Production of human recombinant phenylalanine hydroxylase in Lactobacillus plantarum for gastrointestinal delivery.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by a defective phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr) in presence of the cofactor tetrahydrobiopterin (BH4). Defective PAH causes accumulation of phenylalanine, which has neurotoxic effects and leads to dermatological, behavioral, and neurocognitive problems. Treatments for this disease consist in life-long diets that are hard for patients to keep, or supplementation with BH4. In this study, we propose a system where a probiotic lactic acid bacteria (LAB) can be used as vehicle to express in situ an engineered human PAH. Engineered PAHs contain a secretion peptide, a gastrointestinal signal (GI), the human PAH, and a flexible glycine linker followed by the fluorescence protein mEGFP. Engineered constructs were successfully transformed, expressed, and secreted in Lactobacillus plantarum CM_PUJ411. PAH construct containing either the signal peptide GI1 or GI2 were transported through a Caco-2 cell monolayer. Nevertheless, the one containing GI1 allowed the highest transport through the cell monolayer. Co-culture of L. plantarum and Caco-2 cells showed that engineered PAH is produced in-situ and transported through the cell monolayer. Finally, the activity test showed that the engineered PAH secreted by L. plantarum CM_PUJ411 is active, leading to a reduction in l-Phe and an increase in l-Tyr levels, respectively. These results show the potential of this system as a new therapeutic alternative for the treatment of PKU patients.

Native and heterologous production of bacteriocins from gram-positive microorganisms.

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.