Biology subjects

Jimenez, J. I.

Publications and source records attributed to Jimenez, J. I..

11 recordsLinked to original sources

bioRxiv2025

Pseudomonas putida JM37 as a novel bacterial chassis for ethylene glycol upcycling

Ethylene glycol (EG), one of the main monomers of polyethylene terephthalate (PET), is an attractive target for microbial upcycling. Despite this interest, there is a limited number of described organisms that can efficiently metabolise EG. Here, we report the metabolic and biotechnological potential of Pseudomonas putida JM37 as a novel bacterial chassis for EG valorization. We show that JM37 efficiently grows on EG as the sole carbon and energy source, outperforming other Pseudomonas strains. Genome sequencing and directed mutagenesis revealed that genetic redundancies in the glyoxylate assimilation pathways underlie its robust EG metabolism. Beyond biomass generation, we demonstrated the biotechnological potential of JM37. This strain was able to accumulate medium-chain polyhydroxyalkanoates (mcl-PHAs), dominated by C10 monomers, directly from EG. Moreover, JM37 successfully expressed heterologous biosynthetic pathways, including a violacein biosynthetic operon and a PET-hydrolase which has been secreted actively into the extracellular medium. Together, our results support the use of P. putida JM37 as a versatile synthetic biology chassis for sustainable EG upcycling and as a promising platform for circular bioproduction.

synthetic biology↗

bioRxiv2025

Directed evolution and characterisation of light harvesting complexes with altered energy transfer dynamics in purple non-sulfur bacteria

Purple non-sulfur bacteria (PNSB) are metabolically versatile microorganisms that inhabit diverse environments by taking advantage of a remarkably efficient photosynthetic machinery. In this work, we describe the creation of a workflow for experimental studies that aim to compare the energy transfer mechanisms occurring within structural variants of the light-harvesting complex 2 (LH2) of PNSB. Through the creation of a library of LH2 variants using site-directed mutagenesis, we engineered proteins with different spectral properties to be expressed in a LH2-defficient mutant of the model PNSB Rhodobacter sphaeroides. We validated this approach by reproducing a previously described mutant exhibitng a blue-shift, in addition to identifying a novel mutant exhibiting a red-shift of the B850 absorption peak. We characterised the fluorescence lifetime of the purified LH2 spectral variants in vitro, and performed a bacterial growth assay to assess the fitness of the LH2 variants in vivo under oversaturating light conditions. Our results suggest that the LH2s variants expressed by PNSB in nature reflect the intricate tunning of their quantum properties not towards the fastest energy transfer but towards the optimum light-harvesting efficiency which is defined by diverse environmental factors. Statement of significanceIn this work we report a platform for the systematic investigation of the mutational landscape of light harvesting complexes forming part of the photosynthetic machinery of purple non-sulfur bacteria. By conducting directed evolution of selected residues of the light-harvesting antenna (LH2) of Rhodobacter sphaeroides we identified a novel mutant with distinct and red-shifted spectral properties. We characterised the energy transfer dynamics of this and a previous characterised mutant and demonstrated that the new variant confers a phenotypic growth advantage when cultured with high-intensity light. Our findings offer new insights into the mechanisms of light capture and energy transfer also bridging the in vitro observations with quantifiable fitness advantages under the conditions tested.

biophysics↗

bioRxiv2025

Investigating The Potential of Division of Labour in Synthetic Bacterial Communities for the Production of Violacein

With advancements in synthetic biology and metabolic engineering, microorganisms can now be engineered to perform increasingly complex functions, which may be limited by the resources available in individual cells. Division of labour in synthetic microbial communities offers a promising approach to enhance metabolic efficiency and resilience in bioproduction. By distributing complex metabolic pathways across multiple subpopulations, the resource competition and metabolic burden imposed on an individual cell is reduced, potentially enabling more efficient production of target compounds. Violacein is a high-value pigment with anti-tumour properties that exemplifies such a challenge due to its complex bioproduction pathway, imposing a significant metabolic burden on host cells. In this study we investigated the benefits of division of labour for violacein production by splitting the violacein bioproduction pathway between two subpopulations of Escherichia coli based synthetic communities. We tested several pathway splitting strategies and reported that splitting the pathway into two subpopulations expressing VioABE and VioDC at a final composition of 60:40 yields a 2.5 fold increase in violacein production as compared to a monoculture. We demonstrated that the coculture outperforms the monoculture when both subpopulations exhibit similar metabolic burden levels, resulting in comparable growth rates, and when both subpopulations are present in sufficiently high proportions.

bioengineering↗

bioRxiv2024

Degradation of PET plastic with engineered environmental bacteria

Polyethylene terephthalate (PET) is one of the most widely used plastic materials in the food and textile industry. Consequently, post-consumer PET waste is a common environmental pollutant that leaks into the environment in the form of macro and microplastics with concerning health impacts. There is a pressing need to identify novel and sustainable solutions to process the abundance of PET waste contributing to this pollution. While there is extensive research into enzymes able to hydrolyse PET in vitro, a similar solution for discarded or difficult-to-collect PET based on whole-cell microbial catalysts is missing. In this work we report the engineering of environmental bacteria to use PET as a growth substrate. This was achieved by isolating a strain of Pseudomonas umsongensis able to use the PET monomer terephthalate as carbon source, engineering the strain to effectively secrete the high-activity PET hydrolase PHL7 through the addition of a recombinant TAT secretion leader sequence, and enhancing the bioavailability of PET by transforming it into an amorphous and macroporous structure by pre-treatment with an organic solvent. Our findings demonstrate the direct microbial consumption of PET, which could lead to improved and more sustainable upcycling strategies for this plastic.

synthetic biology↗

bioRxiv2022

Production of selenium nanoparticles occurs through an interconnected pathway of sulfur metabolism and oxidative stress response in Pseudomonas putida KT2440

The soil bacterium Pseudomonas putida KT2440 has been shown to produce selenium nanoparticles aerobically from selenite; however, the molecular actors involved in this process are unknown. Here, through a combination of genetic and analytical techniques, we report the first insights into selenite metabolism in this bacterium. Our results suggest that the reduction of selenite occurs through an interconnected metabolic network involving central metabolic reactions, sulfur metabolism, and the response to oxidative stress. Genes such as sucA, D2HGDH and PP_3148 revealed that the 2-ketoglutarate and glutamate metabolism is important to converting selenite into selenium. On the other hand, mutants affecting the activity of sulfite reductase reduced the bacterias ability to transform selenite. Other genes related to sulfur metabolism (ssuEF, sfnCE, sqrR, sqr and pdo2) and stress response (gqr, lsfA, ahpCF and sadI) were also identified as involved in selenite transformations. Interestingly, suppression of genes sqrR, sqr and pdo2 resulted in the production of selenium nanoparticles at a higher rate than the wild-type strain, which is of biotechnological interest. The data provided in this study brings us closer to understanding the metabolism of selenium in bacteria, and offers new targets for the development of biotechnological tools for the production of selenium nanoparticles.

microbiology↗

bioRxiv2021

Regulatory perturbations of ribosome allocation results in a trade-off between fast growth and adaptation capacity

Bacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655 in glucose minimal medium. Mutants with only 3 of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Higher growth rates due to increased ribosome content affected resource allocation. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) reshape the proteome for growth with a concomitant fitness cost HighlightsMutants with only 3 ribosomal operons grow faster than wild-type in minimal medium {Delta}4 rrn and rpoB mutants share phenotypic traits Faster growth of mutants is achieved by increased ribosome content Fast-growing mutants display reduced hedging expression and adaptation trade-offs Despite similar ribosomal content in rich medium the mutants present growth defects

systems biology↗

bioRxiv2021

The interplay between growth rate and nutrient quality defines gene expression capacity

The gene expression capacity of bacterial cells depends on the interplay between growth and the availability of the transcriptional and translational machinery. Growth rate is widely accepted as the global physiological parameter controlling the allocation of cell resources. This allocation has an impact on the ability of the cell to produce both host and heterologous proteins required for synthetic circuits and pathways. Understanding the relationship between growth and resources is key for the efficient design of artificial genetic constructs, however, it is obscured by the mutual dependence of growth and gene expression on each other. In this work, we investigate the individual contributions of molecular factors, growth rate and metabolism to gene expression by investigating the behaviour of bacterial cells growing in chemostats in growth-limited conditions. We develop a model of the whole cell that captures trade-offs in gene expression arising from the individual contributions of different factors, and validate it by analysing gene couplings which emerge from competition for the gene expression machinery. Our results show that while growth rate and molecular factors, such as the number of rRNA operons, set the abundance of transcriptional and translational machinery available, it is metabolism that governs the usage of those resources by tuning elongation rates. We show that synthetic gene expression capacity can be maximised by using low growth in a high-quality medium. These findings provide valuable insights into fundamental trade-offs in microbial physiology that will inform future strain and bioprocesses optimisation.

synthetic biology↗

bioRxiv2020

The loss of the pyoverdine secondary receptor in Pseudomonas aeruginosa results in a fitter strain suitable for population invasion

The rapid emergence of antibiotic resistant bacterial pathogens constitutes a critical problem in healthcare and requires the development of novel treatments. Potential strategies include the exploitation of microbial social interactions based on public goods, which are produced at a fitness cost by cooperative microorganisms, but can be exploited by cheaters that do not produce these goods. Cheater invasion has been proposed as a Trojan horse approach to infiltrate pathogen populations with strains deploying built-in weaknesses (e.g. sensitiveness to antibiotics). However, previous attempts have been often unsuccessful because population invasion by cheaters was prevented by various mechanisms including the presence of spatial structure (e.g. growth in biofilms), which limits the diffusion and exploitation of public goods. Here we followed an alternative approach and examined whether the manipulation of public good uptake and not its production could result in potential Trojan horses suitable for population invasion. We focused on the siderophore pyoverdine produced by the human pathogen Pseudomonas aeruginosa MPAO1 and manipulated its uptake by deleting and/or overexpressing the pyoverdine primary (FpvA) and secondary (FpvB) receptors. We found that receptor synthesis feeds back on pyoverdine production and uptake rates, which led to strains with altered pyoverdine-associated costs and benefits. Moreover, we found that the receptor FpvB was advantageous under iron-limited conditions but revealed hidden costs in the presence of an antibiotic stressor (gentamicin). As a consequence, FpvB mutants became the fittest strain under gentamicin exposure, displacing the wildtype in liquid cultures, and in biofilms and during infections of the wax moth larvae Galleria mellonella, which both represent structured environments. Our findings reveal that an evolutionary trade-off associated with the costs and benefits of a versatile pyoverdine uptake strategy can be harnessed for devising a Trojan horse candidate for medical interventions.

microbiology↗

bioRxiv2019

A quantitative method for proteome reallocation using minimal regulatory interventions

Engineering resource allocation in biological systems for synthetic biology applications is an ongoing challenge. Wild type organisms allocate abundant cellular resources for ensuring survival in changing environments, reducing the productivity of engineered functions. Here we present a novel approach for engineering the resource allocation of Escherichia coli by rationally modifying the transcriptional regulatory network of the bacterium. Our method (ReProMin) identifies the minimal set of genetic interventions that maximise the savings in cell resources that would normally be used to express non-essential genes. To this end we categorize Transcription Factors (TFs) according to the essentiality of the genes they regulate and we use available proteomic data to rank them based on its proteomic balance, defined as the net proteomic charge they release. Using a combinatorial approach, we design the removal of TFs that maximise the release of the proteomic charge and we validate the model predictions experimentally. Expression profiling of the resulting strain shows that our designed regulatory interventions are highly specific. We show that our resulting engineered strain containing only three mutations, theoretically releasing 0.5% of their proteome, has higher proteome budget and show increased production yield of a molecule of interest obtained from a recombinant metabolic pathway. This approach shows that combining whole-cell proteomic and regulatory data is an effective way of optimizing strains in a predictable way using conventional molecular methods.\n\nImportanceBiological regulatory mechanisms are complex and occur in hierarchical layers such as transcription, translation and post-translational mechanisms. We foresee the use of regulatory mechanism as a control layer that will aid in the design of cellular phenotypes. Our ability to engineer biological systems will be dependent on the understanding of how cells sense and respond to their environment at a system level. Few studies have tackled this issue and none of them in a rational way. By developing a workflow of engineering resource allocation based on our current knowledge of E. colis regulatory network, we pursue the objective of minimizing cell proteome using a minimal genetic intervention principle. We developed a method to rationally design a set of genetic interventions that reduce the hedging proteome allocation. Using available datasets of a model bacterium we were able to reallocate parts of the unused proteome in laboratory conditions to the production of an engineered task. We show that we are able to reduce the unused proteome (theoretically 0.5%) with only three regulatory mutations designed in a rational way, which results in strains with increased capabilities for recombinant expression of pathways of interest.\n\nHighlightsO_LIProteome reduction with minimal genetic intervention as design principle\nC_LIO_LIRegulatory and proteomic data integration to identify transcription factor activated proteome\nC_LIO_LIDeletion of the TF combination that reduces the greater proteomic load\nC_LIO_LIRegulatory interventions are highly specific\nC_LIO_LIDesigned strains show less burden, improved protein and violacein production\nC_LI

synthetic biology↗

bioRxiv2018

Engineering Translational Resource Allocation Controllers: Mechanistic Models, Design Guidelines, and Potential Biological Implementations

The use of orthogonal ribosomes in combination with dynamic resource allocation controllers is a promising approach for relieving the negative effects of cellular resource limitations on the modularity of synthetic gene circuits. Here, we develop a detailed mechanistic model of gene expression and resource allocation, which when simplified to a tractable level of complexity, allows the rational design of translational resource allocation controllers. Analysis of this model reveals a fundamental design trade-off; that reducing coupling acts to decrease gene expression. Through a sensitivity analysis of the experimentally tuneable controller parameters, we identify how each controller design parameter affects the overall closed-loop behaviour of the system, leading to a detailed set of design guidelines for optimally managing this trade-off. Based on our designs, we evaluated a number of alternative potential experimental implementations of the proposed system using commonly available biological components. Finally, we show that the controller is capable of dynamically allocating ribosomes as needed to restore modularity in a number of more complex synthetic circuits, such as the repressilator, and activation cascades composed of multiple interacting modules.

synthetic biology↗

bioRxiv2017

Dynamic Allocation Of Orthogonal Ribosomes Facilitates Uncoupling Of Co-Expressed Genes

Introduction of synthetic circuits into host microbes creates competition between circuit and host genes for shared cellular resources, such as RNA polymerases and ribosomes. This can lead to the emergence of unwanted coupling between the expression of different genes, complicating circuit design and potentially leading to circuit failure. Here we demonstrate the ability of orthogonal ribosomes to alleviate the effects of this resource competition. We partition the ribosome pool by expressing an engineered 16S RNA with altered specificity, and use this division of specificity to build simple resource allocators which reduce the level of ribosome-mediated gene coupling. We then design and implement a dynamic resource allocation controller, which acts to increase orthogonal ribosome production as the demand for translational resources by a synthetic circuit increases. Our results highlight the potential of dynamic translational resource allocation as a means of minimising the impact of cellular limitations on the function of synthetic circuitry.

synthetic biology↗