Elimination of GlnKAmtB affects serine biosynthesis and improves growth and stress tolerance of Escherichia coli under nutrient-rich conditions

Nitrogen is a most important nutrient resource for Escherichia coli and other bacteria that harbor the glnKamtB operon, a high-affinity ammonium uptake system highly interconnected with cellular metabolism. Although this system confers an advantage to bacteria when growing under nitrogen-limiting conditions, little is known about the impact of these genes on microbial fitness under nutrient-rich conditions. Here, the genetically tractable E. coli BW25113 strain and its glnKamtB-null mutant (JW0441) were used to analyze the impact of GlnK-AmtB on growth rates and oxidative stress tolerance. Strain JW0441 showed a shorter initial lag phase, higher growth rate, higher citrate synthase activity, higher oxidative stress tolerance and lower expression of serA than strain BW25113 under nutrient-rich conditions, suggesting a fitness cost to increase metabolic plasticity associated with serine metabolism. The overexpression of serA in strain JW0441 resulted in a decreased growth rate and stress tolerance in nutrient-rich conditions similar to that of strain BW25113, suggesting that the negative influence on bacterial fitness imposed by GlnK-AmtB can be traced to the control of serine biosynthesis. Finally, we discuss the potential applications of glnKamtB mutants in bioproduction processes.Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; ArgentinaFil: Stritzler, Margarita. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; ArgentinaFil: Pascuan, Cecilia Gabriela. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; ArgentinaFil: Liebrenz, Karen Ivana. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; ArgentinaFil: Galindo Sotomonte, Luisa. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; ArgentinaFil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; ArgentinaFil: Nikel, Pablo Iván. Technical University of Denmark; DinamarcaFil: Ayub, Nicolas Daniel. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular. Grupo Vinculado Instituto de Genetica "ewald A. Favret" Al Iabimo.; Argentin

Quantitative Physiology Approaches to Understand and Optimize Reducing Power Availability in Environmental Bacteria

The understanding of how carbon fluxes are distributed through a metabolic network offers an overview of the pathways that a given microorganism uses to produce energy, reducing power, and biomass. These invaluable data are related to the physiological state of the cell and provide information about the metabolic potential of microorganisms for specific environmental and biotechnological applications such as the degradation of toxic compounds (e.g., hydrocarbons) or the targeted production of high value-added products (e.g., lipids). Here, we propose a general approach to explore the pathways involved in NADPH balance in bacteria, which are in turn responsible for maintaining redox homeostasis and endowing the microorganism with the ability to counteract oxidative stress. We focus on the fluxes catalyzed by NADP+-dependent enzymes in the metabolic network of the model soil bacterium Pseudomonas putida KT2440. This environmental microorganism is a promising cell factory for a number of NADPH-dependent biotransformations, including industrial and bioremediation processes. The relevant enzymes involved in redox balance in strain KT2440 are (1) glucose-6-phosphate dehydrogenase, (2) 6-phosphogluconate dehydrogenase, (3) isocitrate dehydrogenase, (4) malic enzyme, and (5) 2-keto-6-phosphogluconate reductase. NADPH can be generated or consumed by other enzymatic reactions depending on the microorganism; however, the first four enzymes listed above are recognized as a major source of reducing power in a wide variety of microorganisms. The present protocol includes a first stage in which the NADPH balance is derived from fluxomic data and in vitro enzymatic assays. A second step is then proposed, where the redox ratios of pyridine dinucleotides and the cell capacity to counter oxidative stress are qualitatively correlated.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

From Rags to Riches:Exploiting the Calvin-Benson-Bassham Cycle for Biomanufacturing

Industrial chemical production largely relies on fossil fuels, resulting in the unavoidable release of carbon dioxide (CO2) into the atmosphere. The concept of a circular carbon bioeconomy has been proposed to address this issue, wherein CO2 is captured and used as a raw material for manufacturing new chemicals. Microbial cell factories and, in particular, autotrophic microorganisms capable of utilizing CO2 as the sole carbon source, emerged as potential catalysts for upcycling CO2 to valuable products. The Calvin-Benson-Bassham cycle (CBBc), the best-known CO2 fixation pathway, is widely distributed in Nature. While extensively studied, microbial engineering programmes based on the CBBc remains relatively underexplored. In this review, we discuss avenues towards biotechnological exploitation of the CBBc to engineer CO2-utilizing microbial cell factories, with a focus on chemically-derived electron donors. We also highlight the advantages and challenges of implementing the CBBc in heterotrophic microbial hosts and its potential to advance a true circular carbon bioeconomy. Moreover, based on the pathway's architecture, we argue about the ideal value-added products to generate from this metabolic route. Altogether, studying and engineering the CBBc in both natural- and synthetic-autotrophs will enhance our understanding on this CO2 fixation pathway, enabling further exploration of biomanufacturing avenues with CO2 as feedstock

A Hitchhiker’s guide to CRISPR editing tools in bacteria:CRISPR can help unlock the bacterial world, but technical and regulatory barriers persist

Join us on a journey through the complex and ever-expanding universe of CRISPR approaches for genome editing in bacteria. Discover what is available, current technical challenges, successful implementation of these tools and the regulatory framework around their use. (Figure presented.)</p

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 provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity toward NADP(+), some variants accept 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 genomic, genetic, and biochemical perspectives. P. putida represents an ideal model to tackle this endeavor, as its genome harbors 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. The abundance of G6PDH isoforms in these species goes hand in hand with low NADP(+) affinity, at least in one isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy toward balancing the relative production of NADPH and NADH. IMPORTANCE Protein families have likely arisen during evolution by gene duplication and divergence followed by neofunctionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the coexistence 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 coexisting glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs 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 toward achieving redox balance according to the growth conditions

Automating the design-build-test-learn cycle towards next-generation bacterial cell factories

Automation is playing an increasingly significant role in synthetic biology. Groundbreaking technologies, developed over the past 20 years, have enormously accelerated the construction of efficient microbial cell factories. Integrating state-of-the-art tools (e.g. for genome engineering and analytical techniques) into the design-build-test-learn cycle (DBTLc) will shift the metabolic engineering paradigm from an almost artisanal labor towards a fully automated workflow. Here, we provide a perspective on how a fully automated DBTLc could be harnessed to construct the next-generation bacterial cell factories in a fast, high-throughput fashion. Innovative toolsets and approaches that pushed the boundaries in each segment of the cycle are reviewed to this end. We also present the most recent efforts on automation of the DBTLc, which heralds a fully autonomous pipeline for synthetic biology in the near future

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida

Background: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. Results: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL−1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L−1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g−1 DCW h−1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)–polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. Conclusions: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones. </div

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida

Abstract Background Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. Results The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL− 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L− 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g− 1 DCW h− 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)–polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. Conclusions Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones. Graphical abstrac

Enhanced chaotrope tolerance and (S)‐2‐hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans

Abstract Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α‐hydroxyketones, such as (S)‐2‐hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine‐tuned gene expression was achieved using an expression plasmid under the control of the LacIQ/Ptrc system, and the cross‐protective role of PprI was assessed against multiple stress treatments. Moreover, the stress‐tolerant P. putida strain was tested for 2‐hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2‐hydroxypropiophenone more efficiently than the parental P. putida strain. 2‐Hydroxypropiophenone concentration reached 1.6 g L−1 upon a 3‐h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL−1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2‐HPP g−1 benzaldehyde and 0.089 g 2‐HPP g cell dry weight−1 h−1, respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2‐HPP production in P. putida ATCC 12633

Transcriptional control of 2,4-dinitrotoluene degradation in Burkholderia sp. R34 bears a regulatory patch that eases pathway evolution

The dnt pathway of Burkholderia sp. R34 is in the midst of an evolutionary journey from its ancestral, natural substrate (naphthalene) towards a new xenobiotic one [2,4-dinitrotoluene (DNT)]. The gene cluster encoding the leading multicomponent ring dioxygenase (DntA) has activity on the old and the new substrate, but it is induced by neither. Instead, the transcriptional factor encoded by the adjacent gene (dntR) activates expression of the dnt cluster upon addition of salicylate, one degradation intermediate of the ancestral naphthalene route but not any longer a substrate/product of the evolved DntA enzyme. Fluorescence of cells bearing dntA-gfp fusions revealed that induction of the dnt genes by salicylate was enhanced upon exposure to bona fide DntA substrates, i.e., naphthalene or DNT. Such amplification was dependent on effective dioxygenation of these pathway-specific head compounds, which thereby fostered expression of the cognate catabolic operon. The phenomenon seems to happen not through direct binding to a cognate transcriptional factor but through the interplay of a non-specific regulator with a substrate-specific enzyme. This regulatory scenario may ease transition of complete catabolic operons (i.e. enzymes plus regulatory devices) from one substrate to another without loss of fitness during the evolutionary roadmap between two optimal specificities.Sociedad Española de Trombosis y Hemostasia/[RTI2018-095584-B-C42]/SETH/EspañaSynthetic microbial communities for the production of limonene derived products/[ERA-COBIOTECH 2018 - PCI2019- 111859-2]/SyCoLiM/Reino UnidoMadonna University/[H2020-FET-OPEN-RIA-2017-1-766975]/MADONNA/Estados UnidosBioRoboost/[H2020-NMBP-BIO-CSA-2018-820699]//Unión EuropeaSynBio4Flav/[H2020-NMBP-TR-IND/H2020-NMBP-BIO-2018- 814650]//Unión EuropeaIngeniería Microbiana, Salud y Calidad de Vida/[S2017/BMD-3691]/InGEMICS-CM/EspañaMIX-UP/[MIX-UP H2020-BIO-CN-2019-870294]//Unión EuropeaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA