Biology subjects

Nikel, P. I.

Publications and source records attributed to Nikel, P. I..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Disentangling the regulatory response of Agrobacterium tumefaciens CHLDO to glyphosate for engineering whole-cell phosphonate biosensors

AO_SCPLOWBSTRACTC_SCPLOWPhosphonates (PHTs), organic compounds with a stable C--P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as the source of genetic parts for constructing PHT biosensors. In this species, the phn gene cluster, encoding the C--P lyase pathway, is regulated by the PhnF transcriptional repressor and is part of the Pho regulon. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise optimization of the transcriptional cascade, we created a biosensor capable of detecting GP in the 0.25-50 M range in various samples, including soil and water.

synthetic biology↗

bioRxiv2024

Long-term evolution reveals the role of the circadian cycle in the environmental adaptation of cyanobacteria

Circadian clocks synchronize internal cellular states with diurnal rhythms. Widespread in bacteria and eukaryotes, they regulate a variety of physiological processes, from hormone secretion in animals to carbon fixation in photosynthetic organisms. The adaptive role of circadian clocks is assumed to stem from their ability to anticipate environmental change, yet their impact on ecological adaptation remains unclear. Here, we use experimental evolution to study the interplay between fitness and circadian regulation in the model cyanobacterium Synechococcus elongatus PCC 7942. After 1,200 generations under continuous, high-intensity illumination, we obtained a strain that grew six times faster than its ancestral counterpart. Genome sequencing revealed three mutations fixed in the population, two of which replicated the fast-growing phenotype in the wild-type. A deletion in SasA, a key circadian regulator, was essential for fast growth. Transcriptomic and metabolomic analyses revealed that this mutation perturbed the rhythmicity of the cycle, while simultaneously locking the cell in a transcriptomic response to high intensity illumination. A comparison with another fast- growing isolate, UTEX 2973, showed convergent transcriptomic states despite different driving mutations. Our results indicate that the clock acts not only as a timekeeping device, but also as an adaptive mechanism to optimize growth across diverse ecological conditions.

microbiology↗

bioRxiv2024

Leveraging engineered Pseudomonas putida minicells for bioconversion of organic acids into short-chain methyl ketones

Methyl ketones, key building-blocks widely used in diverse industrial applications, largely depend on oil-derived chemical methods for their production. Here, we investigated bio-based production alternatives for short-chain ketones, adapting the solvent-tolerant soil bacterium Pseudomonas putida as a host for ketone biosynthesis either by whole-cell biocatalysis or using engineered minicells, chromosome-free bacterial vesicles. Organic acids (acetate, propanoate and butyrate) were selected as the main carbon substrate to drive the biosynthesis of acetone, 2-butanone and 2-pentanone. Pathway optimization identified efficient enzyme variants from Clostridium acetobutylicum and Escherichia coli, which were tested under both constitutive and inducible expression of the cognate genes. By implementing these optimized pathways in P. putida minicells, which can be prepared through a simple 3-step purification protocol, the feedstock was converted into the target short-chain methyl ketones, remaining catalytically functional for >4 months. These results highlight the value of combining morphology and pathway engineering of non-canonical bacterial hosts to establish alternative bioprocesses for toxic chemicals that are difficult to produce by conventional approaches. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/574483v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@14db69eorg.highwire.dtl.DTLVardef@1104c0corg.highwire.dtl.DTLVardef@f0c725org.highwire.dtl.DTLVardef@1a28c2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Release of extracellular DNA by Pseudomonas species as a major determinant for biofilm switching and an early indicator for cell population control

Withdrawal StatementThe authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2021/430776. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: 10.1101/2021.02.11.430776

microbiology↗

bioRxiv2023

High-yield enzymatic synthesis of mono- and trifluorinated alanine enantiomers

Fluorinated amino acids are a promising entry point for incorporating new-to-Nature chemistries in biological systems. Hence, novel methods are needed for the selective synthesis of these building blocks. In this study, we focused on the enzymatic synthesis of fluorinated alanine enantiomers. To this end, the alanine dehydrogenase from Vibrio proteolyticus and the diaminopimelate dehydrogenase from Symbiobacterium thermophilum were applied to the in vitro production of (R)-3-fluoroalanine and (S)-3-fluoroalanine, respectively, using 3-fluoropyruvate as the substrate. Additionally, an alanine racemase from Streptomyces lavendulae, originally selected for setting an alternative enzymatic cascade leading to the production of these non-canonical amino acids, had an unprecedented catalytic efficiency in the {beta}-elimination of fluorine from the monosubstituted fluoroalanine. The in vitro enzymatic cascade based on the dehydrogenases of V. proteolyticus and S. thermophilum included a cofactor recycling system, whereby a formate dehydrogenase from Pseudomonas sp. 101 (either native or engineered) coupled formate oxidation to NAD(P)H formation. Under these conditions, the reaction yields for (R)-3-fluoroalanine and (S)-3-fluoroalanine reached >85% on the fluorinated substrate and proceeded with complete enantiomeric excess. Moreover, the selected dehydrogenases were also able to catalyze the conversion of trifluoropyruvate into trifluorinated alanine, as a first-case example of biocatalysis with amino acids carrying a trifluoromethyl group.

synthetic biology↗

bioRxiv2023

Time-resolved, deuterium-based fluxomics uncovers the hierarchy and dynamics of sugar processing by Pseudomonas putida

Pseudomonas putida, a soil bacterium widely used for synthetic biology and metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. Such a periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we have developed a novel methodology for metabolic flux analysis, D-fluxomics, based on deuterated sugar substrates. D-Fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate--unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.

systems biology↗

bioRxiv2023

The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Pseudomonas species

A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) and an adenine base-editor (ABE) have been optimized for precise single-nucleotide modification of plasmid and genome targets. CBE comprises a cytidine deaminase conjugated to a Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting in C[->]T (or G[->]A) substitutions. Conversely, ABE consists of an adenine deaminase fused to SpnCas9 for A[->]G (or T[->]C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems and a novel protospacer adjacent motif (PAM)-relaxed SpnCas9 (SpRY) variant. Base-editing was validated in Pseudomonas putida and other Gram-negative bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins and metabolic (antibiotic-resistance) functions. The functional knockouts obtained by engineering STOP codons via CBE were reverted to the wild-type genotype using ABE. Additionally, a series of induction-responsive vectors was developed to facilitate the curing of the base-editing platform in a single cultivation step, simplifying complex strain engineering programs without relying on homologous recombination and yielding plasmid-free, modified bacterial cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/537106v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1ac5e70org.highwire.dtl.DTLVardef@16c0efforg.highwire.dtl.DTLVardef@14caaf4org.highwire.dtl.DTLVardef@6b968f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

Pseudomonas putida KT2440 is an attractive bacterial host for biotechnological production of valuable chemicals from renewable lignocellulosic feedstocks as it can valorize lignin-derived aromatics or cellulosic glucose. P. putida EM42, a genome-reduced variant of P. putida KT2440 endowed with advantageous physiological properties, was recently engineered for growth on cellobiose, a major cellooligosaccharide product of enzymatic cellulose hydrolysis. Co-utilization of cellobiose with glucose was achieved in a mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the observed co-utilization was not understood and the {Delta}gcd strain suffered from a significant growth defect. In this study, we aimed to investigate the basis of the simultaneous uptake of the two sugars and accelerate the growth of P. putida EM42 {Delta}gcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion abolished the inhibition of the exogenous {beta}-glucosidase BglC from Thermobifida fusca by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of the hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore the rapid growth on glucose. The reduced growth rate of the {Delta}gcd mutant was partially compensated by the implantation of heterologous glucose (Glf from Zymomonas mobilis) and cellobiose (LacY from Escherichia coli) transporters. Remarkably, this intervention resulted in the accumulation of pyruvate in aerobic P. putida cultures. We demonstrated that the excess of this key metabolic intermediate can be redirected to the enhanced biosynthesis of ethanol and lactate. The overproduction of pyruvate was then unveiled by an upgraded genome-scale metabolic model constrained with proteomic and kinetic data. The model pointed to the saturation of glucose catabolism enzymes due to unregulated substrate uptake and it predicted improved bioproduction of pyruvate-derived chemicals by the engineered strain. This work sheds light on the co-metabolism of cellulosic sugars in an attractive biotechnological host and introduces a novel strategy for pyruvate overproduction in bacterial cultures under aerobic conditions. HighlightsO_LICo-utilization of glucose and cellobiose achieved in P. putida EM42 {Delta}gcd mutant. C_LIO_LIGrowth defect of the mutant compensated by implanting exogenous sugar transporters. C_LIO_LIEnhanced influx of carbon caused aerobic overproduction of pyruvate and acetate. C_LIO_LICarbon from excess pyruvate streamed into ethanol or L-lactate. C_LIO_LIPyruvate overproduction unveiled by a mathematical model of P. putida metabolism. C_LI

bioengineering↗

bioRxiv2022

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways of a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Inspired by this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-guided engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route--the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic auxotroph to integrate the PKT shunt to restore C2 prototrophy. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff and the synthetic PKT pathway for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

synthetic biology↗

bioRxiv2022

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains empowers synthetic formate assimilation

AO_SCPLOWBSTRACTC_SCPLOWFormate is a promising, water-soluble C1 feedstock for biotechnology since it can be efficiently produced from CO2--but very few industrially-relevant hosts have been engineered towards formatotrophy. Here, the non-pathogenic soil bacterium Pseudomonas putida was adopted as a platform for synthetic formate assimilation. The metabolism of genome-reduced variants of P. putida was rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. The rGly pathway mediates the formate [->] glycine [->] serine transformations that yield pyruvate, ultimately assimilated into biomass. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylorubrum extorquens) and native reactions. Promoter engineering of chromosomally-encoded functions coupled to modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate for energy supply. Adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs significantly reduced doubling times to ca. 15 h. During evolution, two catabolic regimes became predominant in independently evolved clones, either via glycine hydroxymethylation (GlyA) or oxidation (ThiO). Taken together, our results expand the landscape of microbial platforms for C1-based biotechnological production towards supporting a formate bioeconomy. GO_SCPLOWRAPHICALC_SCPLOW AO_SCPLOWBSTRACTC_SCPLOW O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/499488v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@10b12edorg.highwire.dtl.DTLVardef@7a4eaorg.highwire.dtl.DTLVardef@1dc101aorg.highwire.dtl.DTLVardef@a52d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

A non-conventional archaeal fluorinase identified by in silico mining catalyzes the fastest natural nucleophilic fluorination

Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential biocatalysts for the sustainable synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorinases retrieved from systematic in silico mining and identified a non-conventional archaeal enzyme from Methanosaeta sp. that mediates the highest fluorination rate reported to date. Furthermore, we demonstrate enhanced production of fluoronucleotides in vivo in a bacterial host engineered with this archaeal fluorinase, paving the way towards synthetic metabolism for efficient biohalogenations. Fastest biological SN2 fluorination

synthetic biology↗

bioRxiv2021

Parameterization of regulatory nodes for engineering broad host range heterologous gene expression

By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacIq-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3 and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in an Escherichia coli strain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of cross-regulation offer a panoply of choices to potential users and help interoperability of the specific constructs.

synthetic biology↗

bioRxiv2021

Extracellular DNA (eDNA) enables early detection of the phenotypic switch of Pseudomonas sp. during biofilm development

The different steps involved in biofilm formation have been the subjects of intensive researches. However, the very early cell decision-making process related to the switch from planktonic to sessile state still remains uncharacterized. Based on the use of Pseudomonas putida KT2440 and derivatives with varying biofilm-forming capabilities, we observed a subpopulation of cells bound to extracellular DNA (eDNA) in the planktonic phase, as indicated by propidium iodide (PI) staining. Strikingly, the size of this eDNA-bound/PI-positive subpopulation correlated with the overall biofilm forming capability of the bacterial population. This finding challenges the conventional view of phenotypic switching and suggests that, in Pseudomonas, biofilm switching is determined collectively based on the quantity of eDNA released in the supernatant. The whole process can be followed based on automated flow cytometry, and the appearance of PI-positive cells was considered as an early-warning indicator for biofilm formation. For this purpose, automated glucose pulsing was used successfully to interfere with the proliferation of PI-positive cells, resulting in a reduction of biofilm formation. This study provides insights into the collective determinants of biofilm switching in Pseudomonas species and introduces a potential strategy for controlling biofilm formation.

microbiology↗

bioRxiv2021

Spatiotemporal manipulation of the mismatch repair system of Pseudomonas putida accelerates phenotype emergence

Developing complex phenotypes in industrially-relevant bacteria is a major goal of metabolic engineering, which encompasses the implementation of both rational and random approaches. In the latter case, several tools have been developed towards increasing mutation frequencies--yet the precise spatiotemporal control of mutagenesis processes continues to represent a significant technical challenge. Pseudomonas species are endowed with one of the most efficient DNA mismatch repair (MMR) systems found in bacteria. Here, we investigated if the endogenous MMR system could be manipulated as a general strategy to artificially alter mutation rates in Pseudomonas species. To bestow a conditional mutator phenotype in the platform bacterium Pseudomonas putida, we constructed inducible mutator devices to modulate the expression of the dominant-negative mutLE36K allele. Regulatable overexpression of mutLE36K in a broad-host-range, easy-to-cure plasmid format resulted in a transitory inhibition of the MMR machinery, leading to a significant increase (up to 438-fold) in mutation frequencies and a heritable fixation of genome mutations. Following such accelerated mutagenesis-followed-by selection approach, three phenotypes were successfully evolved: resistance to antibiotics streptomycin and rifampicin and reversion of a synthetic uracil auxotrophy. Thus, these mutator devices could be applied to accelerate evolution of metabolic pathways in long-term evolutionary experiments, alternating cycles of (inducible) mutagenesis coupled to selection schemes.

synthetic biology↗

bioRxiv2021

Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria

Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic gatekeepers for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH. ImportanceProtein families have likely arisen during evolution by gene duplication and divergence followed by neo-functionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the co-existence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically-versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of co-existing glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs encoded in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+- or NADP+-specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy towards achieving redox balance according to the growth conditions.

systems biology↗

bioRxiv2020

Redox stress reshapes carbon fluxes of Pseudomonas putida for cytosolic glucose oxidation and NADPH generation

The soil bacterium and metabolic engineering platform Pseudomonas putida tolerates high levels of endogenous and exogenous oxidative stress, yet the ultimate reason of such property remains unknown. To shed light on this question, NADPH generation routes--the metabolic currency that fuels redox stress responses--were assessed when P. putida KT2440 was challenged with H2O2 as proxy of oxidative conditions. 13C-tracer experiments, metabolomics and flux analysis, together with inspection of physiological parameters and measurement of enzymatic activities, revealed a substantial flux reconfiguration under oxidative stress. In particular, periplasmic glucose processing was rerouted to cytoplasmic oxidation, and cyclic operation of the pentose phosphate pathway led to significant NADPH fluxes, exceeding biosynthetic demands by ~50%. This NADPH surplus, in turn, fuelled the glutathione system for H2O2 reduction. These properties not only contribute to the tolerance of P. putida to environmental stresses, but they also highlight the value of this host for harsh biotransformations.

microbiology↗

bioRxiv2018

Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida

In contrast to the current paradigm of using microbial monocultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed-species consortia to perform functions that are difficult to program into individual strains. Additionally, the division of labor between specialist species found in natural consortia can lead to increased catalytic efficiency and stability relative to a monoculture or a community composed of generalists. In this work, we have designed a synthetic co-culture for phototrophic degradation of xenobiotics, composed of a cyanobacterium, (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). Cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose as the only carbon source and to degrade the environmental pollutant 2,4-dinitrotoluene (2,4-DNT). The synthetic consortium is able to degrade 2,4-DNT with only light and CO2 as inputs for the system, and it was stable over time through repeated backdilutions. Furthermore, cycling this consortium through low nitrogen medium promoted the accumulation of polyhydroxyalkanoate (PHA)-an added-value biopolymer-in P. putida, thus highlighting the versatility of this production platform. Altogether, the synthetic consortium allows for simultaneous bioproduction of PHA and remediation of the industrial pollutant 2,4-DNT, using light and CO2 as inputs.\n\nImportanceIn this study, we have created an artificial consortium composed of two bacterial species that enables the degradation of the industrially-produced environmental pollutant 2,4-DNT while simultaneously producing PHA bioplastic. In these co-cultures, the photosynthetic cyanobacteria fuel an engineered P. putida strain programmed both to use sucrose as a carbon source and to perform the biotransformation of 2,4-DNT. The division of labor in this synthetic co-culture is reminiscent of that commonly observed in microbial communities and represents a proof-of-principle example of how artificial consortia can be employed for bioremediation purposes. Furthermore, this co-culture system enabled the utilization of freshwater sources that could not be utilized in classical agriculture settings, reducing the potential competition of this alternative method of bioproduction with current agricultural practices, as well as remediation of contaminated water streams.

synthetic biology↗

bioRxiv2018

Complete decoupling of bacterial growth from biopolymer production through proteolytic control of enzyme levels

Most current methods for controlling the rate of formation of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of targeted proteins. The device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. The protein degradation process can be reversed by activating the system via addition of an orthogonal inducer (e.g. 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry) and further exploited for engineering poly(3-hydroxybutyrate) (PHB) accumulation completely uncoupled from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively directed into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation with a polymer content of ca. 72% (w/w) in glucose cultures set in the growth-decoupled mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/389809_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (34K):\norg.highwire.dtl.DTLVardef@1d9ec08org.highwire.dtl.DTLVardef@1722feorg.highwire.dtl.DTLVardef@fa77b2org.highwire.dtl.DTLVardef@1c8624a_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

synthetic biology↗