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The globally expanding threat of antibiotic resistance calls for the development of new strategies for abating Gram-negative bacterial infections. The use of extracorporeal blood cleansing devices with affinity sorbents to selectively capture bacterial lipopolysaccharide (LPS), which is the major constituent of Gram-negative bacterial outer membranes and the responsible agent for eliciting an exacerbated innate immune response in the host during infection, has received outstanding interest. For that purpose, molecules that bind tightly to LPS are required to functionalize the affinity sorbents. Particularly, anti-LPS factors (ALFs) are promising LPS-sequestrating molecules. Hence, in this work, molecular dynamics (MD) simulations are used to investigate the interaction mechanism and binding pose of the ALF isoform 3 from Penaeus monodon (ALFPm3), which is referred to as “AL3” for the sake of simplicity, and lipid A (LA, the component of LPS that represents its endotoxic principle). We concluded that hydrophobic interactions are responsible for AL3–LA binding and that LA binds to AL3 within the protein cavity, where it buries its aliphatic tails, whereas the negatively charged phosphate groups are exposed to the medium. AL3 residues that are key for its interaction with LA were identified, and their conservation in other ALFs (specifically Lys39 and Tyr49) was also analyzed. Additionally, based on the MD-derived results, we provide a picture of the possible AL3–LA interaction mechanism. Finally, an in vitro validation of the in silico predictions was performed. Overall, the insights gained from this work can guide the design of novel therapeutics for treating sepsis, since they may be significantly valuable for designing LPS-sequestrating molecules that could functionalize affinity sorbents to be used for extracorporeal blood detoxification.

The globally expanding threat of antibiotic resistance calls for the development of new strategies for abating Gram-negative bacterial infections. The use of extracorporeal blood cleansing devices with affinity sorbents to selectively capture bacterial lipopolysaccharide (LPS), which is the major constituent of Gram-negative bacterial outer membranes and the responsible agent for eliciting an exacerbated innate immune response in the host during infection, has received outstanding interest. For that purpose, molecules that bind tightly to LPS are required to functionalize the affinity sorbents. Particularly, anti-LPS factors (ALFs) are promising LPS-sequestrating molecules. Hence, in this work, molecular dynamics (MD) simulations are used to investigate the interaction mechanism and binding pose of the ALF isoform 3 from Penaeus monodon (ALFPm3), which is referred to as “AL3” for the sake of simplicity, and lipid A (LA, the component of LPS that represents its endotoxic principle). We concluded that hydrophobic interactions are responsible for AL3–LA binding and that LA binds to AL3 within the protein cavity, where it buries its aliphatic tails, whereas the negatively charged phosphate groups are exposed to the medium. AL3 residues that are key for its interaction with LA were identified, and their conservation in other ALFs (specifically Lys39 and Tyr49) was also analyzed. Additionally, based on the MD-derived results, we provide a picture of the possible AL3–LA interaction mechanism. Finally, an in vitro validation of the in silico predictions was performed. Overall, the insights gained from this work can guide the design of novel therapeutics for treating sepsis, since they may be significantly valuable for designing LPS-sequestrating molecules that could functionalize affinity sorbents to be used for extracorporeal blood detoxification.

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix–turn–helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

There is an increasing demand for astaxanthin in food, feed, cosmetics and pharmaceutical applications because of its superior anti-oxidative and coloring properties. However, naturally produced astaxanthin is expensive, mainly due to low productivity and limited sources. Reprogramming of microorganisms for astaxanthin production via metabolic engineering is a promising strategy. We primarily focus on the application of synthetic biology, enzyme engineering and metabolic engineering in enhancing the synthesis and accumulation of astaxanthin in microorganisms in this review. We also discuss the biosynthetic pathways of astaxanthin within natural producers, and summarize the achievements and challenges in reprogramming microorganisms for enhancing astaxanthin production. This review illuminates recent biotechnological advances in microbial production of astaxanthin. Future perspectives on utilization of new technologies for boosting microbial astaxanthin production are also discussed.

Endotoxin, or lipopolysaccharide (LPS), is recognized as the most powerful microbial mediator implicated in the pathogenesis of sepsis, a life-threatening organ disfunction whose overall prognosis is still poor. Therefore, the need to detect and remove LPS has led some authors to focus their efforts on the design of pathogen cleansing microdevices. Micromagnetophoretic cleansing involves a first step for the selective binding and sequestration of LPS on non-toxic particles followed by the removal of the loaded particles from the fluid medium. While outstanding progress has been made in magnetic separation, the state of the art of the first step still needs to fill important gaps in knowledge to advance towards the development of detection systems. Thus, this work reports an integrated methodology to advance the design of the LPS sequestration stage to promote its separation from biofluids. The methodology combines protein and separation fundamentals and starts with the synthesis of an anti LPS factor protein (LALF), followed by the quantitative determination of its binding strength to LPS. For this analysis LALF was supported on agarose beads and the variables affecting LPS binding, as binding and capture temperature, the optimum bead:protein and protein:LPS ratios, have been experimentally studied. The methodology and results here reported constitute the information needed to advance the knowledge for the correct design of LPS separation devices.

Relaxases are essential proteins for plasmid conjugation. They process the DNA to be transferred by means of a covalent intermediate. Thus, the characterization of these covalent complexes is essential to understand the biological role of this reaction and to improve it for biotechnological applications. In this article, we describe the use of the polyacrylamide electrophoresis techniques for the identification of relaxase-DNA covalent complexes, being SDS-PAGE a simple and reliable method for the detection of protein-DNA covalent adducts. Relaxases also perform a strand transfer reaction to recircularize the DNA and finish the DNA transfer process in the recipient cell. Urea-PAGE allows us the analysis of oligonucleotides generated by the strand transfer reaction. These methods could also be used for the analysis of other HUH endonucleases.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon–carbon bond formation. We report the X-ray crystal structure of the KS–CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS–CLF didomain as well as its interaction with a cognate acyl carrier protein partner.

Plasmids, when transferred by conjugation in natural environments, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida. Expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. ardC was also required if the hsdRMS system was present in E. coli recipient cells. Thus, ArdC has antirestriction activity against the HsdRMS system and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity was not needed for the antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggered an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies and settle into new hosts.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, often pose a serious risk not only when delivered in the bloodstream but also in air, the environment and several industrial fields such as pharmaceutics or food. LPS is constituted of three regions; the O-specific chain, the core region and the lipid A, which is the responsible segment of the toxicity. Previous literature dealt with the study of lipid A, its potential ligands as well as the mechanisms of Lipid A interactions that, among other applications, establish the basis for detection methods such as Limulus Amebocyte Lysate (LAL) assays and emerging biosensoring techniques. However, quantifying LPS binding affinity is an urgent need that still requires thorough studies. In this context, this work reviews the molecules that bind LPS, highlighting quantitative affinity parameters. Moreover, state of the art methods to analyze the affinity and kinetics of lipid-ligand interactions are also reviewed and different techniques have been briefly described. Thus, first, we review existing information on LPS ligands, classifying them into three main groups and targeting the comparison of molecules in terms of their interaction affinities and, second, we establish the basis for further research aimed at the development of effective methods for LPS detection and removal.

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

Some marine bacteria, such as Moritella marina, are able to produce certain amounts of the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Moreover, E.coli heterologously expressing this pfa gene cluster from M. marina is also able to produce DHA [1]. The aim of this study was to find genetic or metabolic conditions to improve DHA production in E. coli or any other microorganism. Firstly, we studied the expression of Pfa promoters in E. coli and replaced these promoters by inducible pBAD promoters in order to increase the production of the final product. Secondly, we altered the canonical carbon flux in E. coli to improve the availability of substrates for Pfa complex by two strategies: using the exogenous compound cerulenin, and deleting a initiation enzyme of a competing pathway, FabH [2]. Both strategies exploit a substrate competition mechanism between the native fatty acid synthase from E. coli and the heterologous Pfa complex from M. marina. Finally, we improved E. coli growth at low temperature by introducing two psychrophilic chaperonins, Cpn10 and Cpn60 from Oleispira antarctica [3], in order to improve protein folding. These approaches have increased the overall efficiency of DHA synthesis in E. coli, and could be used for biotechnological optimization in different organisms to synthesize DHA or other polyunsaturated fatty acids.

Omega-3 polyunsaturated fatty acids (PUFA) are produced in some unicellular organisms, such as marine gammaproteobacteria, myxobacteria, and thraustochytrids, by large enzyme complexes called PUFA synthases. These enzymatic complexes resemble bacterial antibiotic-producing proteins known as polyketide synthases (PKS). One of the PUFA synthase subunits is a conserved large protein (PfaA in marine proteobacteria) that contains three to nine tandem acyl carrier protein (ACP) domains as well as condensation and modification domains. In this work, a study of the PfaA architecture and its ability to initiate the synthesis by selecting malonyl units has been carried out. As a result, we have observed a self-acylation ability in tandem ACPs whose biochemical mechanism differ from the previously described for type II PKS. The acyltransferase domain of PfaA showed a high selectivity for malonyl-CoA that efficiently loads onto the ACPs domains. These results, together with the structural organization predicted for PfaA, suggest that this protein plays a key role at early stages of the anaerobic pathway of PUFA synthesis.

[Background]: Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specifc enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to fnd genetic or metabolic conditions to increase DHA production in E. coli., [Results]: First, we analysed the efect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations afecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most efective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is afected in its frst catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA., [Conclusions]: DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in diferent organisms.

[EN]: This thesis compromises two chapters. The first chapter describes a number of efforts to express and purify the acyltransferase of module 4 (fkAT4) involved in the biosynthesis of the medically important compound tacrolimus. Such efforts were attempted using E. coli and P. pastoris expression systems. Although it was possible to obtain the fkAT4 domain refolding inclusion bodies formed by E. coli, the recombinant domain purified didn’t keep the correct folding pattern, and thus in-vitro assays couldn’t be accomplished. The second chapter describes the development of Streptomyces rimosus platform as an alternative expression system for production of extracellular heterologous proteins. Results demonstrated the capacity of Streptomyces rimosus to produce phytase extracellularly, thus facilitating downstream processing. In addition, throughout a comparative analysis of S. rimosus transcriptome, the genetic information related for protein production, such as the secretion pathways (Sec and Tat) and production of endogenous proteases which can degrade the target protein, are described. Information gathered form the transcriptome enhanced the understanding of S. rimosus genetics, this way facilitating further efforts to develop the chassis of S. rimosus specific for production of heterologous proteins., [ES]: La presente tesis contiene dos capítulos, el primero describe diferentes estrategias empleadas para purificar el dominio aciltransferasa del 4to modulo (fkAT4) implicado en la biosíntesis de tacrolimus, fármaco empleado como agente inmunosupresor. Para la obtención del dominio fkAT4 se usaron los sistemas de expresión basados en E. coli y P. pastoris, no obstante solo fue posible la obtención de cuerpos de inclusión, los cuales no demostraron actividad enzimática luego de ser replegados. El segundo capítulo describe el desarrollo del kit genético en Streptomyces rimosus específico para la producción de proteínas heterólogas. El kit incluye una serie de plásmidos y promotores que permitieron usar a S. rimosus como huésped en la producción extracelular de fitasa, de este modo facilitando la purificación de la encima recombinante. Además, mediante el transcriptoma de S. rimosus se pudieron establecer los diferentes mecanismos por los cuales S. rimosus secreta las proteínas al medio extracelular. El sistema desarrollado en esta tesis basado en S. rimosus ofrece una alternativa a los sistemas de expresión actuales.

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3- fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.

[EN]: Fatty acids are carboxylic acids with an aliphatic chain and an even number of carbon atoms. When they have two or more unsaturations are called polyunsaturated fatty acids and have many beneficial properties for health. Fatty acids can be accumulated in the form of triglycerides, which are the main form of energy storage in living cells and can be easily converted into biofuels and other valuable products. In this doctoral thesis an in vitro evolution protocol has been developed for a bacterial protein that is able to synthesize triglycerides, achieving in this way a significant improvement in its catalytic capacity. In addition, the synthesis of polyunsaturated fatty acids in marine bacteria has been studied from a biochemical and structural biology point of view, improving in this way the general understanding of the complex of proteins responsible for this biological process., [ES]: Los ácidos grasos son ácidos carboxílicos con una cadena alifática y con un número par de átomos de carbono. Cuando éstos poseen dos o más insaturaciones se denominan ácidos grasos poliinsaturados y poseen muchas propiedades beneficiosas para la salud. Los ácidos grasos se pueden acumular en forma de triglicéridos, que constituyen la forma principal de almacenamiento de energía en los seres vivos y además pueden ser fácilmente convertidos en biocombustibles y otros productos valiosos. En esta tesis doctoral se ha desarrollado un método de evolución in vitro para una proteína bacteriana productora de triglicéridos, consiguiéndose una mejora significativa en su capacidad catalítica. Además es esto se ha estudiado la síntesis de ácidos grasos poliinsaturados en bacterias marinas desde un punto de vista bioquímico y de la biología estructural, mejorando de esta forma el entendimiento del complejo de proteínas responsable de este proceso biológico.