Decrease of energy spilling in Escherichia coli continuous cultures with rising specific growth rate and carbon wasting

<p>Abstract</p> <p>Background</p> <p>Growth substrates, aerobic/anaerobic conditions, specific growth rate (μ) etc. strongly influence <it>Escherichia coli </it>cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA.</p> <p>Results</p> <p>We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor <it>E. coli </it>K-12 MG1655 flux and energy metabolism dynamic responses to change of μ and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing μ and carbon wasting to non-CO₂by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of <it>E. coli </it>pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on μ, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway.</p> <p>Conclusions</p> <p>It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.</p

Survival and synergistic growth of mixed cultures of bifidobacteria and lactobacilli combined with prebiotic oligosaccharides in a gastrointestinal tract simulator

Background: Probiotics, especially in combination with non-digestible oligosaccharides, may balance the gut microflora while multistrain preparations may express an improved functionality over single strain cultures. In vitro gastrointestinal models enable to test survival and growth dynamics of mixed strain probiotics in a controlled, replicable manner. Methods: The robustness and compatibility of multistrain probiotics composed of bifidobacteria and lactobacilli combined with mixed prebiotics (galacto-, fructo- and xylo-oligosaccharides or galactooligosaccharides and soluble starch) were studied using a dynamic gastrointestinal tract simulator (GITS). The exposure to acid and bile of the upper gastrointestinal tract was followed by dilution with a continuous decrease of the dilution rate (de-celerostat) to simulate the descending nutrient availability of the large intestine. The bacterial numbers and metabolic products were analyzed and the growth parameters determined. Results: The most acid- and bile-resistant strains were Lactobacillus plantarum F44 and L. paracasei F8. Bifidobacterium breve 46 had the highest specific growth rate and, although sensitive to bile exposure, recovered during the dilution phase in most experiments. B. breve 46, L. plantarum F44, and L. paracasei F8 were selected as the most promising strains for further studies. Conclusions: De-celerostat cultivation can be applied to study the mixed bacterial cultures under defined conditions of decreasing nutrient availability to select a compatible set of strains

Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase

<p>Abstract</p> <p>Background</p> <p>The biotechnology industry has extensively exploited <it>Escherichia coli </it>for producing recombinant proteins, biofuels etc. However, high growth rate aerobic <it>E. coli </it>cultivations are accompanied by acetate excretion <it>i.e</it>. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.</p> <p>Results</p> <p>In the current work, growth rate dependent acetate overflow metabolism of <it>E. coli </it>was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h^-1) is the repression of acetyl-CoA synthethase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h^-1with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for <it>E. coli </it>chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of <it>E. coli </it>decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h^-1leading to total Acs inhibition and fast accumulation of acetate.</p> <p>Conclusion</p> <p>This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in <it>E. coli </it>shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in <it>E. coli </it>is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.</p

Multi-omics approach to study the growth efficiency and amino acid metabolism in Lactococcus lactis at various specific growth rates

<p>Abstract</p> <p>Background</p> <p><it>Lactococcus lactis </it>is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth.</p> <p>Results</p> <p>Using glucose limited continuous cultivations, specific growth rate dependent metabolism of <it>L. lactis </it>including utilization of amino acids was studied based on extracellular metabolome, global transcriptome and proteome analysis. A new growth medium was designed with reduced amino acid concentrations to increase precision of measurements of consumption of amino acids. Consumption patterns were calculated for all 20 amino acids and measured carbon balance showed good fit of the data at all growth rates studied. It was observed that metabolism of <it>L. lactis </it>became more efficient with rising specific growth rate in the range 0.10 - 0.60 h^-1, indicated by 30% increase in biomass yield based on glucose consumption, 50% increase in efficiency of nitrogen use for biomass synthesis, and 40% reduction in energy spilling. The latter was realized by decrease in the overall product formation and higher efficiency of incorporation of amino acids into biomass. <it>L. lactis </it>global transcriptome and proteome profiles showed good correlation supporting the general idea of transcription level control of bacterial metabolism, but the data indicated that substrate transport systems together with lower part of glycolysis in <it>L. lactis </it>were presumably under allosteric control.</p> <p>Conclusions</p> <p>The current study demonstrates advantages of the usage of strictly controlled continuous cultivation methods combined with multi-omics approach for quantitative understanding of amino acid and energy metabolism of <it>L. lactis </it>which is a valuable new knowledge for development of balanced growth media, gene manipulations for desired product formation etc. Moreover, collected dataset is an excellent input for developing metabolic models.</p

Preparation of onion-like multilayered particles comprising mainly poly(iso-butyl methacrylate)-block-polystyrene by two-step AGET ATRP

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses

KUPERJANOVI PATALJONI AJATEENIJATE TOITUMINE JA SELLE MÕJU NENDE TERVISENÄITAJATELE

2021. aastal uurisid Tallinna Tehnikaülikooli teadlased Kaitseministeeriumi tellimusel Kaitseväe 2. jalaväebrigaadi Kuperjanovi pataljoni ajateenijate toitumist koos nende vere ja soolemikrobioota analüüsidega, et leida seoseid sööklatoidu/kuiv toiduratsioonide ja tervisenäitajate vahel. Ajateenijate toiduvalikutes oli suuri erine vusi, kuid levinud oli see, et söödi vähe täisteraviljatooteid ja köögivilju. Uuringu tulemused on üldistatavad Eesti ajateenijatele laiemalt. Toidupäevikute analüüsid kinnitasid, et kiudainevaene ja rasvarikas toit sisaldab oluliselt vähem mitmeid mineraalaineid ja vitamiine. Vere ja mikrobioomianalüüsid näitasid, et rafineeritud toidu tarbijatel oli kõrgem üld ja LDL kolesteroolitase veres ning suurem põletikega seostatud bakterite, kuid madalam kasulikke happeid tootvate bakterite hulk soo lestiku mikrobiootas. Kuivtoidupakkides eelistati konservtoitudele külmkuivatatud toite. Sööklatoidud peaksid sisaldama rohkem köögivilju, marju ja täisteravilju, mis on head kiudainete ja mikrotoitainete allikad

Advanced continuous cultivation methods for systems microbiology

Increasing the throughput of systems biology-based experimental characterization of in silico designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state – growth space analysis (GSA) – can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories

Simplified scheme of carbon flux rates.

<p>Fluxes are shown in C-mmol (gdw*h)⁻¹ from A-stat experiments of <i>Lactococcus lactis</i>. Blue line represents average values of three independent experiments and red lines represent upper and lower values of standard deviations. Originally input values were experimentally measured at 20 time points and the other points were extrapolated between the measured values to calculate metabolic fluxes at interval of 0.01 h⁻¹. Violet boxes are substrates, orange boxes are products and blue boxes intracellular metabolites. Diamonds illustrates proteins and pathways involved in the given conversion of metabolites. ace - acetate, lact - lactate, etOH - ethanol, Glc - glucose, Orn - ornithine, Glx - glutamate + glutamine, Asx - aspartate + asparagine, PPP - peptose phosphate pathway, Pyr - pyrimidine synthesis, Pur - purine synthesis, I_AA_Glu_Group - the sum of consumption of arginine, proline, glutamine and glutamate, I_AA_Ala_Group - the sum of consumption of alanine, asparagine, aspartate, cysteine, glycine, serine and threonine, I_AA_His_Group - the sum of consumption of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine and valine, X_prod_Pyr - unmeasured products to balance carbon in the calculations, X_prod_AA3 - unmeasured products from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, tyrosine and valine to balance carbon in the calculations and X_prod_Glu - unmeasured products from arginine, proline, glutamine and glutamate to balance carbon in the calculations.</p

Distribution of proteins.

<p>All proteins quantified at all specific growth rates has been divided into different groups by function. Proteins in the network covers enzymes that are involved in the reactions used for metabolic flux analysis and listed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048223#pone.0048223.s001" target="_blank">File S1</a>, Table S1. Lists of all proteins can be seen in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048223#pone.0048223.s002" target="_blank">File S2</a>, Table S8.</p

Increased Biomass Yield of <em>Lactococcus lactis</em> by Reduced Overconsumption of Amino Acids and Increased Catalytic Activities of Enzymes

<div><p>Steady state cultivation and multidimensional data analysis (metabolic fluxes, absolute proteome, and transcriptome) are used to identify parameters that control the increase in biomass yield of <em>Lactococcus lactis</em> from 0.10 to 0.12 C-mol C-mol⁻¹ with an increase in specific growth rate by 5 times from 0.1 to 0.5 h⁻¹. Reorganization of amino acid consumption was expressed by the inactivation of the arginine deiminase pathway at a specific growth rate of 0.35 h⁻¹ followed by reduced over-consumption of pyruvate directed amino acids (asparagine, serine, threonine, alanine and cysteine) until almost all consumed amino acids were used only for protein synthesis at maximal specific growth rate. This balanced growth was characterized by a high glycolytic flux carrying up to 87% of the carbon flow and only amino acids that relate to nucleotide synthesis (glutamine, serine and asparagine) were consumed in higher amounts than required for cellular protein synthesis. Changes in the proteome were minor (mainly increase in the translation apparatus). Instead, the apparent catalytic activities of enzymes and ribosomes increased by 3.5 times (0.1 vs 0.5 h⁻¹). The apparent catalytic activities of glycolytic enzymes and ribosomal proteins were seen to follow this regulation pattern while those of enzymes involved in nucleotide metabolism increased more than the specific growth rate (over 5.5 times). Nucleotide synthesis formed the most abundant biomonomer synthetic pathway in the cells with an expenditure of 6% from the total ATP required for biosynthesis. Due to the increase in apparent catalytic activity, ribosome translation was more efficient at higher growth rates as evidenced by a decrease of protein to mRNA ratios. All these effects resulted in a 30% decrease of calculated ATP spilling (0.1 vs 0.5 h⁻¹). Our results show that bioprocesses can be made more efficient (using a balanced metabolism) by varying the growth conditions.</p> </div