Bacterial Lighthouses-Real-Time Detection of Yersinia enterocolitica by Quorum Sensing.

Foodborne zoonotic pathogens have a severe impact on food safety. The demand for animal-based food products (meat, milk, and eggs) is increasing, and therefore faster methods are necessary to detect infected animals or contaminated food before products enter the market. However, conventional detection is based on time-consuming microbial cultivation methods. Here, the establishment of a quorum sensing-based method for detection of foodborne pathogens as Yersinia enterocolitica in a co-cultivation approach using a bacterial biosensor carrying a special sensor plasmid is described. We combined selective enrichment with the simultaneous detection of pathogens by recording autoinducer-1-induced bioluminescent response of the biosensor. This new approach enables real-time detection with a calculated sensitivity of one initial cell in a sample after 15.3 h of co-cultivation, while higher levels of initial contamination can be detected within less than half of the time. Our new method is substantially faster than conventional microbial cultivation and should be transferrable to other zoonotic foodborne pathogens. As we could demonstrate, quorum sensing is a promising platform for the development of sensitive assays in the area of food quality, safety, and hygiene.

Plant-Derived Cell-Free Biofactories for the Production of Secondary Metabolites.

Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot in vitro transcription-translation reaction and contains highly active energy and cofactor regeneration pathways. Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 µg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.

Production, Quality Control, Stability and Pharmacotoxicity of a Malaria Vaccine Comprising Three Highly Similar PfAMA1 Protein Molecules to Overcome Antigenic Variation.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.