Manuel Cánovas

Ectoines in cell stress protection: uses and biotechnological production.

Microorganisms produce and accumulate compatible solutes aiming at protecting themselves from environmental stresses. Among them, the wide spread in nature ectoines are receiving increasing attention by the scientific community because of their multiple applications. In fact, increasing commercial demand has led to a multiplication of efforts in order to improve processes for their production. In this review, the importance of current and potential applications of ectoines as protecting agents for macromolecules, cells and tissues, together with their potential as therapeutic agents for certain diseases are analyzed and current theories for the understanding of the molecular basis of their biological activity are discussed. The genetic, biochemical and environmental determinants of ectoines biosynthesis by natural and engineered producers are described. The major limitations of current bioprocesses used for ectoines production are discussed, with emphasis on the different microorganisms, environments, molecular engineering and fermentation strategies used to optimize the production and recovery of ectoines. The combined application of both bioprocess and metabolic engineering strategies, allowing a deeper understanding of the main factors controlling the production process is also stated. Finally, this review aims to summarize and update the state of the art in ectoines uses and applications and industrial scale production using bacteria, emphasizing the importance of reactor design and operation strategies, together with the metabolic engineering aspects and the need for feedback between wet and in silico work to optimize bioproduction.

An insight into the role of phosphotransacetylase (pta) and the acetate/acetyl-CoA node in Escherichia coli

BackgroundAcetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii) the inhibition of cellular growth and protein production by acetate and iii) the need for cofactor recycling (namely redox coenzymes and free CoASH) to sustain balanced growth and cellular homeostasis.ResultsThis work analyzes the effect of mutations in the acetate excretion/assimilation pathways, acetyl-CoA synthethase (acs) and phosphotransacetylase (pta), in E. coli BW25113 grown on glucose or acetate minimal media. Biomass and metabolite production, redox (NADH/NAD+) and energy (ATP) state, enzyme activities and gene expression profiles related to the central metabolism were analyzed. The knock-out of pta led to a more altered phenotype than that of acs. Deletion of pta reduced the ability to grow on acetate as carbon source and strongly affected the expression of several genes related to central metabolic pathways.ConclusionResults showed that pta limits biomass yield in aerobic glucose cultures, due to acetate production (overflow metabolism) and its inefficient use during glucose starvation. Deletion of pta severely impaired growth on acetate minimal medium and under anaerobiosis due to decreased acetyl-coenzyme A synthethase, glyoxylate shunt and gluconeogenic activities, leading to lower growth rate. When acetate is used as carbon source, the joint expression of pta and acs is crucial for growth and substrate assimilation, while pta deletion severely impaired anaerobic growth. Finally, at an adaptive level, pta deficiency makes the strain more sensitive to environmental changes and de-regulates the central metabolism.

cAMP-CRP co-ordinates the expression of the protein acetylation pathway with central metabolism in Escherichia coli.

Lysine acetylation is a well‐established post‐translational modification widely conserved and distributed in bacteria. Although multiple regulatory roles have been proved, little is known about its regulation. Here, we present evidence that the transcription of the Gcn5‐like acetyltransferase YfiQ of Escherichia coli (proposed name: PatZ) is regulated by cAMP‐CRP and its implications on acetate metabolism regulation. The acetate scavenging acetyl‐CoA synthetase (Acs) is regulated at the transcriptional and post‐translational levels. Post‐translational regulation depends on a protein acetyltransferase (yfiQ) and an NAD+‐dependent deacetylase (cobB). We have studied their expression under different environmental conditions. cobB is constitutively expressed from a promoter located upstream nagK. The expression of yfiQ occurs from its own promoter; it is upregulated in the stationary phase and in the presence of non‐PTS carbon sources and is positively regulated by cAMP‐CRP. Two putative CRP binding sites are necessary for its full activity. Gene deletion revealed that cobB is essential for growth on acetate, yfiQ deletion restoring growth of the cobB mutant. The fine tuning of metabolic enzymes results from the integration of multiple mechanisms, and redundant systems may exist. Despite the existence of divergent catabolite repression systems, this may be a conserved strategy common to both Gram‐positive and ‐negative bacteria.

Protein acetylation affects acetate metabolism, motility and acid stress response in Escherichia coli

Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin‐like deacetylase, and patZ, the best‐known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl‐CoA synthetase, we found that CobB‐controlled acetylation of isocitrate lyase contributes to the fine‐tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation.

Regulation of bacterial physiology by lysine acetylation of proteins.

Post-translational modification of proteins is a reversible mechanism of cellular adaptation to changing environmental conditions. In eukaryotes, the physiological relevance of N-ɛ-lysine protein acetylation is well demonstrated. In recent times, important roles in the regulation of metabolic processes in bacteria are being uncovered, adding complexity to cellular regulatory networks. The aim of this mini-review is to sum up the current state-of-the-art in the regulation of bacterial physiology by protein acetylation. Current knowledge on the molecular biology aspects of known bacterial protein acetyltransferases and deacetylases will be summarized. Protein acetylation in Escherichia coli, Salmonella enterica, Bacillus subtilis, Rhodopseudomonas palustris and Mycobacterium tuberculosis, will be explained in the light of their physiological relevance. Progress in the elucidation of bacterial acetylomes and the emerging understanding of chemical acylation mechanisms will be discussed together with their regulatory and evolutionary implications. Fundamental molecular studies detailing this recently discovered regulatory mechanism pave the way for their prospective application for the construction of synthetic regulation networks.

High-density Escherichia coli cultures for continuous L(-)-carnitine production.

Abstract The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l−1 h−1). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used.

Acetate scavenging activity in Escherichia coli: interplay of acetyl–CoA synthetase and the PEP–glyoxylate cycle in chemostat cultures

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl−CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl−CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl−CoA synthetase are upregulated. A mutant in acs (encoding acetyl−CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl−CoA synthetase by cAMP−CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl−CoA and acetyl−phosphate pools, although establishing an energy-dissipating substrate cycle.

Biotransformation of D(+)–carnitine into L(−)–carnitine by resting cells of Escherichia coli O44 K74

l(−)‐carnitine was produced from d(+)‐carnitine by resting cells of Escherichia coli O44 K74. Oxygen did not inhibit either the carnitine transport system or the enzymes involved in the biotransformation process. Aerobic conditions led to higher product yield than anaerobic conditions. The biotransformation yield depended both on biomass and initial substrate concentrations used; the selected values for these variables were 4·30 g l−1 cells and 100 mmol l−1d(+)‐carnitine. Under these conditions the l(−)‐carnitine production rate was 0·55 g l−1 h−1, the process yield was 44%, and the productivity was 0·22 g l−1 h−1 after a 30 h incubation period. Crotonobetaine production, besides l(−)‐carnitine, showed that the action of more than one enzyme occurred during the biotransformation process. On the other hand, the addition of fumarate at high substrate concentrations (250 and 500 mmol l−1) led to a higher metabolic activity, which meant an increment of l(−)‐carnitine production.

Role of central metabolism in the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens

Background: Chromohalobacter salexigens synthesizes and accumulates ectoines. Results: High ratio of the anaplerotic and catabolic fluxes involved in ectoines synthesis supports high biosynthetic fluxes at high salinity and leads to metabolite overflow at low salinity. Conclusion: Evolution optimized the metabolism of C. salexigens to support high production of ectoines. Significance: Metabolic adaptations in a compatible solute-accumulating halophile are described for the first time. Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-13C]-, [2-13C]-, [6-13C]-, and [U-13C6]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.

2,3,5-triphenyltetrazolium chloride as a viability assay for immobilized plant cells

Grape (V. vinifera L. cv Gamay Freaux) cells were entrapped in calcium alginate beads. The effect of bead diameter and gel concentration on the viability of the cells was checked. The reduction of 2,3,5-triphenyltetrazolium chloride as well as O2 consumption were used as viability test, and their results compared. The existence of diffusional limitations for O2 introduces an unaccuracy as high as 25% for the O2 consumption method. The reduction assay is more simple, precise and reliable.