Food-grade Lactobacilli expression systems for recombinant enzymes

In addition to the well-established recombinant microbial expression systems, such as Escherichia coli, Bacillus subtilis and others, increased attention has been paid to lactic acid bacteria, such as Lactobacilli, as expression host systems in the last few years. Lactobacillus spec. are a diverse genus of Gram-positive lactic acid bacteria which are widely used in the food industry. Different species of this genus have been applied since the 1990s as recombinant expression hosts. They have been used as so-called food-grade cell factories for the production of recombinant proteins, such as enzymes and antigens, as well as other pharmaceuticals. The major advantages of Lactobacilli as food-associated microorganisms used for recombinant protein production are the safe and sustainable use as overall safety food-grade expression systems. Some progress has been made over the past few years in the development of these promising bacterial expression systems, mostly on a laboratory scale. In this general introduction, an overview of some well-characterized tools for heterologous gene expression in Lactobacilli (host organisms, promoters, selection markers) is presented with a particular regard to food-grade approaches. 1 General Introduction and Thesis Outline 10 Lactic acid bacteria Lactic acid bacteria (LAB) are extensively used in the food industry for the production and the preservation of fermented foods. The LAB group includes about 20 different genera; those mainly important for technical applications in the food industry are the genera Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus. LAB are a phylogenetically heterogeneous group of Gram-positive, facultative anaerobic microorganisms that are clustered due to similarities in their metabolic pathway and physiological characteristics [Canchaya et al. 2006; Claesson et al. 2007; Hugenholtz and Smid 2002; Mozzi 2010]. The genus Bifidobacteria, as another important genus for industrial application, also has some of their typical features, but is phylogenetically unrelated to them [Sonomoto and Yokota 2011]. All of those mentioned are characterized due to a good degradation of different kinds of carbohydrates, which are metabolized to the predominant end-product – lactic acid. Because of that, they have been used for thousands of years for the acidification of food and feed, which has a preservative effect. This results in a good growth and viability even at low pH values of about 3.5 to 4 [de Vries et al. 2006]. They are inhabitants in different environments with a high level of carbohydrates, such as different kinds of fermented foods, as well as plant-derived substrates. LAB are used for the production of a variety of food and feed raw materials and are added as starter or adjunct cultures in different food products, such as yogurt, cheese, sausages, and fermented vegetables [Bron and Kleerebezem 2011; Pedersen et al. 2005]. They are involved in effecting the texture, flavour and shelf life of this food. Therefore, they are a common part of the human diet and generally harmless to humans. They do not generate endotoxins and are non-sporulating. Thus, they have a long history of safe use and many LAB have obtained the GRAS status [Bernardeau et al. 2008]. Additionally, specific LAB strains, mainly of the genus Lactobacillus, are known for their health-promoting capacity by the production of specific metabolites with a beneficial effect on healthy human gut microbiota and are used in probiotic products. The genus Lactobacillus belongs to the phylum Firmicutes, class Bacilli, order Lactobacillales, and family Lactobacillaceae. They are rod-shaped bacteria with either homoor heterofermentative lactic acid production. Thereby, the major fermentation pathway of glucose resulting in the end-product lactate or lactate, ethanol and/or acetic acid in equimolar amounts. The genus Lactobacillus contains 1 General Introduction and Thesis Outline 11 over 100 recognised species and is characterized by a high level of diversity [Canchaya et al. 2006]. This diversity is reflected by the G + C content of the genomic DNA of species included in the genus. This range is twice the span usually accepted for one genus and between 32-55% [Axelsson 2004]. The high diversity of the genus Lactobacillus is also obvious in the different kinds of habitat of various species. Some Lactobacillus sp. appear to be highly specialized only in a limited number of habitats, such as Lactobacillus delbrueckii which is found only in dairy environments. Other species, such as Lactobacillus plantarum, have been isolated from a diverse range of habitats including vegetables, meat, fish, and dairy products, as well as the gastro-intestinal tract of humans. The high adaption to habitats rich in carbohydrates and other nutrients leads to a poor biosynthetic capability and to high nutrient requirements for the cultivation of Lactobacilli. Due to a broad range of carbon sources which can be metabolised, Lactobacillus sp. have been used for the production of lactic acid from whey for decades [Kulozik and Wilde 1999]. A lot is known about the cultivation of Lactobacilli in a bioreactor [Schiraldi et al. 2003]. The industrial importance of Lactobacilli leads to the fact that this LAB has been extensively studied and is nowadays one of the best investigated microorganisms. A huge amount of knowledge has been obtained in research fields, such as microbiology, physiology, genetics, gene modification, and metabolic engineering, by applying biochemical, molecular biological and bioinformatic techniques. For these reasons, interest in Lactobacilli as suitable production hosts for recombinant proteins has increased over the last two decades. Different kinds of applications are conceivable using recombinant LAB. They have, for example, potential as delivery systems for valuable proteins, such as antibodies and antigens, and are used as live vaccines [Diep et al. 2009]. Additionally, due to the particular demands of the food industry, Lactobacilli are desirable cell factories for the production of food-grade enzymes [Garcia-Fruitos 2012; Peterbauer et al. 2011]. All these reasons explain why different Lactobacilli expression systems have been