Staphylococcus carnosus: from starter culture to protein engineering platform

Appl Microbiol Biotechnol DOI 10.1007/s00253-017-8528-6 MINI-REVIEW Staphylococcus carnosus: from starter culture to protein engineering platform John Löfblom 1 & Ralf Rosenstein 2 & Minh-Thu Nguyen 2 & Stefan Ståhl 1 & Friedrich Götz 2 Received: 3 July 2017 / Revised: 8 September 2017 / Accepted: 11 September 2017 # The Author(s) 2017. This article is an open access publication Abstract Since the 1950s, Staphylococcus carnosus is used as a starter culture for sausage fermentation where it contributes to food safety, flavor, and a controlled fermentation process. The long experience with S. carnosus has shown that it is a harmless and Bfood grade^ species. This was confirmed by the genome sequence of S. carnosus TM300 that lacks genes involved in pathogenicity. Since the development of a cloning system in TM300, numerous genes have been cloned, expressed, and characterized and in particular, virulence genes that could be functionally validated in this non-pathogenic strain. A secretion system was developed for production and secretion of industrially important proteins and later modified to also enable display of heterologous proteins on the surface. The display system has been employed for various purposes, such as development of live bacterial delivery vehicles as well as microbial biocatalysts or bioadsorbents for potential environmental or biosensor applications. Recently, this surface display system has been utilized for display of peptide and protein libraries for profiling of protease substrates and for generation of various affinity proteins, e.g., Affibody molecules and scFv antibodies. In addition, by display of fragmented antigen-encoding genes, the surface expression system has been successfully used for epitope mapping of * Stefan Ståhl * Friedrich Götz 1 Division of Protein Technology, School of Biotechnology, KTH-Royal Institute of Technology, AlbaNova University Center, Roslagstullsbacken 21, 106 91 Stockholm, Sweden 2 Microbial Genetics, Interfaculty Institute of Microbiology and Infection Medicine and Infection Medicine (IMIT), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany antibodies. Reviews on specific applications of S. carnosus have been published earlier, but here we provide a more extensive overview, covering a broad range of areas from food fermentation to sophisticated methods for protein-based drug discovery, which are all based on S. carnosus. Keywords Bacterial surface display . Combinatorial protein engineering . Epitope mapping . Food fermentation . Starter culture . Virulence factors Introduction This review article is unique in its nature in that it describes the use of the food grade G ram-positive bacterium, Staphylococcus carnosus, evolving over several decades, from being an important strain in food fermentation (Götz 1990c) to becoming a versatile and powerful microbial tool in modern microbiology and biotechnology. When the genome sequence was deciphered (Rosenstein and Götz 2010; Rosenstein et al. 2009), the different characteristics of S. carnosus were better understood, and as will be described, its non-pathogenic nature made it suitable for characterization of virulence factors. The development of a host-vector system for efficient and secreted recombinant production inspired the development of also a surface display system for S. carnosus. The use of these systems in a wide variety of application areas will be reviewed. S. carnosus as a starter culture Some of the most well-investigated staphylococcal species (e.g., S. aureus) are pathogens. However, like many other genera, Staphylococcus is composed of many species (> 40) with a vast Appl Microbiol Biotechnol diversity, of which only few are associated with pathogenicity. The majority has never been associated with infection, and some species are even used as starter cultures in sausage fermentation (Götz et al. 2006). The first reports on using S. carnosus in sausage fermentation came in the 1950s (Lerche and Sinell 1955; Niinivaara and Pohja 1956). At that time, they were regarded as micrococci, a group of Gram-positive cocci that are facultative anaerobic and catalase-positive. However, a systematic analysis of the starter cultures in various fermented dry sausages revealed that most of these micrococci were incorrectly classified and are in fact S. carnosus (Schleifer and Fischer 1982). S. carnosus and S. xylosus are the two main staphylococcal species worldwide that are used as starter cultures in food fermentation, either alone or in combination with defined lactobacilli or other microorganisms. Starter cultures protect the food from undesirable bacteria and make the fermentation process more reliable. They also suppress food spoilage and poisoning by unwanted microorganisms and the whole fermentation process can be better controlled. S. carnosus has several functions during the ripening process of dry sausage (Barriere and Leroy-Setrin 2001; Corbiere Morot-Bzot et al. 2007; Liepe and Porobic 1983); nitrate is reduced to nitrite which, together with myoglobin, forms the red colored nitrosomyoglobin (Neubauer and Götz 1996; Götz 1990c). Subsequently, nitrite is further reduced to ammonia which leads to regeneration of NAD+ that is needed for glycolysis (Neubauer et al. 1999). S. carnosus also contributes to flavor and to detoxification of hydrogen peroxide that is produced by lactobacilli (Barriere and Leroy-Setrin 2001). Because of its use as a starter culture since the 1950s, S. carnosus is regarded as a Bfood grade^ species (Fig. 1a). Dissimilatory nitrate fermentation Beside flavor, one of the main functions of S. carnosus as a starter culture is its ability to reduce nitrate and nitrite. Nitrate and/or nitrite are curing agents that play a decisive role in obtaining the specific sensory properties, stability, and hygienic safety of products such as fermented sausages, ham, and more recently, emulsion type of sausages (Hammes 2012). The intermediary presence of nitrite is important as it prevents the growth of food-spoiling bacteria such as Clostridium. On the other hand, at the end of the fermentation process, both nitrate and nitrite should be decreased below a certain threshold level. As many lactobacilli are unable to reduce nitrate, S. carnosus has an important function in the process. In S. carnosus, the reduction of nitrate to ammonia involves several steps (Fig. 1b) (Neubauer and Götz 1996): (i) nitrate is taken up and reduced to nitrite, and nitrite is subsequently excreted, (ii) after depletion of nitrate, the externally accumulated nitrite is taken up by the cells and reduced to ammonia, which again is excreted into the medium. The nitrate reduction by the nitrate reductase is connected with energy gain and is therefore also referred to as Banaerobic respiration^ or Bdissimilatory nitrate reduction^ (Fast et al. 1996; Fedtke et al. 2002). The nitrate reductase is a membranebound enzyme, whereas nitrite reductas (...truncated)