iBB

Lactococcus lactis is a food-grade, and generally recognized as safe, bacterium, which making it ideal for producing plasmid DNA (pDNA) or recombinant proteins for industrial or pharmaceutical applications. A paper published in Microorganisms by Sofia Duarte and Gabriel Monteiro from BERG-iBB reviews the major findings from L. lactis transcriptome and proteome studies, with an overexpression of native or recombinant proteins. These studies provide important insights on how to engineer the plasmid vectors and/or the strains in order to achieve high pDNA or recombinant proteins yields, with high quality standards. 

While Lactococcus lactis is traditionally associated with the fermented food industry, applications of these bacteria have been spreading to the pharmaceutical industry. The goal is to use L. lactis as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. In a recent review in International Molecular Sciences, Sofia Duarte and Gabriel Monteiro from BERG-iBB critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. The work is funded by the LactoSynt project.

Cell-based therapies can enhance the specificity of anti-cancer therapeutic agents. In this context, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anti-cancer proteins due to their innate tropism for tumors. iBB researchers Marília Silva, Gabriel Monteiro, Arsénio Fialho, Nuno Bernardes and Cláudia Lobato da Silva, engineered human MSC through non-viral methods to secrete a human codon-optimized version of azurin (hazu), a bacterial protein with demonstrated anti-cancer activity towards different cancer models in vitro and in vivo. Upon treatment with conditioned media (CM) from these engineered cells, a decrease in cancer cell proliferation, migration and invasion was seen, and an increase in cell death was observed for breast and lung cancer cell models. The results achieved by SCERG- and BSRG-iBB researchers were published in Frontiers in Cell and Developmental Biology, Stem Cell Research section.

BERG-iBB researchers led by Gabriel Monteiro are engineering lactic acid bacteria and plasmid vectors in order to develop a flexible platform for biomolecule production. The cover image of the August issue of Biotechnology Journal highlights a recent contribution of the group. The work is part of the FCT-funded project LactoSynt.

Peripheral artery disease (PAD) is a debilitating condition characterized by the blockage of arteries, which leads to limb amputation in more severe cases. Researchers from SCERG- and BERG-iBB propose the use of human bone marrow (BM) MSC transiently transfected with minicircles encoding for VEGF as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The data shows that VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays.These results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach to improve angiogenesis in the context of PAD. The work was published in Human Gene Therapy.

The bacterial genome can be modified to favour the synthesis of recombinant protein. Such productivity gains can contribute to lower costs and increase the affordabiliy of protein pharmaceuticals. BERG-iBB researchers and colleagues from the Institute of Technology Guwahati (India) have shown that deletion of the pgi (phosphoglucose isomerase) gene in Escherichia coli favours the production of the pharmaceutically important protein interferon gamma (IFN-gamma). Specific IFN-gamma yields were 3.0-fold higher in mutant compared to native strain. Furthermore, a metabolic model analysis showed that pgi deletion raises flux efficiency towards IFN-gamma synthesis. The work was published in Enzyme Microbial Technology.

Photo details: 3D model of pgi by Emw, own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=8814547

The determination of the recombination efficiency and the monitoring of parental plasmids (PP), minicircles (MC) and miniplasmid (MP) species during processing and in the final product are critical aspects of minicircle manufacturing. A paper published in Analytical Biochemistry by BERG-iBB researchers Cláudia Alves, Miguel Prazeres and Gabriel Monteiro describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The results presented indicate that the method can be applied for the determination of recombination efficiency in pure DNA samples.

Description: plate for screening CRISPR modifications, featured photo by Cláudia Alves, Copyright BERG-iBB and University of Florida 2019.

Context: BIOTecnico PhD student Cláudia Alves, under the supervision of BERG-iBB researchers Gabriel Monteiro and Miguel Prazeres and in collaboration with Christopher Reisch from the University of Florida, is using CRISPR/Cas9 to create mutations in E. coli that improve minicircle production.

The lipopolysaccharide-free Lactococcus lactis is an interesting alternative to E. coli for plasmid production. A key requirement for L. lactis‐based plasmid manufacturing is the availability of high‐copy number plasmids. In a recent publication in Biotechnology Journal, BERG-iBB researchers led by Gabriel Monteiro describe how engineering of the repDE Ribosome Binding Site led to an increase in the copy number  of plasmid pTRKH3 in L. lactis LMG19460 cells.  The number of copies of mutant pTRKH3‐b increased up to 215 copies per chromosome, which corresponds to a 3.5 fold increase when compared to the non‐modified pTRKH3. The new mutant is an important step towards the establishment of lactic acid bacteria as viable plasmid producers. The work was developed in the context of the FCT-funded, LactoSynt project.

The ability to purify supercoiled minicircles (sc MC) from related miniplasmid (MP) and parental plasmid (PP) impurities is critical for a wider application of MC vectors in gene therapy research. BERG-iBB researchers developed a process that relies on multimodal chromatography with a 2-step NaCl elution to separate sc MC from open circular impurities. A detailed study of the DNA-ligand interactions underlying the separation shows that the order of elution reflects the increasing degree of base exposure of the species. Data further suggests that nucleic acid bases most likely engage in cation-π, π-π stacking and H-bonding with ligand. The work was published in Separation and Purification Technology.

The project "LactoSynt: Lactic Acid Bacteria as Cell Factories: a Synthetic Biology Approach for Plasmid DNA and Recombinant Protein Production" has been recommended for funding by FCT (2017 Call for SR&TD Project Grants). The goal of LactoSynt is to engineer lactic acid bacteria and plasmid vectors in order to develop a flexible platform for biomolecule production. Applications in the pharmaceutical field (DNA vaccines, recombinant proteins) are envisaged. The project, which falls within the scientific area of Medical Biotechnology, is headed by Gabriel Monteiro from BERG-iBB and Leonilde Moreira from BSRG-iBB.