Papers by Karel Olavarria
Data in Brief, Dec 1, 2020
Parameters for data collection Kinetic data were recorded at 30 °C. Continuous cell cultures were... more Parameters for data collection Kinetic data were recorded at 30 °C. Continuous cell cultures were studied at 37 °C. Further details are explained in the section Experimental Design, Materials and Methods. Description of data collection Kinetic data were obtained by spectrophotometry in a plate reader equipped with monochromator, temperature control and a built-in software controlled by the user through the software Gen5. Data from continuous cultures were obtained by the integration of data obtained by different means. More details in the section Experimental Design, Materials and Methods. Data source location
Metabolic Engineering, Sep 1, 2018
Halomonas bluephagenesis has been developed as a platform strain for the next generation industri... more Halomonas bluephagenesis has been developed as a platform strain for the next generation industrial biotechnology (NGIB) with advantages of resistances to microbial contamination and high cell density growth (HCD), especially for production of polyhydroxyalkanoates (PHA) including poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) and poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). However, little is known about the mechanism behind PHA accumulation under oxygen limitation. This study for the first time found that H. bluephagenesis utilizes NADH instead of NADPH as a cofactor for PHB production, thus revealing the rare situation of enhanced PHA accumulation under oxygen limitation. To increase NADH/NAD + ratio for enhanced PHA accumulation under oxygen limitation, an electron transport pathway containing electron transfer flavoprotein subunits D and E encoded by etf operon was blocked to increase NADH supply, leading to 90% PHB accumulation in the cell dry weight (CDW) of H. bluephagenesis compared with 84% by the wild type. Acetic acid, a cost-effective carbon source, was used together with glucose to balance the redox state and reduce inhibition on pyruvate metabolism, resulting in 22% more CDW and 94% PHB accumulation. The cellular redox state changes induced by the addition of acetic acid increased 3HV ratio in its copolymer PHBV from 4% to 8%, 4HB in its copolymer P34HB from 8% to 12%, respectively, by engineered H. bluephagenesis. The strategy of systematically modulation on the redox potential of H. bluephagenesis led to enhanced PHA accumulation and controllable monomer ratios in PHA copolymers under oxygen limitation, reducing energy consumption and scale-up complexity.
FEBS Journal, May 24, 2012
In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first... more In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcalAEmol)1. In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the x-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD ⁄ NADH (rR NAD) and NADP ⁄ NADPH (rR NADP). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR NADP , but NADH production remains low within the range feasible for rR NAD. Nevertheless, we show that certain combinations of rR NADP and rR NAD sustain greater levels of NADH production over NADPH.
Applied and Environmental Microbiology, Nov 24, 2020
Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategie... more Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. "Candidatus Accumulibacter phosphatis," a polyphosphateaccumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the nonavailability of isolates has led to controversial conclusions on the metabolic pathways used. In this study, we experimentally determined the redox cofactor preferences of different oxidoreductases in the central carbon metabolism of a highly enriched "Ca. Accumulibacter phosphatis" culture. Remarkably, we observed that the acetoacetyl coenzyme A reductase engaged in polyhydroxyalkanoate (PHA) synthesis is NADH preferring instead of showing the generally assumed NADPH dependency. This allows rethinking of the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. Based on previously published metaomics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-toglycogen consumption ratios were simulated, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt, or branches of the tricarboxylic acid (TCA) cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compound availability (polyphosphate/glycogen). This NADH-related metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under various environmental substrate conditions, with high carbon conservation and lower energetic costs than for NADPH-dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain the competitiveness of PAOs under oxygen-fluctuating environments. IMPORTANCE Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of "Ca. Accumulibacter phosphatis" to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.
bioRxiv (Cold Spring Harbor Laboratory), Jan 9, 2021
Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first... more 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 1 , some variants accept NAD 1 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 1 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 1 or NADP 1 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.
Scientific Reports, Mar 8, 2022
The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentatio... more The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentation product. A fundamental trait to accomplish this feature is to express a functional NADH-preferring acetoacetyl-CoA reductase, engaged in PHB accumulation. One way to obtain such a reductase is by engineering the cofactor preference of the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator (AAR Cn1). Aiming to have a deeper understanding of the structural determinants of the cofactor preference in AAR Cn1 , and to obtain an NADH-preferring acetoacetyl-CoA reductase derived from this protein, some engineered enzymes were expressed, purified and kinetically characterized, together with the parental AAR Cn1. One of these engineered enzymes, Chimera 5, experimentally showed a selectivity ratio ((k cat /K M) NADH /(k cat /K M) NADPH) ≈ 18, which is 160 times higher than the selectivity ratio experimentally observed in the parental AAR Cn1. A thermodynamic-kinetic approach was employed to estimate the cofactor preference and flux capacity of Chimera 5 under physiological conditions. According to this approach, Chimera 5 could prefer NADH over NADPH between 25 and 150 times. Being a derivative of AAR Cn1 , Chimera 5 should be readily functional in Escherichia coli and C. necator. Moreover, with the expected expression level, its activity should be enough to sustain PHB accumulation fluxes similar to the fluxes previously observed in these biotechnologically relevant cell factories. Due to its properties, poly-3-hydroxybutyrate (PHB) could replace some fossil-fuel based plastics. A welldescribed PHB production pathway consists of only three reactions, catalyzed by the enzymes β-ketothiolase (E.C. 2.3.1.9), acetoacetyl-CoA reductase (E.C. 1.1.1.36) and PHB synthase (E.C. 2.3.1.304) (Supplementary Material 1). In many species, these enzymes are respectively encoded by the genes phaA, phaB and phaC (Fig. S1). Given its simplicity, PHB accumulation accomplished through the operation of this pathway is also a model to understand the production of other biopolymers. However, PHB production is still more expensive than fossilfuel based plastics with similar properties. One of factors that could potentially decrease its cost is the generation of PHB as a fermentation product. However, to generate PHB as a fermentation product, PHB accumulation has to be coupled to NADH reoxidation 1,2. Given its high PHB accumulation titers, ability of autotrophic growth on H 2 and CO 2 , oxygen tolerance and genetic tractability, Cupriavidus necator is commonly used as a platform for PHB accumulation 3. Moreover, many of the engineered cell factories aimed at PHB production are based on the expression of the phaCAB1 operon from C. necator. However, the phaB1 gene from C. necator encodes for an acetoacetyl-CoA reductase, AAR Cn1 , generally regarded as an NADPH-preferring enzyme 4,5. Thus, to favor the NADPH-driven PHB accumulation, biomass formation is inhibited through nutrient limitation 6,7. However, this approach could affect cellular metabolism at different levels 8 , hampering productivity 9,10 and it requires case-specific fine-tuning of the growth conditions to properly balance biomass and PHB formations 7 .
Applied Microbiology and Biotechnology, May 31, 2019
Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap ... more Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomassspecific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.
Journal of Biotechnology, 2021
Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, g... more Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus Accumulibacter phosphatis-enriched mixed culture, showing a (kcatNADH/KMNADH)/(kcatNADPH/KMNADPH)>500. Further kinetic analyses indicate that, at physiological concentrations, this enzyme clearly prefers NADH, presenting the strongest NADH preference so far observed among the acetoacetyl-CoA reductases. Structural and kinetic analyses indicate that residues between E37 and P41 have an important role for the observed NADH preference. Moreover, an operon was assembled combining the phaCA genes from Cupriavidus necator and the gene encoding for this NADH-preferring acetoacetyl-CoA reductase. Escherichia coli cells expressing that assembled operon showed continuous accumulation of PHB under oxygen limiting conditions and PHB titer increased when decreasing the specific oxygen consumption rate. Taken together, these results show that it is possible to generate PHB as a fermentation product in E. coli, opening opportunities for further protein/metabolic engineering strategies envisioning a more efficient anaerobic production of PHB.
Microbiology, Dec 1, 2014
In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the ... more In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Dpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH R46E,Q47E. Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Dpgi E. coli strains were replaced by DNA encoding LmG6PDH R46E,Q47E. Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Dpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.
Candidatus Accumulibacter phosphatis, a polyphosphate accumulating organism (PAO) growing under a... more Candidatus Accumulibacter phosphatis, a polyphosphate accumulating organism (PAO) growing under alternating anaerobic/aerobic conditions in enhanced biological phosphorus removal (EBPR) systems, displays an intricate metabolic strategy. The organism relies on combining features from either anaerobic- or aerobic-only metabolisms and connecting phases using different reserve polymers. Analysis of the metabolic functions is not trivial, specially without isolates of this microorganism available and without employing advanced metabolic modelling techniques. So far, there is no consensus regarding the type of glycolysis nor the operating mode of the tricarboxylic acid cycle anaerobically. Here, we simulated different conditions using flux balance analyses and a reduced central carbon metabolic network based on previously published meta-omics data and validated with enzyme activity assays. Interestingly, we observed that the polyhydroxyalkanoates (PHA) production was NADH preferring inste...
Scientific Reports, 2022
The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentatio... more The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentation product. A fundamental trait to accomplish this feature is to express a functional NADH-preferring acetoacetyl-CoA reductase, engaged in PHB accumulation. One way to obtain such a reductase is by engineering the cofactor preference of the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator (AARCn1). Aiming to have a deeper understanding of the structural determinants of the cofactor preference in AARCn1, and to obtain an NADH-preferring acetoacetyl-CoA reductase derived from this protein, some engineered enzymes were expressed, purified and kinetically characterized, together with the parental AARCn1. One of these engineered enzymes, Chimera 5, experimentally showed a selectivity ratio ((kcat/KM)NADH/(kcat/KM)NADPH) ≈ 18, which is 160 times higher than the selectivity ratio experimentally observed in the parental AARCn1. A thermodynamic-kinetic approach was emp...
Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first... more 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 mos...
These folders contain experimental records, calculation datasheets and scripts to simulate or fin... more These folders contain experimental records, calculation datasheets and scripts to simulate or find best-fitted kinetic parameters from certain enzymatically catalyzed reactions. These reactions were catalyzed by four engineered enzymes derived from the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator. The DNA and amino acid sequences of these enzymes are provided. It is also provided the DNA sequence map of the vector where the amino acid encoding sequences for these engineered enzymes were inserted. Inside the folder named "kinetic data" it is also possible to find a TXT file with a detailed description of the content. The experimental conditions of the enzymatic assays is carefully explained. Briefly, this folder contains (1) the raw experimental data recorded with the software Gen 5 (Biotek). (2) It is also possible to find Microsoft Excel datasheets with the records of raw absorbance in time together with initial substrates, initial enzyme a...
Biochemical Journal
In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participate... more In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The mo...
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Papers by Karel Olavarria