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Advances in bacterial hosts

8th June 2015

Posted By Paul Boughton


Electron microscopic image of B. megaterium (large cells) and E. coli (small cells). Photo courtesy of Prof. Dr. M. Rohde, TU Braunschweig, Germany
High-density expression of GFP in B. megaterium. Photo courtesy of Prof. Dr. M. Rohde, TU Braunschweig, Germany

Non-E. coli expression hosts represent a heyday for Gram-positive bacteria, says Arne Schulz

Proteins are a group of organic molecules indispensable for life. They are essential for the structure of all living organisms, and proteins also largely regulate the function of cells. Proteins include all enzymes and most hormones. The main source of proteins is our daily food, deriving from plants and animals. The human body breaks the proteins down to amino acids that then are used to form new proteins – in a process called protein synthesis. Dysregulation of this process is responsible for many diseases, for instance cancers and neurological disorders. Using bacteria as cellular factories and thus alternate sources of essential proteins is one of the major aims of gene expression.

A different aspect of artificial protein synthesis by means of bacteria is the production and supply of industrially relevant proteins. Bacillus subtilis is a major source of enzymes capable to degrade a variety of substrates. Popular examples are proteins naturally secreted by Bacillus species into the growth medium, such as alkaline proteases as washing agent or amylases for the starch industry.

A major benefit of using bacteria as cellular factories for the production of proteins is that you can have them producing exactly the protein you want, and only a few steps are required from the genetically modified bacterium to the protein of interest in pure form.

Recent advances in preferred hosts

Over the past decades, the intestinal Gram-negative bacterium Escherichia coli (commonly abbreviated to E. coli) has been the preferred host for artificial gene expression, mainly because it can be grown cost-effectively on widely available cheap substrates with high protein yields. However, its Gram-negative status, giving cause to problems related to the presence of endotoxins, renders it unable to efficiently secrete heterologous proteins into the medium. Therefore, the availability of alternate hosts has become indispensable.

This gap could be filled by Gram-positive bacteria such as Bacillus megaterium, Bacillus subtilis and Lactococcus lactis. These bacterial hosts for gene expression and protein production are free of endotoxins, and efficiently working secretion pathways are present. In addition, Bacillus subtilis is classified by the FDA as GRAS (generally recognised as safe). A great variety of expression vectors for either bacterial host allows for the expression of a protein of interest.

Current state of play

At present, about 60% of the commercially available enzymes are produced by Bacillus species, most notably Bacillus subtilis(1). Vectors carrying signal peptides enable direct secretion of the protein into the growth medium, and different purification tags, for instance, His tag or Myc tag, facilitate easy purification(2). Work focusing on promoter optimisation enhanced the expression yields significantly (Dr. Nguyen, Duc Hoang, pers. comm.). A recent publication has shown that the surface of spores of Bacillus subtilis can be used as, for example, biocatalysts or antigen-carriers in vaccine formulations(3).

A widely used alternative

The Gram-positive bacterium Bacillus megaterium (see Fig.1) represents an increasingly used alternative for high yield intra- and extracellular protein production. During the past two decades, multiple tools, including gene expression plasmids and production strains, have been developed. Introduction of free replicating and integrative plasmids into B. megaterium is possible via protoplasts transformation or transconjugation. Using His- and Strep-affinity tags, the intra- or extracellularly produced proteins can easily be purified in one-step procedures.

Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase driven systems (for instance, T7) enable B. megaterium to produce up to 1.25 g of recombinant protein per litre(4).

Lactococcus lactis, a food-grade bacterial host that has also been validated as suitable for the production of GRAS substances, is used in the production of foreign proteins that are applied to the food and pharmaceutical industry. 

Lactococcus lactis is a further Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications.

In the past 25 years great progress has been made in the development of genetic engineering tools and the molecular characterisation of this species. Most notable is the versatile and tightly controlled gene expression system, based on the auto-regulation mechanism of the bacteriocin nisin – the NIsin Controlled gene Expression system, called NICE.

This system has become one of the most successful and widely used food-grade tools for regulated gene expression in Gram-positive bacteria(5).

For more information at www.scientistlive.com/eurolab

Arne Schulz is with MoBiTec in Germany. 

References: 1  L Westers, H Westers, and WJ Quax Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta, Nov 2004; 1694(1-3): 299-310; 2  Phan, T.T.P., Nguyen, H.D. and Schumann, W. (2006). Novel plasmid-based expression vectorsfor intra- and extracellularproduction of recombinantproteins in Bacillus subtilis. Protein Expr. Purif.; 2006 Apr; 46(2): 189-95; 3  Iwanicki A, Piątek I, Stasiłojć M, Grela A, Lęga T, Obuchowski M, Hinc K. A system of vectors for Bacillus subtilis spore surface display. Microb Cell Fact. 2014 Feb 24;13(1):30. doi: 10.1186/1475-2859-13-30; 4  Simon Stammen, Britta Katrin Müller, Claudia Korneli, Rebekka Biedendieck, Martin Gamer, Ezequiel Franco-Lara, and Dieter Jahn High-Yield Intra- and Extracellular Protein Production Using Bacillus megaterium Appl. Envir. Microbiol., Jun 2010; 76: 4037 - 4046; 5  Mierau, I. and M. Kleerebezem (2005). “10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis.” Applied Microbiology and Biotechnology 9: 1-13.





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