Use of the mCherry fluorescent protein to optimize the expression of class I lanthipeptides in Escherichia coli.

BACKGROUND: Lanthipeptides are a rapidly expanding family of ribosomally synthesized and post-translationally modified natural compounds with diverse biological functions. Lanthipeptide structural and biosynthetic genes can readily be identified in genomic datasets, which provides a substantial repository for unique peptides with a wide range of potentially novel bioactivities. To realize this potential efficiently optimized heterologous production systems are required. However, only a few class I lanthipeptides have been successfully expressed using Escherichia coli as heterologous producer. This may be attributed to difficulties experienced in the co-expression of structural genes and multiple processing genes as well as complex optimization experiments. RESULTS: Here, an optimized modular plasmid system is presented for the complete biosynthesis for each of the class I lanthipeptides nisin and clausin, in E. coli. Genes encoding precursor lanthipeptides were fused to the gene encoding the mCherry red fluorescent protein and co-expressed along with the required synthetases from the respective operons. Antimicrobially active nisin and clausin were proteolytically liberated from the expressed mCherry fusions. The mCherry-NisA expression system combined with in vivo fluorescence monitoring was used to elucidate the effect of culture media composition, promoter arrangement, and culture conditions including choice of growth media and inducer agents on the heterologous expression of the class I lanthipeptides. To evaluate the promiscuity of the clausin biosynthetic enzymes, the optimized clausin expression system was used for the heterologous expression of epidermin. CONCLUSION: We succeeded in developing novel mCherry-fusion based plug and play heterologous expression systems to produce two different subgroups of class I lanthipeptides. Fully modified Pre-NisA, Pre-ClausA and Pre-EpiA fused to the mCherry fluorescence gene was purified from the Gram-negative host E. coli BL21 (DE3). Our study demonstrates the potential of using in vivo fluorescence as a platform to evaluate the expression of mCherry-fused lanthipeptides in E. coli. This allowed a substantial reduction in optimization time, since expression could be monitored in real-time, without the need for extensive and laborious purification steps or the use of in vitro activity assays. The optimized heterologous expression systems developed in this study may be employed in future studies for the scalable expression of novel NisA derivatives, or novel genome mined derivatives of ClausA and other class I lanthipeptides in E. coli.

Recent advances in genetic tools for engineering probiotic lactic acid bacteria.

Synthetic biology has grown exponentially in the last few years, with a variety of biological applications. One of the emerging applications of synthetic biology is to exploit the link between microorganisms, biologics, and human health. To exploit this link, it is critical to select effective synthetic biology tools for use in appropriate microorganisms that would address unmet needs in human health through the development of new game-changing applications and by complementing existing technological capabilities. Lactic acid bacteria (LAB) are considered appropriate chassis organisms that can be genetically engineered for therapeutic and industrial applications. Here, we have reviewed comprehensively various synthetic biology techniques for engineering probiotic LAB strains, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated genome editing, homologous recombination, and recombineering. In addition, we also discussed heterologous protein expression systems used in engineering probiotic LAB. By combining computational biology with genetic engineering, there is a lot of potential to develop next-generation synthetic LAB with capabilities to address bottlenecks in industrial scale-up and complex biologics production. Recently, we started working on Lactochassis project where we aim to develop next generation synthetic LAB for biomedical application.

Therapeutic Application of Lantibiotics and Other Lanthipeptides: Old and New Findings.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides, with modifications that are incorporated during biosynthesis by dedicated enzymes. Various modifications of the peptides are possible, resulting in a highly diverse group of bioactive peptides that offer a potential reservoir for use in the fight against a plethora of diseases. Their activities range from the antimicrobial properties of lantibiotics, especially against antibiotic-resistant strains, to antiviral activity, immunomodulatory properties, antiallodynic effects, and the potential to alleviate cystic fibrosis symptoms. Lanthipeptide biosynthetic genes are widespread within bacterial genomes, providing a substantial repository for novel bioactive peptides. Using genome mining tools, novel bioactive lanthipeptides can be identified, and coupled with rapid screening and heterologous expression technologies, the lanthipeptide drug discovery pipeline can be significantly sped up. Lanthipeptides represent a group of bioactive peptides that hold great potential as biotherapeutics, especially at a time when novel and more effective therapies are required. With this review, we provide insight into the latest developments made toward the therapeutic applications and production of lanthipeptides, specifically looking at heterologous expression systems.

Molecular insights into probiotic mechanisms of action employed against intestinal pathogenic bacteria.

Gastrointestinal (GI) diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species that have developed resistance to antibiotics. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Several studies have documented a reduction, or prevention, of GI diseases by probiotic bacteria. Since the activities of probiotic bacteria are closely linked with conditions in the host's GI-tract (GIT) and changes in the population of enteric microorganisms, a deeper understanding of gut-microbial interactions is required in the selection of the most suitable probiotic. This necessitates a deeper understanding of the molecular capabilities of probiotic bacteria. In this review, we explore how probiotic microorganisms interact with enteric pathogens in the GIT. The significance of probiotic colonization and persistence in the GIT is also addressed.

Development of a novel selection/counter-selection system for chromosomal gene integrations and deletions in lactic acid bacteria.

BACKGROUND: The underlying mechanisms by which probiotic lactic acid bacteria (LAB) enhance the health of the consumer have not been fully elucidated. Verification of probiotic modes of action can be achieved by using single- or multiple-gene knockout analyses of bacterial mutants in in vitro or in vivo models. We developed a novel system based on an inducible toxin counter-selection system, allowing for rapid and efficient isolation of LAB integration or deletion mutants. The Lactococcus lactis nisin A inducible promoter was used for expression of the Escherichia coli mazF toxin gene as counter-selectable marker. RESULTS: The flippase (FLP)/flippase recognition target (FRT) recombination system and an antisense RNA transcript were used to create markerless chromosomal gene integrations/deletions in LAB. Expression of NisR and NisK signalling proteins generated stable DNA integrations and deletions. Large sequences could be inserted or deleted in a series of steps, as demonstrated by insertion of the firefly bioluminescence gene and erythromycin resistance marker into the bacteriocin operons or adhesion genes of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA. CONCLUSIONS: The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. Although this work focuses on two probiotic LAB strains, L. plantarum 423 and E. mundtii ST4SA, the system developed could be adapted to most, if not all, LAB species.

In vivo bioluminescence imaging of the spatial and temporal colonization of lactobacillus plantarum 423 and enterococcus mundtii ST4SA in the intestinal tract of mice.

BACKGROUND: Lactic acid bacteria (LAB) are major inhabitants and part of the normal microflora of the gastrointestinal tract (GIT) of humans and animals. Despite substantial evidence supporting the beneficial properties of LAB, only a few studies have addressed the migration and colonization of probiotic bacteria in the GIT. The reason for this is mostly due to the limitations, or lack of, efficient reporter systems. Here we describe the development and application of a non-invasive in vivo bioluminescence reporter system to study, in real-time, the spatial and temporal persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the intestinal tract of mice. RESULTS: This study reports on the application of the firefly luciferase gene (ffluc) from Photinus pyralis to develop luciferase-expressing L. plantarum 423 and E. mundtii ST4SA, using a Lactococcus lactis NICE system on a high copy number plasmid (pNZ8048) and strong constitutive lactate dehydrogenase gene promoters (Pldh and STldh). The reporter system was used for in vivo and ex vivo monitoring of both probiotic LAB strains in the GIT of mice after single and multiple oral administrations. Enterococcus mundtii ST4SA reached the large intestine 45 min after gavage, while L. plantarum 423 reached the cecum/colon after 90 min. Both strains predominantly colonized the cecum and colon after five consecutive daily administrations. Enterococcus mundtii ST4SA persisted in faeces at higher numbers and for more days compared to L. plantarum 423. CONCLUSIONS: Our findings demonstrate the efficiency of a high-copy number vector, constitutive promoters and bioluminescence imaging to study the colonization and persistence of L. plantarum 423 and E. mundtii ST4SA in the murine GIT. The system allowed us to differentiate between intestinal transit times of the two strains in the digestive tract. This is the first report of bioluminescence imaging of a luciferase-expressing E. mundtii strain to study colonization dynamics in the murine model. The bioluminescence system developed in this study may be used to study the in vivo colonization dynamics of other probiotic LAB.

Reporter systems for in vivo tracking of lactic acid bacteria in animal model studies.

Bioluminescence (BLI) and fluorescence imaging (FI) allow for non-invasive detection of viable microorganisms from within living tissue and are thus ideally suited for in vivo probiotic studies. Highly sensitive optical imaging techniques detect signals from the excitation of fluorescent proteins, or luciferase-catalyzed oxidation reactions. The excellent relation between microbial numbers and photon emission allow for quantification of tagged bacteria in vivo with extreme accuracy. More information is gained over a shorter period compared to traditional pre-clinical animal studies. The review summarizes the latest advances in in vivo bioluminescence and fluorescence imaging and points out the advantages and limitations of different techniques. The practical application of BLI and FI in the tracking of lactic acid bacteria in animal models is addressed.

Use of the mCherry Fluorescent Protein To Study Intestinal Colonization by Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in Mice.

Lactic acid bacteria (LAB) are natural inhabitants of the gastrointestinal tract (GIT) of humans and animals, and some LAB species receive considerable attention due to their health benefits. Although many papers have been published on probiotic LAB, only a few reports have been published on the migration and colonization of the cells in the GIT. This is due mostly to the lack of efficient reporter systems. In this study, we report on the application of the fluorescent mCherry protein in the in vivo tagging of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423. The mCherry gene, encoding a red fluorescent protein (RFP), was integrated into a nonfunctional region on the genome of L. plantarum 423 by homologous recombination. In the case of E. mundtii ST4SA, the mCherry gene was cloned into the pGKV223D LAB/Escherichia coli expression vector. Expression of the mCherry gene did not alter the growth rate of the two strains and had no effect on bacteriocin production. Both strains colonized the cecum and colon of mice.