Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli.

Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA ß-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.

Engineering an acetoacetyl-CoA reductase from Cupriavidus necator toward NADH preference under physiological conditions.

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 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 AARCn1, 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.

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.

An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli.

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.

NADH-driven poly-3-hydroxybutyrate accumulation in Escherichia coli: Data from enzymatic assays and oxygen-limited continuous cultures.

Biosynthesis of poly-3-hydroxybutyrate (PHB) as a fermentation product enables the coupling of growth and product generation. Moreover, the reduction of oxygen supply should reduce operative cost and increase product yield. Generation of PHB as a fermentation product depends on the in vivo activity of an NADH-preferring acetoacetyl-CoA reductase. Proof of this concept requires (i) quantification of the cofactor preference, in physiologically relevant conditions, of a putative NADH-preferring acetoacetyl-CoA reductase and (ii) verification of PHB accumulation using an NADH-preferring acetoacetyl-CoA reductase in a species naturally incapable of doing so, for example, Escherichia coli. This dataset contains kinetic data obtained by spectrophotometry and data from a continuous culture of an engineered E. coli strain accumulating PHB under oxygen-limiting conditions. In this dataset it is possible to find (1) enzyme stability assays; (2) initial rates and progress curves from reactions catalyzed by two acetoacetyl-CoA reductases; (3) estimations of the relative use of NADH and NADPH by two acetoacetyl-CoA reductases; (4) estimations of the flux capacity of the reaction catalyzed by an acetoacetyl-CoA reductase; (5) biomass composition of an engineered E. coli strain transformed with a plasmid; (6) calculation of reconciled specific rates of this engineered strain growing on sucrose as the sole carbon source under oxygen limitation and (7) metabolic fluxes distributions during the continuous growth of this engineered strain. Because a relatively small number of acetoacetyl-CoA reductases have been kinetically characterized, data and scripts here provided could be useful for further kinetic characterizations. Moreover, the procedure described to estimate biomass composition could be interesting to estimate plasmid and protein burden in other strains. Application of data reconciliation to fermentations should help to obtain specific rates consistent with the principle of mass and electron conservation. All the required data and scripts to perform these analyses are deposited in a Mendeley Data repository. This article was co-submitted with the manuscript entitled "An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichiasia. coli".

Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools.

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 an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 µM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

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. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-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 model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

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. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-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 model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Metabolism of sucrose in a non-fermentative Escherichia coli under oxygen limitation.

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 biomass-specific 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.

Engineering NADH/NAD+ ratio in Halomonas bluephagenesis for enhanced production of polyhydroxyalkanoates (PHA).

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(3-hydroxybutyrate-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 α and ß 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.

Quantifying NAD(P)H production in the upper Entner-Doudoroff pathway from Pseudomonas putida KT2440.

Despite the lack of biochemical information, all available in silico metabolic models of Pseudomonas putida KT2440 consider NADP as the only cofactor accepted by the glucose-6-phosphate dehydrogenases. Because the Entner-Doudoroff pathway is the main glycolytic route in this bacterium, determining how much NADH and NADPH are produced in the reaction catalyzed by these enzymes is very important for the correct interpretation of metabolic flux distributions. To determine the actual cofactor preference of the glucose-6-phosphate dehydrogenase encoded by the zwf-1 gene (PputG6PDH-1), the major isoform during growth on glucose, we purified this protein and studied its kinetic properties. Based on simple kinetic principles, we estimated the in vivo relative production of NADH and NADPH during the oxidation of glucose-6-phosphate (G6P). Contrary to the general assumption, our calculations showed that the reaction catalyzed by PputG6PDH-1 yields around 1/3 mol of NADPH and 2/3 mol of NADH per mol of oxidized G6P. Additionally, we obtained data suggesting that the reaction catalyzed by the 6-phosphogluconate dehydrogenase is active during growth on glucose, and it also produces NADH. These results indicate that the stoichiometric matrix of in silico models of P. putida KT2440 must be corrected and highlight the importance of considering the physiological concentrations of the involved metabolites to estimate the actual proportion of NADH and NADPH produced by a dehydrogenase.

The cofactor preference of glucose-6-phosphate dehydrogenase from Escherichia coli--modeling the physiological production of reduced cofactors.

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 kcal·mol(-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 ω-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.

Scaffolding proteins in highly purified rat olfactory cilia membranes.

Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.