The Main Aim of The Study

Key characteristics that make Lactococcus lactis and Bacillus subtilis ideal microorganisms for the food industry are that are Generally Recognized As Safe (GRAS), they have annotated genomes, they can be cultivated easily and various tools for their genetic manipulation are available. The last years these two bacteria have proven to be highly potent on secreting heterologous and homologous proteins. Strains with the ability of secreting high amounts of proteins are of major interest for the industry for various reasons.

This study investigates the possibility of improving protein secretion in Lactococcus lactis and Bacillus subtilis using adaptive evolution and random mutagenesis. The first step for improving the secretion capacity in Lactococcus lactis was to create recombinant strains with the ability of secreting the heterologous enzymes β-galactosidase and β-glucosidase. The enzyme β-galactosidase is the product of the gene LacZ which is a part of the Lac operon in Escherichia coli. In its natural form the enzyme is found in the cytoplasm so in order to render Lactococcus lactis able to secrete the enzyme a sequence coding for a signal peptide should be added upstream of the enzyme’s sequence. The chosen signal leader sequence was of Usp45, the main Sec-depended protein in Lactococcus lactis. Both of the sequences would be cloned downstream of the gapB promoter which naturally controls the expression of the enzyme Glyceraldehyde-3-phosphate dehydrogenase, a very important enzyme involved in glycolysis. The enzyme β-glucosidase originated from Saccharophagus degradans and it would be cloned in an expression vector which offered a signal peptide. After successfully acquiring recombinant L.lactis strains the secretion capacity would be tested using droplet-based microfluidics screening. The sorting of droplets in which β-galactosidase would be found extracellularly was based on Fluorescence Resonance Energy Transfer (FRET). The development of a protocol for the detection of β-galactosidase in the droplets using FRET was also one of the goals in this project. Cells with the ability to secrete the enzyme β-glucosidase would be chosen based on fluorescence produced by catabolizing a fluorogenic substrate. The cells that would have been chosen from the general population would be subjected in multiple rounds of adaptive laboratory evolution and droplet-based microfluidics screening in order to further improve the secretion efficiency.

Bacillus subtilis naturally secretes the enzyme α-amylase thus there was no necessity on inserting any heterologous protein in the bacterium. The microorganism was subjected to treatment with 3 different concentrations of the mutagenesis agent Ethyl Methanesulfate for inducing mutations that could be proven beneficial. Furthermore, the secretion capacity was put under test by harnessing the power of evolution. The microorganism was transferred daily for a period of 7 weeks in new media with the ultimate purpose of creating strains with accumulated beneficial mutations and improved growth characteristics. The strains acquired with both of the approaches were examined using turbidimetric monitoring of the bacterial growth. The growth rates were supplemented with the starch degradation rates as well for testing how growth is correlated to starch degradation. In both cases no improvement was noticed either in the growth rates or the rates of starch degradation. This thesis is a partial fulfilment of the requirements for acquiring the M.Sc. degree within Biotechnology, at the Technical University of Denmark (DTU). The thesis covers 30 ECTS points and has been a product of development in collaboration with the National Food Institute at DTU.

I would like to express my upper gratitude to my supervisors Senior Researcher Claus Heiner Bang-Berthelsen and Postdoc Vinoth Wigneswaran for accepting me in their research group and making this study feasible. They were always available to help me and guide me throughout the project. Except of great supervisors they are great mentors as well. Their support, trust and understanding during some hard times were really important for the completion of the project. I would also like to thank Mike Vestergaard who helped me tremendously in the formulation of experiments during the project. Big thanks to the lab technician Tine Suhr and all the members of the research group for the kindness and willingness to help. Finally, my biggest thanks go to my father, my mother and my sister for their support.Heterologous and homologous protein secretion constitutes a field of great interest for the food industry. A little less than half of all the enzymes sold in a world-wide scale correspond to food and feed enzymes (Fernandes, 2010). Regardless of how potent an enzyme is, its utilization in the industry is directly related to possessing a host organism from which the protein can be isolated successfully and in large quantities. Thus, the search for microorganisms with the ability to secrete considerable amounts of proteins is a field of intense research.

Two highly potent bacteria that are under examination for their ability to secrete homologous and heterologous proteins are Bacillus subtilis and Lactococcus lactis (García-Fruitós, 2012). Protein secretion is a multifactorial process that needs further detailed elucidation in order to exploit the foul potential of these microorganisms. The variables that constitute the equation of protein secretion are the type of promoter, the translation efficiency of the mRNA, the number and type of intracellular and extracellular proteases, the type of signal peptide, the existence of a pro-peptide, the type of secretion system, the correct folding of the protein, the various chaperones and the possibility of a stress response due to protein over-expression [(Nijland & Kuipers, 2008),(Morello et al., 2007)].