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 glucose obtainable from cellulose. P. putida EM42, a genome-reduced variant of strain 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 and glucose was achieved in a mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the co-utilization phenotype remained to be understood and the Δgcd strain had a significant growth defect. In this study, we investigated the basis of the simultaneous uptake of the two sugars and accelerated the growth of P. putida EM42 Δgcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion lifted the inhibition of the exogenous β-glucosidase BglC from Thermobifida fusca exerted by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore rapid growth on glucose. The reduced growth rate of the Δgcd mutant was partially compensated by the implantation of heterologous glucose and cellobiose transporters (Glf from Zymomonas mobilis and LacY from Escherichia coli, respectively). 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 pyruvate overproduction phenotype 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.

• Klíčová slova
• Cellobiose, Co-utilization of sugars, Glucose, Metabolic engineering, Metabolic model, Pseudomonas putida, Pyruvate,
• MeSH
• celobiosa metabolismus   MeSH
• celulosa metabolismus   MeSH
• Escherichia coli metabolismus   MeSH
• glukosa metabolismus   MeSH
• kyselina pyrohroznová metabolismus   MeSH
• metabolické inženýrství MeSH
• proteiny přenášející monosacharidy genetika   metabolismus   MeSH
• proteiny z Escherichia coli * genetika   MeSH
• proteomika MeSH
• Pseudomonas putida * genetika   metabolismus   MeSH
• symportéry * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celobiosa MeSH
• celulosa MeSH
• glukosa MeSH
• kyselina pyrohroznová MeSH
• LacY protein, E coli MeSH Prohlížeč
• proteiny přenášející monosacharidy MeSH
• proteiny z Escherichia coli * MeSH
• symportéry * MeSH

Bacteria of the phylum Verrucomicrobia are ubiquitous in marine environments and can be found as free-living organisms or as symbionts of eukaryotic hosts. Little is known about host-associated Verrucomicrobia in the marine environment. Here we reconstructed two genomes of symbiotic Verrucomicrobia from bacterial metagenomes derived from the Atlanto-Mediterranean sponge Petrosia ficiformis and three genomes from strains that we isolated from offshore seawater of the Eastern Mediterranean Sea. Phylogenomic analysis of these five strains indicated that they are all members of Verrucomicrobia subdivision 4, order Opitutales. We compared these novel sponge-associated and seawater-isolated genomes to closely related Verrucomicrobia. Genomic analysis revealed that Planctomycetes-Verrucomicrobia microcompartment gene clusters are enriched in the genomes of symbiotic Opitutales including sponge symbionts but not in free-living ones. We hypothesize that in sponge symbionts these microcompartments are used for degradation of l-fucose and l-rhamnose, which are components of algal and bacterial cell walls and therefore may be found at high concentrations in the sponge tissue. Furthermore, we observed an enrichment of toxin-antitoxin modules in symbiotic Opitutales. We suggest that, in sponges, verrucomicrobial symbionts utilize these modules as a defence mechanism against antimicrobial activity deriving from the abundant microbial community co-inhabiting the host.

• MeSH
• cukry metabolismus   MeSH
• fylogeneze MeSH
• mikrobiota MeSH
• mořská voda mikrobiologie   MeSH
• Porifera mikrobiologie   MeSH
• symbióza * MeSH
• systémy toxin-antitoxin genetika   MeSH
• Verrucomicrobia klasifikace   genetika   metabolismus   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Středozemní moře MeSH
• Názvy látek
• cukry MeSH

This study suggests an approach for the comparison and evaluation of particular compartments with modest experimental setup costs. A glucose level prediction model was used to evaluate the compartment's glucose transport rate across the blood capillary membrane and the glucose utilization rate by the cells. The glucose levels of the blood, subcutaneous tissue, skeletal muscle tissue, and visceral fat were obtained in experiments conducted on hereditary hypertriglyceridemic rats. After the blood glucose level had undergone a rapid change, the experimenter attempted to reach a steady blood glucose level by manually correcting the glucose infusion rate and maintaining a constant insulin infusion rate. The interstitial fluid glucose levels of subcutaneous tissue, skeletal muscle tissue, and visceral fat were evaluated to determine the reaction delay compared with the change in the blood glucose level, the interstitial fluid glucose level predictability, the blood capillary permeability, the effect of the concentration gradient, and the glucose utilization rate. Based on these data, the glucose transport rate across the capillary membrane and the utilization rate in a particular tissue were determined. The rates obtained were successfully verified against positron emission tomography experiments. The subcutaneous tissue exhibits the lowest and the most predictable glucose utilization rate, whereas the skeletal muscle tissue has the greatest glucose utilization rate. In contrast, the visceral fat is the least predictable and has the shortest reaction delay compared with the change in the blood glucose level. The reaction delays obtained for the subcutaneous tissue and skeletal muscle tissue were found to be approximately equal using a metric based on the time required to reach half of the increase in the interstitial fluid glucose level.

• Klíčová slova
• Glucose, Skeletal muscle, Subcutaneous tissue, Transport rate, Utilization rate, Visceral fat,
• MeSH
• glukosa analýza   metabolismus   MeSH
• hypertriglyceridemie MeSH
• kapilární permeabilita fyziologie   MeSH
• kosterní svaly metabolismus   MeSH
• krevní glukóza analýza   metabolismus   MeSH
• krysa rodu Rattus MeSH
• nitrobřišní tuk metabolismus   MeSH
• podkožní tuk metabolismus   MeSH
• statistické modely MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glukosa MeSH
• krevní glukóza MeSH

The impact of some types of antihypertensives on carbohydrate metabolism and their association with type 2 diabetes is well-known. In this respect, ACE inhibitors, AT1 blockers or I1 imidazoline agonists, known to improve insulin sensitivity, are the first line therapeutic choice. Metabolism-neutral calcium channel blockers, particularly the dihydropyridines, are the second in line of therapeutic options. On the other hand, beta-blockers and diuretics, thiazide in particular, exert negative effect on carbohydrate metabolism. Should their use be still desirable, e.g. due to co-morbidities, it is advisable to select cardio-selective beta-blockers or beta-blockers with ISA activity, since the effect of these agents on carbohydrate and lipid metabolism is less significant. Diuretics should then be used only in combination therapy and in small doses; potassium-sparing or metabolically neutral indapamide derivatives should be selected. The benefit of cardioprotectivity, gained from the treatment with cardioselective beta-blockers will, particularly in patients with ischemic heart disease, usually outweigh the risk of metabolic adverse effects. Combination therapy, using AT1 blockers or ACE inhibitors in combination with calcium channel blockers or diuretics, should be utilized to its full potential in order to ensure that target values are achieved. Recently completed studies provide the evidence to support this approach. Hypertension in patients with lower limb ischemia increases the already high cardiovascular risk in these patients. Blood pressure reduction as such is more important than a specific antihypertensive.

• MeSH
• antihypertenziva MeSH
• diabetes mellitus 2. typu krev   komplikace   MeSH
• dolní končetina krevní zásobení   MeSH
• hypertenze komplikace   farmakoterapie   MeSH
• ischemie komplikace   farmakoterapie   MeSH
• krevní glukóza MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antihypertenziva MeSH
• krevní glukóza MeSH