Multilayer precision-based screening of potential inhibitors targeting Mycobacterium tuberculosis acetate kinase using in silico approaches.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a major threat to global health, with the emergence of multi-drug and extensively drug-resistant strains posing a serious challenge. Thereby, understanding the molecular basis of MTB virulence and disease pathogenesis is critical for developing effective therapeutic strategies. Targeting proteins involved in central metabolism has been recognized as a promising therapeutic approach to combat MTB. In this regard, the enzyme AckA of the acetate metabolic pathway which produces acetate from acetyl phosphate, is an important drug target for various pathogenic organisms. Therefore, this study aimed to identify potential AckA inhibitors through in silico methods, including molecular modeling, molecular dynamics simulation (MDS), and high-throughput virtual screening (HTVS) followed by ADMETox, MMGBSA, Density Functional Theory (DFT) calculations. HTVS of one million compounds from the ZINC database against AckA resulted in the top five hits (ZINC82048449, ZINC1219737510, ZINC1771921358, ZINC119699567, and ZINC1427100376) with better binding affinity and optimal binding free energy. MDS studies on complexes revealed that key residues, Asn195, Asp266, Phe267, Gly314, and Asn318 played a significant role in stable interactions of the top-ranked compounds to AckA. These outcomes provide insights into the optimal binding of the leads to inhibit the acetate pathway and aid in the rational design of novel therapeutic agents. Thus, the identified leads may act as promising compounds for targeting AckA and may serve as a potential therapeutic modality for treating TB. Our findings offer valuable insights into the inhibition of the acetate pathway, while also serving as a blueprint for rational drug design. The identified leads hold promise as compelling compounds for targeting AckA, thereby offering a potential therapeutic avenue for tackling TB. Thus, our study uncovers a pathway toward promising TB therapeutics by elucidating AckA inhibitors. By leveraging in silico methodologies, potent compounds that hold the potential to thwart AckA's role in MTB's acetate pathway have been unveiled. This breakthrough fosters optimism in the quest for novel and effective TB treatments, addressing a global health challenge with renewed vigor.

Reduced acetic acid formation using NaHSO3 as a steering agent by Actinobacillus succinogenes GXAS137.

The high production of acetic acid (AC) as a by-product leads to difficult separation and purification of succinic acid (SA) and increases production costs in SA fermentation by Actinobacillus succinogenes. NaHSO3 as a steering agent was used to reduce AC production. Herein, the optimum fermentation conditions were achieved by single-factor and orthogonal tests as follows: glucose 60 g/L; MgCO3 60 g/L; NaHSO3 0.15% (w/v); and NaHSO3 addition time, 8 h after inoculation. After optimization, the SA and AC contents were 44.42 and 5.73 g/L. The SA improved by 100.72%, the AC decreased by 21.18% compared with the unfermented. The acetate kinase activity decreased by 14.36% and acetyl-CoA content improved by 97.55% in the group of NaHSO3 addition compared with control check (CK). The mechanism of NaHSO3 is formation acetaldehyde-sodium bisulfite compound and reduction the activity of acetate kinase. These findings indicated a new way of using NaHSO3 as a steering agent to reduce AC generation and may help promote the development of SA industrial production.

Noise in a Metabolic Pathway Leads to Persister Formation in Mycobacterium tuberculosis.

Tuberculosis is difficult to treat due to dormant cells formed in response to immune stress and stochastically formed persisters, both of which are tolerant of antibiotics. Bactericidal antibiotics kill by corrupting their energy-dependent targets. We reasoned that stochastic variation, or noise, in the expression of an energy-generating component will produce rare persister cells. In sorted M. tuberculosis cells grown on acetate, there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing ackA sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters. Entrance into a low-energy state driven by noise in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters. IMPORTANCE M. tuberculosis infection requires the administration of multiple antibiotics for a prolonged period of time. Treatment difficulty is generally attributed to M. tuberculosis entrance into a nonreplicative, antibiotic-tolerant state. M. tuberculosis enters this nonreplicative state in response to immune stress. However, a small population of cells enter a nonreplicative, multidrug-tolerant state under normal growth conditions, absent any stress. These cells are termed persisters. The mechanisms by which persisters enter a nonreplicative state are largely unknown. Here, we show that, as with other bacteria, M. tuberculosis persisters are low-energy cells formed stochastically during normal growth. Additionally, we identify the natural variation in the expression of energy producing genes as a source of the stochastic entrance of M. tuberculosis into the low-energy persister state. These findings have important implications for understanding the heterogeneous nature of M. tuberculosis infection and will aid in designing better treatment regimens against this important human pathogen.

Coordinated expression of acetyl CoA synthetase and the ace operon enzymes in Escherichia coli in preparation for adaptation to acetate.

Successful adaptation of Escherichia coli to constant environmental challenges demands the operation of a wide range of regulatory control mechanisms, some of which are global, while others are specific. Here, we show that the ability of acetate-negative phenotype strains of E. coli devoid of acetate kinase (AK) and phosphotransacetylase (PTA) to assimilate acetate when challenged at the end of growth on acetogenic substrates is explicable by the co-expression of acetyl CoA-synthetase (AcCoA-S) and acetate permease (AP). Furthermore, mRNA transcript measurements for acs and aceA, together with the enzymatic activities of their corresponding enzymes, acetyl CoA synthetase (AcCoA-S) and isocitrate lyase (ICL), clearly demonstrate that the expression of the two enzymes is inextricably linked and triggered in response to growth rate threshold signal (0.4 h-1± 0.03: n4). Interestingly, further restriction of carbon supply to the level of starvation led to the repression of acs (AcCoA-S), ackA (AK) and pta (PTA). Further, we provide evidence that the reaction sequence catalysed by PTA, AK and AcCoA-S is not in operation at low growth rates and that the reaction catalysed by AcCoA-S is not merely an ATP-dissipating reaction but rather advantageous, as it elevates the available free energy (ΔG°) in central metabolism. Moreover, the transcriptomic data reinforce the view that the expression of PEP carboxykinase is essential in gluconeogenic phenotypes.

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides.

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.

Visualization of mRNA Expression in Pseudomonas aeruginosa Aggregates Reveals Spatial Patterns of Fermentative and Denitrifying Metabolism.

Gaining insight into the behavior of bacteria at the single-cell level is important given that heterogeneous microenvironments strongly influence microbial physiology. The hybridization chain reaction (HCR) is a technique that provides in situ molecular signal amplification, enabling simultaneous mapping of multiple target RNAs at small spatial scales. To refine this method for biofilm applications, we designed and validated new probes to visualize the expression of key catabolic genes in Pseudomonas aeruginosa aggregates. In addition to using existing probes for the dissimilatory nitrate reductase (narG), we developed probes for a terminal oxidase (ccoN1), nitrite reductase (nirS), nitrous oxide reductase (nosZ), and acetate kinase (ackA). These probes can be used to determine gene expression levels across heterogeneous populations such as biofilms. Using these probes, we quantified gene expression across oxygen gradients in aggregate populations grown using the agar block biofilm assay (ABBA). We observed distinct patterns of catabolic gene expression, with upregulation occurring in particular ABBA regions both within individual aggregates and over the aggregate population. Aerobic respiration (ccoN1) showed peak expression under oxic conditions, whereas fermentation (ackA) showed peak expression in the anoxic cores of high metabolic activity aggregates near the air-agar interface. Denitrification genes narG, nirS, and nosZ showed peak expression in hypoxic and anoxic regions, although nirS expression remained at peak levels deeper into anoxic environments than other denitrification genes. These results reveal that the microenvironment correlates with catabolic gene expression in aggregates, and they demonstrate the utility of HCR in unveiling cellular activities at the microscale level in heterogeneous populations. IMPORTANCE To understand bacteria in diverse contexts, we must understand the variations in behaviors and metabolisms they express spatiotemporally. Populations of bacteria are known to be heterogeneous, but the ways this variation manifests can be challenging to characterize due to technical limitations. By focusing on energy conservation, we demonstrate that HCR v3.0 can visualize nuances in gene expression, allowing us to understand how metabolism in Pseudomonas aeruginosa biofilms responds to microenvironmental variation at high spatial resolution. We validated probes for four catabolic genes, including a constitutively expressed oxidase, acetate kinase, nitrite reductase, and nitrous oxide reductase. We showed that the genes for different modes of metabolism are expressed in overlapping but distinct subpopulations according to oxygen concentrations in a predictable fashion. The spatial transcriptomic technique described here has the potential to be used to map microbial activities across diverse environments.

Regulation of CcpA on the growth and organic acid production characteristics of ruminal Streptococcus bovis at different pH.

BACKGROUND: Catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5. RESULTS: There was a significant interaction between strain and pH for the maximum specific growth rate (µmax) and growth lag period (λ), which caused a lowest µmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5. CONCLUSIONS: Overall, low pH inhibited the growth of S. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.

Involvement of an FNR-like oxygen sensor in Komagataeibacter medellinensis for survival under oxygen depletion.

During acetic acid fermentation, acetic acid bacteria face oxygen depletion stress caused by the vigorous oxidation of ethanol to acetic acid. However, the molecular mechanisms underlying the response to oxygen depletion stress remain largely unknown. Here, we focused on an oxygen-sensing FNR homolog, FnrG, in Komagataeibacter medellinensis. Comparative transcriptomic analysis between the wild-type and fnrG-disrupted strains revealed that FnrG upregulated 8 genes (fold change >3). Recombinant FnrG bound to a specific DNA sequence only when FnrG was reconstituted anaerobically. An operon consisting of acetate kinase and xylulose-5-phosphate/fructose-6-phosphate phosphoketolase genes was found to be an FnrG regulon involved in cell survival under oxygen-limiting conditions. Moreover, a strain that overexpressed these 2 genes accumulated more acetic acid than the wild-type strain harboring an empty vector. Thus, these 2 genes could be new targets for the molecular breeding of acetic acid bacteria with high acetic acid productivity.

Characterization of a Rhodobacter sphaeroides primary fatty acid kinase.

Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2--C8).

Acetate kinase and peptidases are associated with the proteolytic activity of Lactobacillus helveticus isolated from fermented food.

Lactobacillus (L.) helveticus is widely used in food industry due to its high proteolytic activity. However, such activity varies greatly between isolates, and the determining factors regulating the strength of proteolytic activity in L. helveticus are unclear. This study sequenced the genomes of 60 fermented food-originated L. helveticus and systemically examined the proteolytic activity-determining factors. Our analyses found that the strength of proteolytic activity in L. helveticus was independent of the isolation source, geographic location, phylogenetic closeness between isolates, and distribution of cell envelope proteinases (CEPs). Genome-wide association study (GWAS) identified two genes, the acetate kinase (ackA) and a hypothetical protein, and 15 single nucleotide polymorphisms (SNPs) that were associated with the strength of the proteolytic activity. Further investigating the functions of these gene components revealed that ackA and two cysteine peptidases coding genes (pepC and srtA) rather than the highly heterogeneous and intraspecific CEPs were linked to the level of proteolytic activity. Moreover, the sequence type (ST) defined by SNP analysis revealed a total of ten STs, and significantly weaker proteolytic activity was observed among isolates of ST2. This study provides practical information for future selection of L. helveticus of strong proteolytic activity.

Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae.

The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

Effect of acid and alkali stress on extracellular metabolite profile of Lactobacillus plantarum ATCC 14917.

As a multifunctional lactic acid bacterium, Lactobacillus plantarum has been proved to survive in the human gastrointestinal tract, and it can also colonize this tract. In this study, the effects of L. plantarum ATCC 14917 metabolic profile caused by initial acid-base (pH 5.5 and 8.5) stress were investigated using 1 H nuclear magnetic resonance spectroscopy and multivariate data analysis. The results showed that the metabolome mainly consisted of 14 metabolites, including the components like amino acids, sugars, organic acids, and alkaloids. According to the nontargeted principal component analysis, there was a decrease in most of the metabolites in the alkali-treated group (mainly change in PC1) except acetate, whereas the production of lactate and glycine was increased in the acid-treated group (mainly change in PC2). Furthermore, the initial alkali stress inhibits the secretion of lactic acid, as a decrease was observed in the activity of lactate dehydrogenase and acetic dehydrogenase of L. plantarum ATCC 14917 in the alkali group. All these findings revealed that alkali stress could limit the acid environment formation of L. plantarum 14917 in the fermentation process; however, low acid pH is more suitable for the growth of L. plantarum.

Adaptation of Methanosarcina barkeri 227 as acetate scavenger for succinate fermentation by Actinobacillus succinogenes.

Acetate is the main by-product from microbial succinate production. In this study, we performed acetate removal by Methanosarcina barkeri 227 for succinate fermentation by Actinobacillus succinogenes 130Z. The acetoclastic methanogen M. barkeri requires similar environmental factors to A. succinogenes, and the conditions required for co-cultivation were optimized in this study: gas used for anaerobicization, strain adaptation, medium composition, pH adjustment, and inoculation time points. M. barkeri 227 was adapted to acetate for 150 days, which accelerated the acetate consumption to 9-fold (from 190 to 1726 mmol gDW-1 day-1). In the acetate-adapted strain, there was a noticeable increase in transcription of genes required for acetoclastic pathway-satP (acetate transporter), ackA (acetate kinase), cdhA (carbon monoxide dehydrogenase/acetyl-CoA synthase complex), and mtrH (methyl-H4STP:CoM methyltransferase), which was not induced before the adaptation process. The activities of two energy-consuming steps in the pathway-acetate uptake and acetate kinase-increased about 3-fold. This acetate-adapted M. barkeri could be successfully applied to succinate fermentation culture of A. succinogenes, but only after pH adjustment following completion of fermentation. This study suggests the utility of M. barkeri as an acetate scavenger during fermentation for further steps towards genetic and process engineering.

Disruption of the lactate dehydrogenase and acetate kinase genes in Klebsiella pneumoniae HD79 to enhance 2,3-butanediol production, and related transcriptomics analysis.

OBJECTIVES: 2,3-Butanediol (2,3-BD) is widely used in several chemical syntheses as well as the manufacture of plastics, solvents, and antifreeze formulations, and can be manufactured by microbial glucose fermentation. Conventional (2,3-BD) fermentation typically has low productivity, yield, and purity, and is expensive for commercial applications. We aimed to delete the lactate dehydrogenase and acetate kinase (ldhA and ack) genes in Klebsiella pneumoniae HD79 by using λRed homologous recombination technology, to eliminate by-products and thereby improve (2,3-BD) production. We also analyzed the resulting gene changes by using transcriptomics. RESULTS: The yield of (2,3-BD) from the mutant Klebsiella strain was 46.21 g/L, the conversion rate was 0.47 g/g, and the productivity was 0.64 g/L·h, which represented increases of 54.9%, 20.5%, and 106.5% respectively, compared to (WT) strains. Lactate and acetate decreased by 48.2% and 62.8%, respectively. Transcriptomics analysis showed that 4628 genes were differentially expressed (404 significantly up-regulated and 162 significantly down-regulated). Moreover, the (2,3-BD) operon genes were differentially expressed. CONCLUSION: Our data showed that the biosynthesis of (2,3-BD) was regulated by inducers (lactate and acetate), a regulator (BudR), and carbon flux. Elimination of acidic by-products by ldhA and ack knockdown significantly improved (2,3-BD) production. Our results provide a deeper understanding of the mechanisms underlying (2,3-BD) production, and form a molecular basis for the improvement this process by genetic modification in the future.

Improvement of Streptococcus suis glutamate dehydrogenase expression in Escherichia coli through genetic modification of acetate synthesis pathway.

Escherichia coli generates acetate as an undesirable by-product that has several negative effects on protein expression, and the reduction of acetate accumulation by modifying genes of acetate synthesis pathway can improve the expression of recombinant proteins. In the present study, the effect of phosphotransacetylase (pta) or/and acetate kinase (ackA) deletion on glutamate dehydrogenase (GDH) expression was investigated. The results indicated that the disruptions of pta or/and ackA decreased the acetate accumulation and synthesis of per gram cell, and increased cell density, and GDH expression and synthesis of per gram cell. The pta gene was more important for acetate formation than the ackA gene. Using the strain with deletions of pta-ackA (SSGPA) for GDH expression, acetate accumulation (2·61 g l-1 ) and acetate synthesis of per gram cell (0·229 g g-1 ) were lowest, decreasing by 28·29 and 41·43% compared with those of the parental strain (SSG) respectively. The flux of acetate synthesis (6·6%) was decreased by 72·15% compared with that of SSG, and the highest cell density (11·38 g l-1 ), GDH expression (2·78 mg ml-1 ), and GDH formation of per gram cell (0·2442 mg mg-1 ) were obtained, which were 1·22-, 1·43- and 1·17-times higher than the parental strain respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Acetate is the key undesirable by-product in Escherichia coli cultivation, and both biomass and production of desired products are increased by the reduction of acetate accumulation. In the present study, the strains with deletions of pta or/and ackA were constructed to reduce the acetate accumulation and improve the GDH expression, and the highest expression level of GDH was obtained using the strain with lesion in pta-ackA that was 1·17-times higher than that of the parental strain. The construction strategy of recombinant E. coli for decreasing the acetate excretion can be used for high expression level of other desired products.

Implementation of research on potential drug target cloning and characterization in a biochemistry laboratory.

An approach for incorporating preliminary drug discovery research into a biochemistry laboratory is described. During a total of 42 hr (one 3-hr laboratory section a week for 14 weeks), students were exposed to bioinformatics; molecular cloning; protein expression, purification, and characterization; and enzymatic kinetic assays. This research-oriented laboratory not only includes the standard elements of common undergraduate biochemistry laboratory manuals but also emphasizes the logical connections among biochemistry laboratory techniques. Moreover, this approach exposed students to laboratory research and the concept of drug discovery.

In silico-guided engineering of Pseudomonas putida towards growth under micro-oxic conditions.

BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.

Physiological and transcriptional comparison of acetate catabolism between Acinetobacter schindleri ACE and Escherichia coli JM101.

Acinetobacter bacteria preferentially use gluconeogenic substrates instead of hexoses or pentoses. Accordingly, Acinetobacter schindleri ACE reaches a high growth rate on acetate but is unable to grow on glucose, xylose or arabinose. In this work, we compared the physiology of A. schindleri ACE and Escherichia coli JM101 growing on acetate as the carbon source. In contrast to JM101, ACE grew on acetate threefold faster, had a twofold higher biomass yield, and a 45% higher specific acetate consumption rate. Transcriptional analysis revealed that genes like ackA, pta, aceA, glcB, fumA, tktA and talA were overexpressed while acsA, sfcA, ppc and rpiA were underexpressed in ACE relative to JM101. This transcriptional profile together with carbon flux balance analysis indicated that ACE forms acetyl-CoA preferentially by the AckA-Pta (acetate kinase-phosphotransacetylase) pathway instead of Acs (acetyl-CoA synthetase) and that the glyoxylate shunt and tricarboxylic acid cycle are more active in ACE than in JM101. Moreover, in ACE, ribose 5-phosphate and erythrose 4-phosphate are formed from trioses, and NADPH is mainly produced by isocitrate dehydrogenase. This knowledge will contribute to an understanding of the carbon metabolism of Acinetobacter species of medical, biotechnological and microbiological relevance.

Reversibility of enzymatic reactions might limit biotransformation of organic micropollutants.

Biotransformation of many organic micropollutants (OMPs) in sewage treatment plants is incomplete leading to their release into the environment. Recent findings suggest that thermodynamic aspects of the reaction as chemical equilibrium limit biotransformation, while kinetic parameters have a lower influence. Reversibility of enzymatic reactions might result in a chemical equilibrium between the OMP and the transformation product, thus impeding a total removal of the compound. To the best of our knowledge, no study has focused on proving the reversible action of enzymes towards OMPs so far. Therefore, we aimed at demonstrating this hypothesis through in vitro assays with bisphenol A (BPA) in the presence of kinase enzymes, namely acetate kinase and hexokinase, which are key enzymes in anaerobic processes. Results suggest that BPA is phosphorylated by acetate kinase and hexokinase in the presence of ATP (adenosine 5-triphosphate), but when the concentration of this co-substrate decreases and the enzymes loss their activity, the backward reaction occurs, revealing a reversible biotransformation mechanism. This information is particularly relevant to address new removal strategies, which up to now were mainly focused on modifying the kinetic parameters of the reaction.

Biosynthesis of (deoxy)guanosine-5'-triphosphate by GMP kinase and acetate kinase fixed on the surface of E. coli.

(Deoxy)guanosine-5'-triphosphate (5'-(d)GTP), the precursor for synthesizing DNA or RNA in vivo, is an important raw material for various modern biotechnologies based on PCR. In this study, we investigated the application of whole-cell catalysts constructed by bacterial cell surface display in biosynthetic reactions of 5'-(d)GTP from (deoxy)guanosine-5'-monophosphate (5'-(d)GMP). By N-terminal or N- and C-terminal fusion of the ice nucleation protein, we successfully displayed the GMP kinase of Lactobacillus bulgaricus and the acetate kinase of E. coli on the surface of E. coli cells. A large amount of soluble target protein was obtained upon induction with 0.2 mM IPTG at 25 °C for 30 h. The conversion of dGMP was up to 91% when catalysed by the surface-displayed enzymes at 37 °C for 4 h. Up to 95% of the GMP was converted after 3 h of reaction. The stability of the whole-cell catalyst at 37 °C was very good. The enzyme activity was maintained above 50% after 9 rounds of recovery. Our research showed that only one-twentieth of the initial substrate concentration of added ATP was sufficient to meet the reaction requirements.