Eminent Antimicrobial Peptide Resistance in Zymomonas mobilis: A Novel Advantage of Intrinsically Uncoupled Energetics.

Relative to several model bacteria, the ethanologenic bacterium Zymomonas mobilis is shown here to have elevated resistance to exogenous antimicrobial peptides (AMPs)- with regard to both peptide bulk concentration in the medium and the numbers of peptide molecules per cell. By monitoring the integration of AMPs in the bacterial cell membrane and observing the resulting effect on membrane energy coupling, it is concluded that the membranotropic effects of the tested AMPs in Z. mobilis and in Escherichia coli are comparable. The advantage of Z. mobilis over E. coli apparently results from its uncoupled mode of energy metabolism that, in contrast to E. coli, does not rely on oxidative phosphorylation, and hence, is less vulnerable to the disruption of its energy-coupling membrane by AMPs. It is concluded that the high resistance to antimicrobial peptides (AMPs) observed in Z. mobilis not only proves crucial for its survival in its natural environment but also offers a promising platform for AMP production and sheds light on potential strategies for novel resistance development in clinical settings.

Comparative Evaluation of Existing and Rationally Designed Novel Antimicrobial Peptides for Treatment of Skin and Soft Tissue Infections.

Skin and soft tissue infections (SSTIs) and acne are among the most common skin conditions in primary care. SSTIs caused by ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) can range in severity, and treating them is becoming increasingly challenging due to the growing number of antibiotic-resistant pathogens. There is also a rise in antibiotic-resistant strains of Cutibacterium acne, which plays a role in the development of acne. Antimicrobial peptides (AMPs) are considered to be a promising solution to the challenges posed by antibiotic resistance. In this study, six new AMPs were rationally designed and compared to five existing peptides. The MIC values against E. coli, P. aeruginosa, K. pneumoniae, E. faecium, S. aureus, and C. acnes were determined, and the peptides were evaluated for cytotoxicity using Balb/c 3T3 cells and dermal fibroblasts, as well as for hemolytic activity. The interaction with bacterial membranes and the effect on TNF-α and IL-10 secretion were also evaluated for selected peptides. Of the tested peptides, RP556 showed high broad-spectrum antibacterial activity without inducing cytotoxicity or hemolysis, and it stimulated the production of IL-10 in LPS-stimulated peripheral blood mononuclear cells. Four of the novel AMPs showed pronounced specificity against C. acnes, with MIC values (0.3-0.5 µg/mL) below the concentrations that were cytotoxic or hemolytic.

Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by Crypthecodinium cohnii from Glycerol, Glucose and Ethanol.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of C. cohnii metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by C. cohnii, making glycerol an attractive substrate for DHA production.

Syntrophy of Crypthecodinium cohnii and immobilized Zymomonas mobilis for docosahexaenoic acid production from sucrose-containing substrates.

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω-3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh-sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2-3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

Ethanologenesis and respiration in a pyruvate decarboxylase-deficient Zymomonas mobilis.

OBJECTIVE: Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner-Doudoroff (E-D) glycolysis, pyruvate decarboxylase (Pdc) and two alcohol dehydrogenase (ADH) isoenzymes. Pyruvate is the end-product of the E-D pathway and the substrate for Pdc. Construction and study of Pdc-deficient strains is of key importance for Z. mobilis metabolic engineering, because the pyruvate node represents the central branching point, most novel pathways divert from ethanol synthesis. In the present work, we examined the aerobic metabolism of a strain with partly inactivated Pdc. RESULTS: Relative to its parent strain the mutant produced more pyruvate. Yet, it also yielded more acetaldehyde, the product of the Pdc reaction and the substrate for ADH, although the bulk ADH activity was similar in both strains, while the Pdc activity in the mutant was reduced by half. Simulations with the kinetic model of Z. mobilis E-D pathway indicated that, for the observed acetaldehyde to ethanol production ratio in the mutant, the ratio between its respiratory NADH oxidase and ADH activities should be significantly higher, than the measured values. Implications of this finding for the directionality of the ADH isoenzyme operation in vivo and interactions between ADH and Pdc are discussed.

Improvement of Acetaldehyde Production in Zymomonas mobilis by Engineering of Its Aerobic Metabolism.

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers.

Translocation of Zymomonas mobilis pyruvate decarboxylase to periplasmic compartment for production of acetaldehyde outside the cytosol.

Acetaldehyde, a valuable commodity chemical, is a volatile inhibitory byproduct of aerobic fermentation in Zymomonas mobilis and in several other microorganisms. Attempting to improve acetaldehyde production by minimizing its contact with the cell interior and facilitating its removal from the culture, we engineered a Z. mobilis strain with acetaldehyde synthesis reaction localized in periplasm. For that, the pyruvate decarboxylase (PDC) was transferred from the cell interior to the periplasmic compartment. This was achieved by the construction of a Z. mobilis Zm6 PDC-deficient mutant, fusion of PDC with the periplasmic signal sequence of Z. mobilis gluconolactonase, and the following expression of this fusion protein in the PDC-deficient mutant. The obtained recombinant strain PeriAc, with most of its PDC localized in periplasm, showed a twofold higher acetaldehyde yield, than the parent strain, and will be used for further improvement by directed evolution.

Aerobic catabolism and respiratory lactate bypass in Ndh-negative Zymomonas mobilis.

Ability to ferment in the presence of oxygen increases the robustness of bioprocesses and opens opportunity for novel industrial setups. The ethanologenic bacterium Zymomonas mobilis performs rapid and efficient anaerobic ethanol fermentation, yet its respiratory NADH dehydrogenase (Ndh)-deficient strain (ndh-) is known to produce ethanol with high yield also under oxic conditions. Compared to the wild type, it has a lower rate of oxygen consumption, and an increased expression of the respiratory lactate dehydrogenase (Ldh). Here we present a quantitative study of the product spectrum and carbon balance for aerobically growing ndh-. Ldh-deficient and Ldh-overexpressing ndh- strains were constructed and used to examine the putative role of the respiratory lactate bypass for aerobic growth and production. We show that aerobically growing ndh- strains perform fermentative metabolism with a near-maximum ethanol yield, irrespective of their Ldh expression background. Yet, Ldh activity strongly affects the aerobic product spectrum in glucose-consuming non-growing cells. Also, Ldh-deficiency hampers growth at elevated temperature (42 °C) and delays the restart of growth after 10-15 h of aerobic starvation.

The Low Energy-Coupling Respiration in Zymomonas mobilis Accelerates Flux in the Entner-Doudoroff Pathway.

Performing oxidative phosphorylation is the primary role of respiratory chain both in bacteria and eukaryotes. Yet, the branched respiratory chains of prokaryotes contain alternative, low energy-coupling electron pathways, which serve for functions other than oxidative ATP generation (like those of respiratory protection, adaptation to low-oxygen media, redox balancing, etc.), some of which are still poorly understood. We here demonstrate that withdrawal of reducing equivalents by the energetically uncoupled respiratory chain of the bacterium Zymomonas mobilis accelerates its fermentative catabolism, increasing the glucose consumption rate. This is in contrast to what has been observed in other respiring bacteria and yeast. This effect takes place after air is introduced to glucose-consuming anaerobic cell suspension, and can be simulated using a kinetic model of the Entner-Doudoroff pathway in combination with a simple net reaction of NADH oxidation that does not involve oxidative phosphorylation. Although aeration hampers batch growth of respiring Z. mobilis culture due to accumulation of toxic byproducts, nevertheless under non-growing conditions respiration is shown to confer an adaptive advantage for the wild type over the non-respiring Ndh knock-out mutant. If cells get occasional access to limited amount of glucose for short periods of time, the elevated glucose uptake rate selectively improves survival of the respiring Z. mobilis phenotype.

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase.

Hydrogen-producing Escherichia coli strains overexpressing lactose permease: FT-IR analysis of the lactose-induced stress.

The lactose permease gene (lacY) was overexpressed in the septuple knockout mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and as a result stimulate overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay in the recombinant strain on lactose, resembling to some extent the "lactose killing" phenomenon. Therefore, the recombinant strain was subjected to selection on lactose-containing media. Selection on plates with 3% lactose yielded a strain with a decreased content of the recombinant plasmid but with an improved ability to grow and produce hydrogen on lactose. Macromolecular analysis of its biomass by means of Fourier transform-infrared spectroscopy demonstrated that increase of the cellular polysaccharide content might contribute to the adaptation of E. coli to lactose stress.

Effect of ADH II deficiency on the intracellular redox homeostasis in Zymomonas mobilis.

Mutant strain of the facultatively anaerobic, ethanol-producing bacterium Zymomonas mobilis, deficient in the Fe-containing alcohol dehydrogenase isoenzyme (ADH II), showed impaired homeostasis of the intracellular NAD(P)H during transition from anaerobic to aerobic conditions, and also in steady-state continuous cultures at various oxygen supplies. At the same time, ADH II deficiency in aerobically grown cells was accompanied by a threefold increase of catalase activity and by about 50% increase of hydrogen peroxide excretion. It is concluded that ADH II under aerobic conditions functions to maintain intracellular redox homeostasis and to protect the cells from endogenous hydrogen peroxide.

Electron transport and oxidative stress in Zymomonas mobilis respiratory mutants.

The ethanol-producing bacterium Zymomonas mobilis is of great interest from a bioenergetic perspective because, although it has a very high respiratory capacity, the respiratory system does not appear to be primarily required for energy conservation. To investigate the regulation of respiratory genes and function of electron transport branches in Z. mobilis, several mutants of the common wild-type strain Zm6 (ATCC 29191) were constructed and analyzed. Mutant strains with a chloramphenicol-resistance determinant inserted in the genes encoding the cytochrome b subunit of the bc (1) complex (Zm6-cytB), subunit II of the cytochrome bd terminal oxidase (Zm6-cydB), and in the catalase gene (Zm6-kat) were constructed. The cytB and cydB mutants had low respiration capacity when cultivated anaerobically. Zm6-cydB lacked the cytochrome d absorbance at 630 nm, while Zm6-cytB had very low spectral signals of all cytochromes and low catalase activity. However, under aerobic growth conditions, the respiration capacity of the mutant cells was comparable to that of the parent strain. The catalase mutation did not affect aerobic growth, but rendered cells sensitive to hydrogen peroxide. Cytochrome c peroxidase activity could not be detected. An upregulation of several thiol-dependent oxidative stress-protective systems was observed in an aerobically growing ndh mutant deficient in type II NADH dehydrogenase (Zm6-ndh). It is concluded that the electron transport chain in Z. mobilis contains at least two electron pathways to oxygen and that one of its functions might be to prevent endogenous oxidative stress.

NADH dehydrogenase deficiency results in low respiration rate and improved aerobic growth of Zymomonas mobilis.

The respiratory chain of the ethanol-producing bacterium Zymomonas mobilis is able to oxidize both species of nicotinamide cofactors, NADH and NADPH. A mutant strain with a chloramphenicol-resistance determinant inserted in ndh (encoding an NADH : CoQ oxidoreductase of type II) lacked the membrane NADH and NADPH oxidase activities, while its respiratory D-lactate oxidase activity was increased. Cells of the mutant strain showed a very low respiration rate with glucose and no respiration with ethanol. The aerobic growth rate of the mutant was elevated; exponential growth persisted longer, resulting in higher biomass densities. For the parent strain a similar effect of aerobic growth stimulation was achieved previously in the presence of submillimolar cyanide concentrations. It is concluded (i) that the respiratory chain of Z. mobilis contains only one functional NAD(P)H dehydrogenase, product of the ndh gene, and (ii) that inhibition of respiration, whether resulting from a mutation or from inhibitor action, stimulates Z. mobilis aerobic growth due to redirection of the NADH flux from respiration to ethanol synthesis, thus minimizing accumulation of toxic intermediates by contributing to the reduction of acetaldehyde to ethanol.