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Staphylococcus epidermidis is a known opportunistic pathogen and is one of the leading causes of chronic biofilm-associated infections. Biofilm formation is considered as a main strategy to resist antibiotic treatment and help bacteria escape from the human immune system. Understanding the complex mechanisms in biofilm formation can help find new ways to treat resistant strains and lower the prevalence of nosocomial infections. In order to examine the role of RNAIII regulated by the agr quorum sensing system and to what extent it influences biofilm resistance to antimicrobial agents, deletion mutant S. epidermidis RP62a-ΔRNAIII deficient in repressor domains with a re-maining functional hld gene was created. A deletion strain was used to examine the influence of oxacillin in combination with vanillin on biofilm resistance and cell survival was determined. Utilizing real-time qPCR, confocal laser scanning microscopy (CLSM), and crystal violet staining analyses, we found that the RNAIII-independent controlled phenol soluble modulins (PSMs) and RNAIII effector molecule have a significant role in biofilm resistance to antibiotics and phenolic compounds, and it protects the integrity of biofilms. Moreover, a combination of antibiotic and antimicrobial agents can induce methicillin-resistant S. epidermidis biofilm formation and can lead to exceedingly difficult medical treatment.

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

Yeast Candida utilis is considered to be a potentially advantageous expression system for production of recombinant proteins utilizable for industrial and pharmaceutical purposes. As the scientific literature is not consistent in the ploidy of this yeast, in this work, we focused on resolving the problem via several methods such as the copy number determination of maltase gene by multiplex PCR, measuring α-glucosidase activity, the characterization of maltase gene copy number in deletion mutants using qPCR and flow cytometry. In context with the published data and results obtained in this study about the copy number of the maltase gene on C. utilis genome, we attempted to hypothesise and made conclusion about the ploidy of C. utilis. The results of this work, besides the biotechnological aspect, contribute to the elementary knowledge of C. utilis. The exact information about the ploidy or more specifically about the copy number of appropriate gene is essential for expression cassette dosage determination integrated into the chromosome of the host. In this study, we come to the conclusion that the maltase gene is present in C. utilis genome in four alleles, and in combination with flow cytometry, published information and the published genome sequences, the observations support the theory about tetraploidy of C. utilis.

Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations.

Human enterokinase (enteropeptidase, rhEP), a serine protease expressed in the proximal part of the small intestine, converts the inactive form of trypsinogen to active trypsin by endoproteolytic cleavage. The high specificity of the target site makes enterokinase an ideal tool for cleaving fusion proteins at defined cleavage sites. The mature active enzyme is comprised of two disulfide-linked polypeptide chains. The heavy chain anchors the enzyme in the intestinal brush border membrane, whereas the light chain represents the catalytic enzyme subunit. The synthetic gene encoding human enteropeptidase light chain with His-tag added at the C-terminus to facilitate protein purification was cloned into Pichia pastoris expression plasmids under the control of an inducible AOX1 or constitutive promoters GAP and AAC. Cultivation media and conditions were optimized as well as isolation and purification of the target protein. Up to 4 mg/L of rhEP was obtained in shake-flask experiments and the expression level of about 60–70 mg/L was achieved when cultivating in lab-scale fermentors. The constitutively expressing strains proved more efficient and less labor-demanding than the inducible ones. The rhEP was immobilized on AV 100 sorbent (Iontosorb) to allow repeated use of enterokinase, showing specific activity of 4 U/mL of wet matrix.

The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter. The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms. Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 microM; for Staphylococcus aureus 2.31 microM, and for Enterococcus faecalis 5.54 microM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of beta-galactosidase activity in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate quantities of active human antimicrobial peptide LL-37.

Recently, a new gene encoding beta-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37 degrees C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52 degrees C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of beta-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of beta-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified beta-glucuronidase from SEZ glcHis was four times as low as beta-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal beta-glucuronidase from E. coli with E. coli beta-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.

Streptococcus equi subsp. zooepidemicus is known to produce a hyaluronic acid capsule to resist the host immune defense. As the structure of the polysaccharide is identical to the one produced by humans, the bacteria S. equi subsp. zooepidemicus is used in biotechnological production of hyaluronic acid. In our laboratory we prepared mutated strains that are beta-glucuronidase deficient. Comparing the wild-type strain, which is positive in beta-glucuronidase activity, with the mutated strains named clone1 and clone2 in laboratory conditions, we observed that beta-glucuronidase influences the production of hyaluronic acid considerably and the molecular weight of hyaluronan slightly. The production of hyaluronic acid by the mutated strains is higher by approximately 20% and the molecular weight is larger by about 2%. The significant increase in the production of hyaluronic acid and the slight increase in the molecular weight are probably caused by an absence of free beta-glucuronic acid, due to its removal from the non-reducing termini of the polysaccharide by beta-glucuronidase. The presence of free beta-glucuronic acid would likely induce the expression of the beta-glucuronic-acid-utilizing operon, which in turn would reflect into a misuse of energy in the glucose-rich media.

Candida utilis represents a promising expression host, generating relatively high levels of recombinant proteins. The current study presents preliminary characterization of the upstream regulatory regions controlling the carbon source-dependent expression of the C. utilis maltase gene. Cellobiose and soluble starch were recognised as inducers of maltase promoter, besides maltose. We successfully applied the Cre-loxP system to acquire a null mutant strain with disrupted maltase gene and promoter in polyploid yeast C. utilis. Furthermore, the strength and minimal functional region of the promoter was evaluated by measuring β-galactosidase activity. Our results suggest, the qPCR was shown itself a very smart method for relatively easy characterization of the transformants and correlation of the expression levels with gene dosage.