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.

Complex Analysis of Vanillin and Syringic Acid as Natural Antimicrobial Agents against Staphylococcus epidermidis Biofilms.

The presence of Staphylococcus epidermidis biofilms on medical devices is a major cause of nosocomial diseases and infections. Extensive research is directed at inhibiting the formation and maturation of such biofilms. Natural plant-derived phenolic compounds have promising antimicrobial effects against drug-resistant bacteria. The anti-biofilm activity of two selected phenolic compounds (vanillin and syringic acid) was tested against three biofilm-forming methicillin-resistant S. epidermidis strains with different genotypes. Resazurin assay combining crystal violet staining and confocal microscopy was used for biofilm and extracellular polymer substance (EPS) inhibition tests. Effects on EPS compounds such as proteins, extracellular DNA, and polysaccharides were also examined. Combined with quantitative real-time PCR of selected agr quorum-sensing systems and biofilm genetic determinants, our complex analysis of vanillin and syringic acid showed similar biofilm and EPS inhibition effects on S. epidermidis strains, reducing biofilm formation up to 80% and EPS up to 55%, depending on the genotype of the tested strain. Natural antimicrobial agents are thus potentially useful inhibitors of biofilms.

Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in Staphylococcus epidermidis Biofilms.

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.

Enhancement of solubility of recombinant alcohol dehydrogenase from Rhodococcus ruber using predictive tool.

Solubility is one of key factors influencing the heterologous production of recombinant proteins in biotechnology. Among many aggregation-prone proteins, alcohol dehydrogenase (ADH-A) from Rhodococcus ruber (in this work abbreviated RrADH) shows a great potential in processes involved in the biotransformation of natural compounds. As ADH-A is a potentially high value asset in industrial biotransformation processes, improvement of its solubility would be of major commercial benefit. Predictive tools and in silico analysis provide a fast means for improving protein properties, for selecting appropriate changes, and ultimately for saving costs. We have therefore focused on enhancement of the solubility of RrADH using an online accesible predictive tool Aggrescan 3D 2.0. Selected mutations were introduced into the protein amino acid sequence by using site-directed PCR. This led to a 17% increase in the protein solubility of RrADHmut1 and a 98% increase for RrADHmut2. Moreover, the basic kinetics of the enzyme reaction were positively affected, further optimizing the overall performance of the production process.

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.

Functional and structural characterisation of 5 missense mutations of the phenylalanine hydroxylase.

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.

High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

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.

Escherichia coli as a glycoprotein production host: recent developments and challenges.

Chinese Hamster Ovary cells are the most popular host expression system for the large-scale production of human therapeutic glycoproteins, but, the race to engineer Escherichia coli to perform glycosylation is gathering pace. The successful functional transfer of an N-glycosylation pathway from Campylobacter jejuni to Escherichia coli in 2002 can be considered as the crucial first engineering step. Here, we discuss the recent advancements in the field of N-glycosylation of recombinant therapeutic proteins in E. coli cells, from the manipulation of glycan composition, to the improvement in glycosylation efficiency, along with the challenges that remain before E. coli can be available as an industry host cell for economically viable glycoprotein production.