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.

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.