predicted score. These results validate the ranking function of our CAD tool, and open the way to its utilization in the biosynthesis of novel compounds.

BackgroundThe E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes.ResultsThe results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time.ConclusionsIn the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon., This work has been supported by the Spanish MICINN, projects CTQ2008-00578 and CTQ2011-28398-C02-01, and by DURSI 2009SGR281 Generalitat de Catalunya.

The lac-operon and its components have been studied for decades and it is widely used as one of the common systems for recombinant protein production in Escherichia coli. However, the role of the lactose permease, encoded by the lacY gene, when using the gratuitous inducer IPTG for the overexpression of heterologous proteins, is still a matter of discussion. A lactose permease deficient strain was successfully constructed. Growing profiles and acetate production were compared with its parent strain at shake flask scale. Our results show that the lac-permease deficient strain grows slower than the parent in defined medium at shake flask scale, probably due to a downregulation of the phosphotransferase system (PTS). The distributions of IPTG in the medium and inside the cells, as well as recombinant protein production were measured by HPLC-MS and compared in substrate limiting fed-batch fermentations at different inducer concentrations. For the mutant strain, IPTG concentration in the medium depletes slower, reaching at the end of the culture higher concentration values compared with the parent strain. Final intracellular and medium concentrations of IPTG were similar for the mutant strain, while higher intracellular concentrations than in medium were found for the parent strain. Comparison of the distribution profiles of IPTG of both strains in fed-batch fermentations showed that lac-permease is crucially involved in IPTG uptake. In the absence of the transporter, apparently IPTG only diffuses, while in the presence of lac-permease, the inducer accumulates in the cytoplasm at higher rates emphasizing the significant contribution of the permease-mediated transport.