Novel expression system based on enhanced permeability of Vibrio natriegens cells induced by D,D- carboxypeptidase overexpression.

Vibrio natriegens is a fast-growing, non-pathogenic marine bacterium with promising features for biotechnological applications such as high-level recombinant protein production or fast DNA propagation. A remarkable short generation time (< 10 min), robust proteosynthetic activity and versatile metabolism with abilities to utilise wide range of substrates contribute to its establishment as a future industrial platform for fermentation processes operating with high productivity.D,D-carboxypeptidases are membrane-associated enzymes involved in peptidoglycan biosynthesis and cell wall formation. This study investigates the impact of overexpressed D,D-carboxypeptidases on membrane integrity and the increased leakage of intracellular proteins into the growth medium in V. natriegens. Our findings confirm that co-expression of these enzymes can enhance membrane permeability, thereby facilitating the transport of target proteins into the extracellular environment, without the need for secretion signals, tags, or additional permeabilization methods. Using only a single step IMAC chromatography, we were able to purify AfKatG, MDBP or Taq polymerase in total yields of 117.9 ± 56.0 mg/L, 36.5 ± 12.9 mg/L and 26.5 ± 6.0 mg/L directly from growth medium, respectively. These results demonstrate the feasibility of our V. natriegens based system as a broadly applicable extracellular tag-less recombinant protein producer.

Tellurium: A Rare Element with Influence on Prokaryotic and Eukaryotic Biological Systems.

Metalloid tellurium is characterized as a chemical element belonging to the chalcogen group without known biological function. However, its compounds, especially the oxyanions, exert numerous negative effects on both prokaryotic and eukaryotic organisms. Recent evidence suggests that increasing environmental pollution with tellurium has a causal link to autoimmune, neurodegenerative and oncological diseases. In this review, we provide an overview about the current knowledge on the mechanisms of tellurium compounds' toxicity in bacteria and humans and we summarise the various ways organisms cope and detoxify these compounds. Over the last decades, several gene clusters conferring resistance to tellurium compounds have been identified in a variety of bacterial species and strains. These genetic determinants exhibit great genetic and functional diversity. Besides the existence of specific resistance mechanisms, tellurium and its toxic compounds interact with molecular systems, mediating general detoxification and mitigation of oxidative stress. We also discuss the similarity of tellurium and selenium biochemistry and the impact of their compounds on humans.

Evaluation of thermostable catalase-peroxidase AfKatG supplementation on toxicity of residual hydrogen peroxide in cultivation media of lactic acid bacteria from starter cultures.

Lactic acid bacteria (LAB) are exceptionally important strains in food industry. It is a heterogeneous group sharing same metabolic and physiological properties. They are usually catalase-negative strains, which represents a big disadvantage in food production in comparison with pathogenic bacteria as staphylococci and listeria existing in the same environment, because of the use of hydrogen peroxide as a disinfection agent which is utilized by catalases. We focused on increase in LAB surviving through the disinfection without any positive effect on growth of pathogenic bacteria. In our functional test hydrogen peroxide was used for disinfection. Ten mM thermostable catalase-peroxidase AfKatG was added to solid media to cultivate bacteria afterwards. As predicted there was no difference in the growth of pathogenic bacteria with or without catalase-peroxidase addition to media. However, we showed a huge positive effect on surviving LAB. With addition of AfKatG to solid media we gained 2-38 times higher CFU/ml than in control samples without it. We can assume AfKatG as an excellent supplement for growth media of food strains.

Production of Reverse Transcriptase and DNA Polymerase in Bacterial Expression Systems.

DNA amplification and reverse transcription enzymes have proven to be invaluable in fast and reliable diagnostics and research applications because of their processivity, specificity, and robustness. Our study focused on the production of mutant Taq DNA polymerase and mutant M-MLV reverse transcriptase in the expression hosts Vibrio natriegens and Escherichia coli under various expression conditions. We also examined nonspecific extracellular production in V. natriegens. Intracellularly, M-MLV was produced in V. natriegens at the level of 11% of the total cell proteins (TCPs) compared with 16% of TCPs in E. coli. We obtained a soluble protein that accounted for 11% of the enzyme produced in V. natriegens and 22% of the enzyme produced in E. coli. Taq pol was produced intracellularly in V. natriegens at the level of 30% of TCPs compared with 26% of TCPs in E. coli. However, Taq pol was almost non-soluble in E. coli, whereas in V. natriegens, we obtained a soluble protein that accounted for 23% of the produced enzyme. We detected substantial extracellular production of Taq pol. Thus, V. natriegens is a suitable alternative host with the potential for production of recombinant proteins.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation.

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

Comparison of simple expression procedures in novel expression host Vibrio natriegens and established Escherichia coli system.

Marine bacterium Vibrio natriegensis a novel host platform for different applications in molecular biology and biotechnology. It has one of the fastest growth rates of any known microorganisms and its extremely short doubling time indicates a high level of proteosynthetic activity. Regarding the necessity of developing new high-level protein expression systems it represents an extremely interesting subject. V. natriegens fulfills many important features for a suitable host including non- pathogenicity, easy scale-up process, potential for using alternative carbon sources (compared to E. coli), growth media and potential for further genetic and metabolic engineering with employment of a wide range of genetic tools. This work compares V. natriegens as an expression host for production of recombinant human growth hormone (hGH), yeast alcohol dehydrogenase (ADH) and archaeal catalase-peroxidase (AfKatG) to E. coliand establishes the basis for future development of this platform. The selected proteins are of different origins, sizes and intended applications. Our results have shown that cultures of V. natriegens using sucrose as a main carbon source can be used for the production of industrially applicable proteins, where it offers higher biomass productions compared to E. coli. In case of human growth hormone production, produced amounts were lower compared to those of E. coli (38 % of total cell protein (TCP) for V. natriegens vs. 58 % of TCP for E. coli, with similar solubility of around 40 % in both cases). In case of yeast alcohol dehydrogenase, V. natriegens produced 26 % of TCP vs. 42 % of TCP in E. coli, but with severely decreased solubility in case of V. natriegens cultures. Finally V. natriegens cultures were able to produce catalase-peroxidase AfKatG at the level of 33 % of TCP compared to 26 % of TCP in E. coli. Obtained results suggest that there are still significant differences in reliability and ease of use between E. coli and V. natriegens, with latter being more susceptible to condition changes and producing inconsistent results.

Assessment of bioavailability of Mg from Mg citrate and Mg oxide by measuring urinary excretion in Mg-saturated subjects.

BACKGROUND: Low magnesium (Mg) levels are linked to many diseases. Studies suggest that organic salts of Mg are more readily bioavailable than its oxide or inorganic salts used for supplements production. Unfortunately, the plethora of variables in the previous study designs complicates the making of any clear and reliable conclusions. METHODS: 14 healthy males were supplemented for five days with 400 mg Mg to saturate Mg pools before intake of the test products. Bioavailability of 400 mg Mg from Mg citrate (MgC) and Mg oxide (MgO) after single-dose administration was assessed by measuring renal Mg excretion in 24-h urine and blood plasma [Mg] at time points 0, 2, 4, 8, and 24 h. RESULTS: Single-dose MgC supplementation led to a significant (P < 0.05) increase in 24 h urinary Mg excretion, but this was not significant following MgO. Plasma [Mg] was also significantly higher for MgC than for MgO at 4 h (P < 0.05) and 8 h (P < 0.05). Compared with baseline levels, MgC supplementation showed a significant increase in plasma [Mg] at all time points, in contrast to MgO. CONCLUSIONS: MgC shows higher bioavailability compared with MgO. Furthermore, urinary Mg excretion should be determined as the primary endpoint of Mg bioavailability studies.