Nou mètode per mesurar un inductor de proteïnes recombinants

L'IPTG és un sucre sintètic anàleg de la lactosa que s'utilitza per a la inducció de la producció de proteïnes en el bacteri Escherichia coli. En la present tesi doctoral s'ha desenvolupat per primera vegada un mètode analític per mesurar l'IPTG en el medi de cultiu i dins de les cèl·lules que ha permès estudiar el comportament d'inducció en cultius d'alta densitat cel·lular així com optimitzar la seva dosi per obtenir la màxima quantitat de producte.El IPTG es un azúcar sintético análogo de la lactosa que se utiliza para la inducción de la producción de proteínas en la bacteria Escherichia coli. En la presente tesis doctoral se ha desarrollado por primera vez un método analítico para medir el IPTG en el medio de cultivo y dentro de las células que ha permitido estudiar el comportamiento de inducción en cultivos de alta densidad celular así como optimizar su dosis para obtener la máxima cantidad de producto

Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

Background: The 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. Results: The 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. Conclusions: In 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

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

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

Opportunities for the development of cassava waste biorefineries for the production of polyhydroxyalkanoates in Sub-Saharan Africa

The use of plastic materials is forecasted to double by 2030 in Africa. The increase in plastic demand presents an opportunity to develop biopolymers such as polyhydroxyalkanoates (PHA) instead of petroleum-based plastics. However, the high cost of PHA production is closely linked to feedstock price, which will hinder their deployment. On the other hand, the Sub-Saharan Africa (SSA) economy is heavily reliant on agriculture, with cassava being one of the most important crops. Cassava industries in SSA produce 146 Mtpa cassava, generating an estimated 40 Mtpa waste, of which 55% goes to landfill or is incinerated. The use of cassava waste as a carbon source for PHA production, therefore, represents an opportunity to decrease production costs of bioplastics while contributing to waste management solutions. This review critically analyses the potential for developing cassava waste biorefineries for the production of PHA in SSA, a region where the bioplastics industry is in a nascent stage. We conclude that cassava waste is an adequate resource for the production of bioplastics in the SSA region that can also contribute toward the reduction of GHG emissions whilst decreasing the dependence on fossil fuels. We identify cost reduction potential with PHA-overproducing strains or strains capable of utilizing substrates more efficiently and show the economic attractiveness of using waste biomass resources in a circular economy framing. Finally, we make recommendations on the next steps needed to pave the way for sustainable economic development, job creation and industrial activity in the SSA region using circular economy principles

Metabolic characterisation of Magnetospirillum gryphiswaldense MSR-1 using LC-MS-based metabolite profiling

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, an understanding of the intracellular metabolites and 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 ¼ 6) and O2-rich conditions (n = 6) corresponding to magnetosome- and non-magnetosome producing cells, respectively. Multivariate, univariate and pathway enrichment analyses were conducted to identify significantly altered metabolites and pathways. Rigorous metabolite identification was carried out using authentic standards, the 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 ¼ 0.76, R2Y ¼ 0.99 and Q2 ¼ 0.98 in OPLS-DA. As a result, 50 metabolites linked to 45 metabolic pathways were found to be 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 understanding the metabolic mechanisms that affect Mgryph growth and magnetosome formation

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1

Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform

Computer-aided design for metabolic engineering.

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

Digital image analysis of the human bite mark

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN010688 / BLDSC - British Library Document Supply CentreGBUnited Kingdo