Biology subjects

de Lorenzo, V.

Publications and source records attributed to de Lorenzo, V..

4 recordsLinked to original sources

bioRxiv2018

Reverse engineering of an aspirin-responsive regulator in bacteria

Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work we have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, including acetyl salicylic acid to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.

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↗

bioRxiv2018

The metabolic redox regime of Pseudomonas putida tunes its evolvability towards novel xenobiotic substrates

During evolution of biodegradation pathways for xenobiotic compounds, the transition towards novel substrates of Rieske non-heme iron oxygenases borne by environmental bacteria is frequently associated with faulty reactions. Such reactions release reactive oxygen species (ROS), endowed with high mutagenic potential. The present work studies how the operation of a given metabolic network by a bacterial host may either foster or curtail the still-evolving biochemical pathway for catabolism of 2,4-dinitrotoluene (2,4-DNT). To this end, the genetically tractable strain Pseudomonas putida EM173 was chromosomally implanted with a Tn7 construct carrying the whole genetic complement (recruited from the environmental isolate Burkholderia sp. R34) necessary for complete biodegradation of 2,4-DNT. By using reporter technology and direct measurements of ROS formation, we observed that the engineered P. putida strain experienced oxidative stress when catabolizing the nitroaromatic substrate. However, ROS was neither translated into significant activation of the SOS response to DNA damage nor resulted in a mutagenic regime (unlike Burkholderia sp. R34, the original host of the pathway). To inspect whether the tolerance of P. putida to oxidative insults could be traced to its characteristic reductive redox regime, we artificially lowered the pool of NAD(P)H by conditional expression of a water forming, NADH-specific oxidase. Under the resulting low-NAD(P)H status, 2,4-DNT triggered a conspicuous mutagenic and genomic diversification scenario. These results indicate that the background biochemical network of environmental bacteria ultimately determines the evolvability of metabolic pathways. Moreover, the data explains the efficacy of some bacteria such as Pseudomonads to host and evolve new catabolic routes.\n\nIMPORTANCESome environmental bacteria evolve new capacities for aerobic biodegradation of chemical pollutants by adapting pre-existing redox reactions to recently faced compounds. The process typically starts by co-option of enzymes of an available route to act on the chemical structure of the substrates-to-be. The critical bottleneck is generally the first biochemical step and most of the selective pressure operates on reshaping the initial reaction. In Rieske non-heme iron oxygenases, the interim uncoupling of the novel substrate to the old enzymes results in production of highly mutagenic ROS. In this work, we demonstrate that the background metabolic regime of the bacterium that hosts an evolving catabolic pathway (e.g. biodegradation of the xenobiotic 2,4-DNT) determines whether the cells would either adopt a genetic diversification regime or a robust ROS-tolerant state. These results expose new perspectives to contemporary attempts for rational assembly of whole-cell biocatalysts, as pursued by present-day metabolic engineering.

microbiology↗

bioRxiv2018

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of disaccharides, pentoses, and hexoses

Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in {beta}-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

synthetic biology↗