Journal of Biotechnology 161 (2012) 92–103 Contents lists available at SciVerse ScienceDirect Journal of Biotechnology j ournal homepage: www.elsevier.com/locate/jbiotec Autodisplay of enzymes—Molecular basis and perspectives a,∗ b a Joachim Jose , Ruth Maria Maas , Mark George Teese a Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany b Autodisplay Biotech GmbH, Merowingerplatz 1a, D-40225 Düsseldorf, Germany a r t i c l e i n f o a b s t r a c t Article history: To display an enzyme on the surface of a living cell is an important step forward towards a broader use of Received 8 October 2011 biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It Received in revised form 14 February 2012 is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the Accepted 4 April 2012 enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells Available online 30 April 2012 can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major Keywords: advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell Autodisplay Biocatalysis surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay Synthesis enzymes with prosthetic groups can be displayed, expanding the application of surface display to the 5 6 Enzymes industrial important P450 enzymes. Finally, up to 10 –10 enzyme molecules can be displayed on a Whole cells single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes with particular attention to industrial needs and process development. Applications that will provide sustainable solutions towards a bio-based industry are discussed. © 2012 Elsevier B.V. All rights reserved. 1. Introduction (Goldberg et al., 2007b). Despite the advantages, there are some drawbacks that prevent a broader application of enzymes. The Enzymes as biocatalysts show some outstanding advantages for purification of enzymes is often a complex and costly production the synthesis of chemicals (Choi, 2009; Luetz et al., 2008), pharma- process. In most cases purified enzymes cannot be used in repeated ceutical and agrochemical intermediates (Fischer and Pietruszka, reactions, they turn to waste after a single processing step. The 2010; Tufvesson et al., 2011) as well as active pharmaceutical and use of microorganisms as whole cell biocatalysts avoids the costs agrochemical compounds (Ran et al., 2009). Unlike conventional associated with enzyme purification and ensures that the enzyme organic chemistry, enzymes can be used under mild conditions con- is working in an optimal environment, where all co-factors and cerning temperature, pressure and pH and usually they convert a regeneration networks are provided. Moreover, the enzyme as a substrate with high regio- and enantioselectivity without protect- biocatalyst is largely protected from destabilizing and degrading ing and de-protecting steps as necessary in conventional organic effects. However the intracellular location of the enzymes means chemistry. In many cases, the use of enzymes in chemical synthe- that this method will only be successful if both the substrate and sis requires less substrate, less energy and reduces waste. Moreover product can cross the membrane barrier. In addition, there is a enzyme discovery and improvement could lead to completely new tremendous consortium of other enzymes present within the cell. processes, such as the use of cellulose for fuel production, the biore- To obtain the product in a pure and unaltered form, whole cell bio- mediation of contaminated water and soil, or the production of catalysis is also limited to substrates and products that cannot be polymers from non–petroleum sources, which under current con- converted by these native enzymes. ditions are not feasible. For many applications, the display of the enzyme at the cell To date enzymes are already used in a distinct number of indus- surface of the microorganism is an advancement of the whole trial processes (Busch et al., 2006). They are either applied as cell biocatalyst approach. Neither substrate nor product needs preparations of purified proteins (Goldberg et al., 2007a), or as to be membrane permeable, and both could be excluded from microorganisms that produce the desired enzyme within the cell any unwanted attack by other enzymes. Among the systems for the display of recombinant proteins on microorganisms, which include yeast (Kuroda and Ueda, 2011), gram positive (Kronqvist et al., 2010) and gram negative bacteria (van Bloois et al., 2011), ∗ Corresponding author at: Institute for Pharmaceutical and Medicinal Chem- the autodisplay system is a particularly elegant and efficient tool istry, Westfälische Wilhelms-Universität, Münster, Hittorfstraße 58-62, D-48149 with some advantageous features for biotechnological and – if Münster, Germany. Tel.: +49 251 83 32210, fax: +49 251 83 32211. E-mail address: [email protected] (J. Jose). scaled up – industrial applications. 0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2012.04.001 J. Jose et al. / Journal of Biotechnology 161 (2012) 92–103 93 domains remain anchored on the cell surface (Linke et al., 2006). For type Vc autotransporters, the passenger and the transloca- tor domains are provided by separate genes (St Geme and Yeo, 2009). Both domains are transported across the inner membrane by the Sec machinery, and interact in the periplasm via a so-called POTRA (“polypeptide transport associated domain”) domain of the translocator, which initiates transport of the passenger across the outer membrane. This makes type Vc resembling similar transport systems existing in chloroplasts and mitochondria, which are sup- posed to be able to transport very complex and extended folded protein structures (Tommassen, 2007). Although these particular features of type Vb and Vc autotransporters make them interest- ing candidates for the surface display of enzymes, they have not been used for this purpose yet. Therefore this review focuses on the application of classical autotransporters in biotechnology. 3. Display of enzymes by classical autotransporters Before we come to the transport and the display of recombi- Fig. 1. Model of the classical autotransporter secretion mechanism. (A) Autotrans- nant enzymes by the aid of an autotransporter, it appears worth porters are synthesized as precursor protein containing all domains needed to to have a look on their natural passengers, which are enzymes transport the passenger to the cell surface. (B) By the aid of a classical signal peptide as well. The prototype of an autotransporter protein and the first the precursor is transported across the inner membrane, which is cleaved off. Subse- family member to be discovered middle of the eighties – although quently, the C terminal part folds into the outer membrane as a porin-like structure, a so-called ␤-barrel. The passenger is translocated to the cell surface by the aid of not named an autotransporter at that time – was IgA1 protease ␤ the -barrel maintains an unfolded conformation during transport. According to from Neisseria gonorrhoeae (Halter et al., 1984). Together with its this model surface translocation requires the formation of an interim hairpin struc- structural description, the very elegant model for outer membrane ture, which was recently experimentally verified (Ieva and Bernstein, 2009). Surface translocation was proposed, without the requirement of energy or translocation is supported by folding of the passenger at the cell surface. accessory factors (Pohlner et al., 1987), which is still valid as a con- cept today. Almost a decade later, the first publication to mention 2. Autodisplay the term “autotransporter” listed ten first examples of this protein family (or eleven when IgA1 proteases form N. gonorrhoeae and Autodisplay is defined as the recombinant surface display of N. meningitidis are considered to be different examples), among proteins or peptides by means of an autotransporter protein in which five enzymes can be found (Jose et al., 1995). Nowadays any gram negative bacterium (Jose and Meyer, 2007). The auto- the autotransporter family of proteins comprises more than 1000 transporter proteins are a large family of secreted proteins in members, among which a considerable number bear proteases or gram negative bacteria and are divided into three subgroups, the other hydrolases, in particular lipases as natural passengers (Benz classical autotransporters (secretion type Va), the trimeric auto- and Schmidt, 2011; Wells et al., 2010; Wilhelm et al., 2011). At this transporter adhesins (Vb), and the two partner secretion systems point the question arises, for what reason natural autotransporters (Vc) (Henderson et al., 2004). All classical autotransporters are are not used more frequently for catalytic purposes (Wilhelm thought to share a common general structure (Jose et al., 1995). et al., 2011). Autotransporter proteins have been discovered first in They are produced as precursor proteins with a standard signal pathogenic gram negative bacteria and are supposed to represent peptide at the very N terminus, which enables the transport