**4. Engineering membrane trafficking as a novel concept**

Whereas the conventional approaches are focused on reaching high levels of active proteins, the engineering of membrane trafficking is aimed at redesigning the membrane trafficking in the host cell enabling a custom made flow of vesicles which can expand the possible use of the microbial cell factory. This designed vesicle flow will give access to the large diversity of intracellular enzyme activities and will be important to produce toxic compounds without damaging the host cell.

Recently a novel strategy was described to engineer membrane trafficking, this concept is called peroxicretion 42. The peroxicretion concept of engineering membrane trafficking is based on two features: 1) the use of cytosolic domains of SNARE proteins (soluble NSF (Nethylmaleimide-sensitive factor) attachment receptor) which are key for specific membrane trafficking and 2) the use of transmembrane domains, which can reposition the SNARE molecules.

Every cellular compartment of the secretory route contains a specific set of SNARE molecules. They are called v-SNARE or t-SNARE molecules (vesicle or target). SNARE molecules are transmembrane molecules which direct membrane trafficking in eukaryotic cells 43. More recently, SNAREs are classified into Q and R SNAREs based on structural features. This nomenclature is more precise since certain SNARE molecules act as both vand t-SNAREs depending on the direction of vesicle flow. Besides transmembrane domains which serve as membrane anchors also lipid modifications like palmitoylation or farnesylation 44, 45 occur. Combinations of transmembrane domains and palmitoylation, preventing degradation of the SNARE, are also reported 46. SNARE molecules are

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking 143

Fig. 3. The membrane trafficking engineering concept. The concept is based on using a cytosolic SNARE domain (green) which is located on a secretory vesicle A. This SNARE domain is fused to a transmembrane domain (grey) of a transmembrane protein originally located on vesicle B. The fusion protein (grey-green) should then be localized at vesicle B,

The peroxisomal membrane protein PMP22 has been studied in CHO cells 56, in *Arabidopsis* 55, and in COS cells 57. All these studies give a description of the membrane topology of PMP22, PMP22 contains 4 TMDs and has the N- and C-termini placed at the cytosolic side of the peroxisome. The precise role of PMP22 is not known. PMP22 contains two peroxisomal targeting regions with almost identical basic clusters which interact with PEX19 57. The Nterminus of PMP22 is placed towards the cytosolic side and this topology makes PMP22 useful for v-SNARE fusions at the N-terminus. To determine if PMP22 localizes to the peroxisomes a GFP-PMP22 fusion was constructed and expressed in *A. niger* 42. Using fluorescence microscopy the localization of GFP-PMP22 was determined. The pattern of the GFP-PMP22 fusion is similar to the GFP-SKL punctuated pattern, which indicates that the

and transforms this vesicle into a secretory vesicle B.

PMP22 can be used as peroxisomal membrane anchor 42.

characterized by the so-called SNARE motif which is a conserved region of approximately 65 amino acids, this region also determines the specificity of the SNARE molecule 47. This important feature enables modification of the specificity of the SNARE molecules by adapting the SNARE motifs. By fusing these SNARE motifs to transmembrane domains of other proteins it becomes possible to relocalize the SNARE motifs and modify membrane trafficking in eukaryotic systems 42. Hu *et al*. showed that, by flipping SNARE molecules to the outside of cell membranes, it is possible to fuse these cells, again indicating that SNARE molecules and especially the SNARE motifs are determining the specificity of fusion of membranes 48.The N-terminal domains of SNAREs are thought to act like a zipper which acts from the N-terminus towards the transmembrane domain thereby bringing the membranes in close proximity followed by the actual fusion. This model has been reviewed in an excellent paper 43. Most research on SNARE molecules has been performed in the field of neuronal cells 49, 50, in relation to synaptic vesicle transport. In *Saccharomyces cerevisiae* a clear set of orthologous proteins can be found as well as in *Aspergillus niger* (see Figure 5).

The concept of SNARE-pin formation has been reviewed by Jahn 51, 43. In this concept, four helices are interacting to form a so called SNARE pin. The helices are named Q- and Rhelices, determined by the glutamine and arginine residues which are participating. The glutamine residues are specified further as Qa, Qb, and Qc. These SNARE complexes are very efficient in mediating membrane fusion. Recently, it was shown very elegantly, by FRET studies, that one SNARE complex is sufficient to enforce membrane fusion 52. Again, this indicates that modification of these SNARE complexes can have a direct effect on vesicle transport.

It has been described that N-terminal domains and SNARE motifs determine the functionality of SNARE proteins 53. The functions of the transmembrane domains are not very well known, but one obvious function would be to target and anchor the SNARE in the correct membrane. By using the cytosolic domain of the SNARE and fusing this part to a transmembrane domain of a protein which is located in a different membrane it is possible to engineer membrane trafficking *in vivo* 42.

To reposition the SNARE molecules a transmembrane domain is needed of a protein which is located on the vesicles which need to be modified in order to engineer membrane trafficking. The protein of which the transmembrane part is used should have the Nterminus at the cytosolic side (i.e. surface) of the membrane. The concept of this approach is depicted in Figure 3.

The transmembrane domains of proteins as tools for relocalization of SNARE molecules should be selected carefully. For the proof of principle which was aimed at transforming peroxisomes into secretory vesicles in order to secrete proteins which were located in the peroxisome, the *A. niger* ortholog of a peroxisomal membrane protein described in *Arabidopsis* named PMP22 54, 55 was used. The final topology of the fusion protein SNARE-membrane anchor is important. The N-terminal domains and SNARE motifs should be positioned at the cytosolic side of the vesicle in order to interact with their SNARE partner. In order to enable peroxisomal fusion with the plasma membrane, it was crucial to identify a peroxisomal membrane anchor, which could be used to decorate the peroxisome with proteins involved in membrane fusion. In order to determine whether peroxisomal membrane proteins have the correct topology we performed topology predictions at the CBS prediction server, as shown in Figure 4 for the PMP22 ortholog of *A. niger* (An04g09130).

characterized by the so-called SNARE motif which is a conserved region of approximately 65 amino acids, this region also determines the specificity of the SNARE molecule 47. This important feature enables modification of the specificity of the SNARE molecules by adapting the SNARE motifs. By fusing these SNARE motifs to transmembrane domains of other proteins it becomes possible to relocalize the SNARE motifs and modify membrane trafficking in eukaryotic systems 42. Hu *et al*. showed that, by flipping SNARE molecules to the outside of cell membranes, it is possible to fuse these cells, again indicating that SNARE molecules and especially the SNARE motifs are determining the specificity of fusion of membranes 48.The N-terminal domains of SNAREs are thought to act like a zipper which acts from the N-terminus towards the transmembrane domain thereby bringing the membranes in close proximity followed by the actual fusion. This model has been reviewed in an excellent paper 43. Most research on SNARE molecules has been performed in the field of neuronal cells 49, 50, in relation to synaptic vesicle transport. In *Saccharomyces cerevisiae* a clear set of orthologous proteins can be found as well as in *Aspergillus niger* (see Figure 5). The concept of SNARE-pin formation has been reviewed by Jahn 51, 43. In this concept, four helices are interacting to form a so called SNARE pin. The helices are named Q- and Rhelices, determined by the glutamine and arginine residues which are participating. The glutamine residues are specified further as Qa, Qb, and Qc. These SNARE complexes are very efficient in mediating membrane fusion. Recently, it was shown very elegantly, by FRET studies, that one SNARE complex is sufficient to enforce membrane fusion 52. Again, this indicates that modification of these SNARE complexes can have a direct effect on vesicle

It has been described that N-terminal domains and SNARE motifs determine the functionality of SNARE proteins 53. The functions of the transmembrane domains are not very well known, but one obvious function would be to target and anchor the SNARE in the correct membrane. By using the cytosolic domain of the SNARE and fusing this part to a transmembrane domain of a protein which is located in a different membrane it is possible

To reposition the SNARE molecules a transmembrane domain is needed of a protein which is located on the vesicles which need to be modified in order to engineer membrane trafficking. The protein of which the transmembrane part is used should have the Nterminus at the cytosolic side (i.e. surface) of the membrane. The concept of this approach is

The transmembrane domains of proteins as tools for relocalization of SNARE molecules should be selected carefully. For the proof of principle which was aimed at transforming peroxisomes into secretory vesicles in order to secrete proteins which were located in the peroxisome, the *A. niger* ortholog of a peroxisomal membrane protein described in *Arabidopsis* named PMP22 54, 55 was used. The final topology of the fusion protein SNARE-membrane anchor is important. The N-terminal domains and SNARE motifs should be positioned at the cytosolic side of the vesicle in order to interact with their SNARE partner. In order to enable peroxisomal fusion with the plasma membrane, it was crucial to identify a peroxisomal membrane anchor, which could be used to decorate the peroxisome with proteins involved in membrane fusion. In order to determine whether peroxisomal membrane proteins have the correct topology we performed topology predictions at the CBS prediction server, as shown in

transport.

depicted in Figure 3.

to engineer membrane trafficking *in vivo* 42.

Figure 4 for the PMP22 ortholog of *A. niger* (An04g09130).

Fig. 3. The membrane trafficking engineering concept. The concept is based on using a cytosolic SNARE domain (green) which is located on a secretory vesicle A. This SNARE domain is fused to a transmembrane domain (grey) of a transmembrane protein originally located on vesicle B. The fusion protein (grey-green) should then be localized at vesicle B, and transforms this vesicle into a secretory vesicle B.

The peroxisomal membrane protein PMP22 has been studied in CHO cells 56, in *Arabidopsis* 55, and in COS cells 57. All these studies give a description of the membrane topology of PMP22, PMP22 contains 4 TMDs and has the N- and C-termini placed at the cytosolic side of the peroxisome. The precise role of PMP22 is not known. PMP22 contains two peroxisomal targeting regions with almost identical basic clusters which interact with PEX19 57. The Nterminus of PMP22 is placed towards the cytosolic side and this topology makes PMP22 useful for v-SNARE fusions at the N-terminus. To determine if PMP22 localizes to the peroxisomes a GFP-PMP22 fusion was constructed and expressed in *A. niger* 42. Using fluorescence microscopy the localization of GFP-PMP22 was determined. The pattern of the GFP-PMP22 fusion is similar to the GFP-SKL punctuated pattern, which indicates that the PMP22 can be used as peroxisomal membrane anchor 42.

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking 145

Fig. 5. Overview on SNARE molecules in *Aspergillus niger* and their orthologs in

apparatus.

*Saccharomyces cerevisiae.* This overview is based on the *A. niger* sequencing work and the supplementary material of the resulting publication 58. In green, the vesicle flow form Golgi towards cell membrane is shown, dark blue indicates vesicle flow from ER towards Golgi and the orange vesicles are representing retrograde transport form Golgi towards ER. Finally, the light blue vesicles are representing vacuolar vesicle flow derived from the Golgi

More recent papers also very clearly showed that peroxisomes originate from the endoplasmic reticulum 60, 67, 68-70. The recent finding that insertion of some peroxisomal membrane proteins occurs already at the ER 71 clearly underpins that peroxisomes are linked to the secretory pathway in the eukaryotic cell. However, the same study mentioned that no conventional signal sequences can be found in a set of Peroxisomal Membrane Proteins (PMP) in *S. cerevisiae*. A possible mechanism could be that Pex19p, which is described as a chaperone for proper insertion of PMP into peroxisomes 72-74 also acts a receptor for peroxisomal membrane proteins which are inserted at the ER membrane and become part of peroxisomes at a later stage. An alternative model could be that Pex19p is involved in de budding process of (pre)peroxisomes from the ER where PMP have been already inserted. This yields an interesting model which strongly suggest that peroxisomes are derivatives of the secretory pathway. It has been reported that the transportation of peroxisomes in *Arabidopsis* by actin filaments is dependent on MYA2 (Myosin XI isoform) 75, similar to the transportation of secretory vesicles. This common ground makes peroxisomes an attractive vehicle for transporting intracellular proteins towards the plasma membrane.

MSAKFQDEAVTSIREDTKELVHKVGNRLTGDGYLALYLRQLQSNPLRTKMLTSGVLSSLQEILASWIA HDVSKHGHYFSARVPKMALYGMFISAPLGHFLIGILQRVFAGRTSIKAKILQILASNLLVSPIQNAVYL CCMAVIAGARTFHQVRATVRAGFMPVMKVSWVTSPIALAFAQKFLPEHTWVPFFNIVGFVIGTYVNT HTKKKRLEALRKCGLPDMVG

Fig. 4. Predicted topology and sequence of the PMP22 ortholog in *A. niger* (An04g09130)*.* The upper panel displays the predicted topology of the PMP22 ortholog by the CBS prediction servers (http://www.cbs.dtu.dk/services/). Four transmembrane domains are predicted, although the TMD between aa 150 and 175 does not exceed the threshold and therefore, 3 TMD are possible as well. It is clear that in both scenarios the N terminus of An04g09130 is positioned towards the outside (cytosolic side) of the vesicle. The lower panel displays the aminoacid sequence of the *A. niger* ortholog of PMP22 (An04g09130) used as input for the CBS prediction server TMD algorithm.

Crucial in this engineering approach is the final orientation of the novel chimeric SNARE molecule, the SNARE domain should be positioned on the outer side (i.e. surface) of the vesicle as shown in Figure 3. The resulting vesicles are decorated with a set of alternative SNARE molecules which can enable fusion of these vesicles with other cellular compartments which contain the appropriate target SNAREs.

Peroxisomes decorated with SNARE proteins have been used as alternative vesicles for transport of intracellular proteins 42. The use of peroxisomes as alternative vesicles for secretion of proteins which are normally located in the cytosol can be justified by several observations. Peroxisomes are organelles with a single membrane 59 and a very elegant publication shows convincing evidence that peroxisomes originate at the ER 60. In addition, the identification of the small GTPase Rho1p as being localized on peroxisomes by interaction of the peroxisomal membrane protein Pex25p again indicates a link of peroxisomes with the secretory machinery 61, since Rho1p is known to play a role in actin reorganization and membrane dynamics 62, 63. In yeast, polarized growth is regulated by Rho1p 64, and also in fungal systems the link between Rho1 and polarized growth has been confirmed 65 in the filamentous fungus *A. niger*, polarized growth is linked to secretion of proteins 66.

MSAKFQDEAVTSIREDTKELVHKVGNRLTGDGYLALYLRQLQSNPLRTKMLTSGVLSSLQEILASWIA HDVSKHGHYFSARVPKMALYGMFISAPLGHFLIGILQRVFAGRTSIKAKILQILASNLLVSPIQNAVYL CCMAVIAGARTFHQVRATVRAGFMPVMKVSWVTSPIALAFAQKFLPEHTWVPFFNIVGFVIGTYVNT

Fig. 4. Predicted topology and sequence of the PMP22 ortholog in *A. niger* (An04g09130)*.* The upper panel displays the predicted topology of the PMP22 ortholog by the CBS prediction servers (http://www.cbs.dtu.dk/services/). Four transmembrane domains are predicted, although the TMD between aa 150 and 175 does not exceed the threshold and therefore, 3 TMD are possible as well. It is clear that in both scenarios the N terminus of An04g09130 is positioned towards the outside (cytosolic side) of the vesicle. The lower panel displays the aminoacid sequence of the *A. niger* ortholog of PMP22 (An04g09130) used as

Crucial in this engineering approach is the final orientation of the novel chimeric SNARE molecule, the SNARE domain should be positioned on the outer side (i.e. surface) of the vesicle as shown in Figure 3. The resulting vesicles are decorated with a set of alternative SNARE molecules which can enable fusion of these vesicles with other cellular

Peroxisomes decorated with SNARE proteins have been used as alternative vesicles for transport of intracellular proteins 42. The use of peroxisomes as alternative vesicles for secretion of proteins which are normally located in the cytosol can be justified by several observations. Peroxisomes are organelles with a single membrane 59 and a very elegant publication shows convincing evidence that peroxisomes originate at the ER 60. In addition, the identification of the small GTPase Rho1p as being localized on peroxisomes by interaction of the peroxisomal membrane protein Pex25p again indicates a link of peroxisomes with the secretory machinery 61, since Rho1p is known to play a role in actin reorganization and membrane dynamics 62, 63. In yeast, polarized growth is regulated by Rho1p 64, and also in fungal systems the link between Rho1 and polarized growth has been confirmed 65 in the filamentous fungus *A. niger*, polarized growth is linked to secretion of

HTKKKRLEALRKCGLPDMVG

proteins 66.

input for the CBS prediction server TMD algorithm.

compartments which contain the appropriate target SNAREs.

Fig. 5. Overview on SNARE molecules in *Aspergillus niger* and their orthologs in *Saccharomyces cerevisiae.* This overview is based on the *A. niger* sequencing work and the supplementary material of the resulting publication 58. In green, the vesicle flow form Golgi towards cell membrane is shown, dark blue indicates vesicle flow from ER towards Golgi and the orange vesicles are representing retrograde transport form Golgi towards ER. Finally, the light blue vesicles are representing vacuolar vesicle flow derived from the Golgi apparatus.

More recent papers also very clearly showed that peroxisomes originate from the endoplasmic reticulum 60, 67, 68-70. The recent finding that insertion of some peroxisomal membrane proteins occurs already at the ER 71 clearly underpins that peroxisomes are linked to the secretory pathway in the eukaryotic cell. However, the same study mentioned that no conventional signal sequences can be found in a set of Peroxisomal Membrane Proteins (PMP) in *S. cerevisiae*. A possible mechanism could be that Pex19p, which is described as a chaperone for proper insertion of PMP into peroxisomes 72-74 also acts a receptor for peroxisomal membrane proteins which are inserted at the ER membrane and become part of peroxisomes at a later stage. An alternative model could be that Pex19p is involved in de budding process of (pre)peroxisomes from the ER where PMP have been already inserted. This yields an interesting model which strongly suggest that peroxisomes are derivatives of the secretory pathway. It has been reported that the transportation of peroxisomes in *Arabidopsis* by actin filaments is dependent on MYA2 (Myosin XI isoform) 75, similar to the transportation of secretory vesicles. This common ground makes peroxisomes an attractive vehicle for transporting intracellular proteins towards the plasma membrane.

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking 147

Fig. 7. Overview of the peroxicretion concept. Panel A shows the expression of SKL tagged proteins of interest together with the expression of SNC1-PMP22 fusion protein which are localized on the peroxisomal membrane. The transmembrane anchor encoded by the PMP22 is depicted in blue, the SNC1 SNARE module is encoded by red, together they ensure the decoration of peroxisomes with the *A. niger* ortholog of the v-SNARE SNC1 at the cytosolic

peroxisomes fuse with the plasma membrane thereby releasing their luminal content. The *A. niger* ortholog of v-SNARE SNC1 (An12g07570) was placed on the peroxisome using the transmembrane domain of the PMP22 ortholog (An04g09130) as a membrane anchor. The transmembrane domain of SNC1 was omitted, the fusion protein is shown in Figure 8. It has been reported that this transmembrane domain of SNC1 is important for the function of SNC1 85, but the replacement of this TMD by the PMP22 TMD did not abolish the function of SNC1 (An12g07570) in *A. niger* 42. The replacement of the SNC1 TMD by the PMP22 protein does not diminish the potential of SNC1 to enforce membrane fusion because the peroxisomal content is released extracellularly. The paper of Grote *et al*. 2000 85 described inhibition of SNC1 function when the TMD was replaced by geranylgeranyl anchors. Probably membrane fusion is only possible when the TMD domains of the SNARE pairs bring the membrane bilayers in close contact so that spontaneous membrane fusion occurs. Proteins which are produced by using the peroxicretion technology are C-terminally tagged with a PTS1 signal. The most commonly used PTS1 is SKL 87, however variations on this

side. Panel B shows the translocation of the SKL tagged proteins into the decorated peroxisomes. Panel C shows the complete translocation of the SKL tagged proteins in peroxisomes, the formation of peroxisomes can be increased by overexpression of PEX11 orthologs 86. Panel D shows the fusion of the peroxisomes with the plasmamembrane, using the ortholog of the t-SNARE SSO1 (purple). The complete SNAREpin structure is more

complex, and is not described here for the sake of simplicity.

Fig. 6. Peroxisomes originate from the endoplasmic reticulum, taken from Tabak et al. 2003 67. In this 3D- reconstituted SEM picture 67 which was processed in silico, the peroxisomes (green) are attached to the smooth ER (light blue), as a continuous membrane structure. Other peroxisomes have already been released from the smooth ER. Rough ER is indicated by dark blue and the red dots represent ribosomes on the RER.

In addition, these organelles contain an import machinery capable of importing completely folded proteins 76-79, and have a controlled way of proliferation 80-82. So, peroxisomes have the same origin as secretory vesicles and are thus candidates for alternative trafficking of proteins. The first application of such an engineered secretory pathway would be to produce intracellular proteins which show a much broader spectrum of enzymatic activities compared to secreted proteins which have predominantly hydrolase activity (as depicted in Figure 1). Normally, the intracellular proteins are folded in the cytosol, are not N-glycosylated, and disulphide bridges are not formed in this relatively reducing environment 2. The cytosol does not contain a major machinery for the formation of disulphide bridges or for N-glycosylation as the ER does 83,84. These basic differences suggest that the conventional route to secrete these intracellular proteins is not compatible with the folding environment, kinetics and modifications of these proteins.

The demonstration of engineering membrane trafficking is based on the ability of peroxisomes to import completely folded proteins. The peroxisomes containing the proteins of interest are equipped with a v-SNARE, which is normally localized at the Golgi apparatus (the study of Marelli *et al*. 2004 did not identify SNARE-like proteins on peroxisomal membranes). This ensures that peroxisomes are able to bind to t-SNAREs which are localized at the plasmamembrane. After the formation of a so called SNARE-pin the

Fig. 6. Peroxisomes originate from the endoplasmic reticulum, taken from Tabak et al. 2003 67. In this 3D- reconstituted SEM picture 67 which was processed in silico, the peroxisomes (green) are attached to the smooth ER (light blue), as a continuous membrane structure. Other peroxisomes have already been released from the smooth ER. Rough ER is indicated

In addition, these organelles contain an import machinery capable of importing completely folded proteins 76-79, and have a controlled way of proliferation 80-82. So, peroxisomes have the same origin as secretory vesicles and are thus candidates for alternative trafficking of proteins. The first application of such an engineered secretory pathway would be to produce intracellular proteins which show a much broader spectrum of enzymatic activities compared to secreted proteins which have predominantly hydrolase activity (as depicted in Figure 1). Normally, the intracellular proteins are folded in the cytosol, are not N-glycosylated, and disulphide bridges are not formed in this relatively reducing environment 2. The cytosol does not contain a major machinery for the formation of disulphide bridges or for N-glycosylation as the ER does 83,84. These basic differences suggest that the conventional route to secrete these intracellular proteins is not compatible with the folding environment, kinetics and

The demonstration of engineering membrane trafficking is based on the ability of peroxisomes to import completely folded proteins. The peroxisomes containing the proteins of interest are equipped with a v-SNARE, which is normally localized at the Golgi apparatus (the study of Marelli *et al*. 2004 did not identify SNARE-like proteins on peroxisomal membranes). This ensures that peroxisomes are able to bind to t-SNAREs which are localized at the plasmamembrane. After the formation of a so called SNARE-pin the

by dark blue and the red dots represent ribosomes on the RER.

modifications of these proteins.

Fig. 7. Overview of the peroxicretion concept. Panel A shows the expression of SKL tagged proteins of interest together with the expression of SNC1-PMP22 fusion protein which are localized on the peroxisomal membrane. The transmembrane anchor encoded by the PMP22 is depicted in blue, the SNC1 SNARE module is encoded by red, together they ensure the decoration of peroxisomes with the *A. niger* ortholog of the v-SNARE SNC1 at the cytosolic side. Panel B shows the translocation of the SKL tagged proteins into the decorated peroxisomes. Panel C shows the complete translocation of the SKL tagged proteins in peroxisomes, the formation of peroxisomes can be increased by overexpression of PEX11 orthologs 86. Panel D shows the fusion of the peroxisomes with the plasmamembrane, using the ortholog of the t-SNARE SSO1 (purple). The complete SNAREpin structure is more complex, and is not described here for the sake of simplicity.

peroxisomes fuse with the plasma membrane thereby releasing their luminal content. The *A. niger* ortholog of v-SNARE SNC1 (An12g07570) was placed on the peroxisome using the transmembrane domain of the PMP22 ortholog (An04g09130) as a membrane anchor. The transmembrane domain of SNC1 was omitted, the fusion protein is shown in Figure 8. It has been reported that this transmembrane domain of SNC1 is important for the function of SNC1 85, but the replacement of this TMD by the PMP22 TMD did not abolish the function of SNC1 (An12g07570) in *A. niger* 42. The replacement of the SNC1 TMD by the PMP22 protein does not diminish the potential of SNC1 to enforce membrane fusion because the peroxisomal content is released extracellularly. The paper of Grote *et al*. 2000 85 described inhibition of SNC1 function when the TMD was replaced by geranylgeranyl anchors. Probably membrane fusion is only possible when the TMD domains of the SNARE pairs bring the membrane bilayers in close contact so that spontaneous membrane fusion occurs.

Proteins which are produced by using the peroxicretion technology are C-terminally tagged with a PTS1 signal. The most commonly used PTS1 is SKL 87, however variations on this

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking 149

signal sequence like SKL. A combination of the peroxicretion approach with more conventional approaches to improve protein production like removal of proteases and increasing folding capacity of, in this case, the cytosol may further improve protein production. This proof of principle shows that by relocalizing the SNARE molecules to alternative compartments, it is possible to redirect vesicle trafficking in cells, which could also enable production of toxic compounds in micro organisms. This exciting possibility of constructing chimeric proteins of a specific membrane anchor with a specific SNARE

Moreover, it can also be hypothesized that due to the modified peroxisomes the complete biomass could now be used for production of enzymes, and not only the hyphal tips as is being suggested for normal secretory proteins 92. A speculative idea would be to determine the components that allow the peroxisomes to utilize all of the biomass to release their content into the medium and adjust the conventional secretory vesicles in a similar way which would enable secretion of secretory proteins using the complete biomass instead of just the hyphal tips. Using this approach we could even increase fermentation yields of secretory proteins.

The cell factory of the future should be able to produce a wide range of proteins and metabolites with high productivities and yields in a robust process. It is clear that current strategies contribute in an incremental way, which is important to make the bioprocess economically feasible. In order to expand the product range and design a truly versatile cell factory more innovative approaches are needed. Until now, membrane engineering has hardly been used as an additional tool for designing cell factories. Recent developments have shown that membrane engineering can be a key for designing a truly versatile cell factory. The potential of this approach is enormous, especially in combination with

In this era of synthetic biology were bio-bricks are used to engineer microbes for new functionalities the concept of membrane trafficking engineering is very well positioned. It enables a true engineering methodology for intracellular trafficking thereby unlocking a complementary approach to already applied strategies like genetic devices based on biobricks. In addition, the availability of -omics technologies will function as a starting platform for further fine-tuning of the membrane engineering concept. Based on this holistic approach more key components will be identified which can be used to increase the efficiency of membrane engineering and the application of engineered membranes in

This quest for further fine-tuning will be exciting and will have academic impact as well. The membrane trafficking engineering concept can serve the academic society with a new way of studying the complex vesicle flow in biological systems. By combining different components of the secretory pathway into novel chimeric proteins with new functionalities a detailed overview is within reach which can describe the molecular details of the protein

This is a major challenge and as with any technology the combined effort of industry and academia can result in significant progress when strong interaction and mutual creativity

functionality is an important tool to engineer membrane trafficking.

**5. Designing a robust and versatile cell factory, an outlook** 

conventional strategies.

microbial cell factories.

secretion mechanism.

are applied and recognized.

sequence have been described as well 87. Since the Pex5p (PTS1 receptor) is highly conserved between yeasts and *A. niger*, it has been shown that SKL also functions as a PTS1 signal in *A. niger* 42. This raises possibilities for purifying the PTS1 tagged products on an affinity column which specifically binds PTS1 sequences. In principal nature has provided us already with a very efficient PTS1 binding protein Pex5p, which is the natural receptor for PTS1.

MSEQPYDPYIPSGANGAGAGASAAQNGDPRTREIDKKIQETVDTMRSNIFKVSERGERLD SLQDKTDNLATSAQGFRRGANRVRKQMWWKDMKMRSAKFQDEAVTSIREDTKELVHK VGNRLTGDGYLALYLRQLQSNPLRTKMLTSGVLSSLQEILASWIAHDVSKHGHYFSARVP KMALYGMFISAPLGHFLIGILQRVFAGRTSIKAKILQILASNLLVSPIQNAVYLCCMAVIAG ARTFHQVRATVRAGFMPVMKVSWVTSPIALAFAQKFLPEHTWVPFFNIVGFVIGTYVNT HTKKKRLEALRKCGLPDMVG

Fig. 8. The SNC1-PMP22 fusion protein which enables peroxicretion. SNC1 ortholog sequence (no TMD)is depicted in green (based on An12g07570) the PMP22 ortholog sequence (based on An04g09130) is depicted in blue.

Mutational studies to determine whether shortening of the region between SNC1 and PMP22, thereby placing SNC1 closer to the peroxisomal membrane, could enhance peroxicretion were performed by our group. This approach was partially successful, the most efficient peroxicretion occurred when SNC1 was linked to the full length PMP22 (318 aa) or a truncated version of PMP22 (300 aa). When PMP22 was truncated further the peroxicretion decreased dramatically (< 50%). This is in contrast with a previous study 88, which predicts an increase of vesicle fusion when the hinge region of the v-SNARE and the TMD domain is shortened. This discrepancy is most likely caused by the unnatural TMD of the SNC1-PMP22 fusion protein. The TMD of PMP22 was not evolutionarily selected for efficient membrane fusion. Moreover, the localization information of PMP22 is most likely positioned in this hinge region. GFP fusions with the different truncated PMP22 constructs showed a localization, which is not unambiguously peroxisomal. This indicated (partial) mis-localization of SNC1-PMP22 fusion products when PMP22 is truncated to less than 300 aa, explaining the dramatic decrease of peroxicretion.

Additional modifications to fine-tune the peroxicretion concept can be foreseen, like rebuilding the peroxisome in such a way that all components of the secretory vesicles (proteins and lipids) are present on the peroxisome, making them efficient cell organelles for peroxicretion. Subcellular proteomics could supply us with these leads. One of the most promising options is to use Sec4p a protein involved in SNARE-pin formation. Sec4p is a Rab-GTPase which plays an important role in polarized secretion by tethering secretory vesicles to the plasmamembrane 89, 90. Sec4p is localized at Golgi-derived vesicles with a geranylgeranyl anchor. Sec4p GEF (Sec2p) and Sec4p GAP (Msb3p and Msb4p) are described and are controlling the activity of Sec4p 91. This makes Sec4p and its partners potential leads for improving the peroxicretion technology.

In the quest for alternative secretory routes the peroxicretion concept was developed 42, which shows that it is possible to redirect peroxisomes to the cell membrane where they are able to fuse and release their cargo in the extracellular medium, as shown in Figure 7. The peroxisomes can be loaded with intracellular enzymes by adding a C terminal peroxisomal

sequence have been described as well 87. Since the Pex5p (PTS1 receptor) is highly conserved between yeasts and *A. niger*, it has been shown that SKL also functions as a PTS1 signal in *A. niger* 42. This raises possibilities for purifying the PTS1 tagged products on an affinity column which specifically binds PTS1 sequences. In principal nature has provided us already with a very efficient PTS1 binding protein Pex5p, which is the natural receptor for

MSEQPYDPYIPSGANGAGAGASAAQNGDPRTREIDKKIQETVDTMRSNIFKVSERGERLD SLQDKTDNLATSAQGFRRGANRVRKQMWWKDMKMRSAKFQDEAVTSIREDTKELVHK VGNRLTGDGYLALYLRQLQSNPLRTKMLTSGVLSSLQEILASWIAHDVSKHGHYFSARVP KMALYGMFISAPLGHFLIGILQRVFAGRTSIKAKILQILASNLLVSPIQNAVYLCCMAVIAG ARTFHQVRATVRAGFMPVMKVSWVTSPIALAFAQKFLPEHTWVPFFNIVGFVIGTYVNT

Fig. 8. The SNC1-PMP22 fusion protein which enables peroxicretion. SNC1 ortholog sequence (no TMD)is depicted in green (based on An12g07570) the PMP22 ortholog

Mutational studies to determine whether shortening of the region between SNC1 and PMP22, thereby placing SNC1 closer to the peroxisomal membrane, could enhance peroxicretion were performed by our group. This approach was partially successful, the most efficient peroxicretion occurred when SNC1 was linked to the full length PMP22 (318 aa) or a truncated version of PMP22 (300 aa). When PMP22 was truncated further the peroxicretion decreased dramatically (< 50%). This is in contrast with a previous study 88, which predicts an increase of vesicle fusion when the hinge region of the v-SNARE and the TMD domain is shortened. This discrepancy is most likely caused by the unnatural TMD of the SNC1-PMP22 fusion protein. The TMD of PMP22 was not evolutionarily selected for efficient membrane fusion. Moreover, the localization information of PMP22 is most likely positioned in this hinge region. GFP fusions with the different truncated PMP22 constructs showed a localization, which is not unambiguously peroxisomal. This indicated (partial) mis-localization of SNC1-PMP22 fusion products when PMP22 is truncated to less than 300

Additional modifications to fine-tune the peroxicretion concept can be foreseen, like rebuilding the peroxisome in such a way that all components of the secretory vesicles (proteins and lipids) are present on the peroxisome, making them efficient cell organelles for peroxicretion. Subcellular proteomics could supply us with these leads. One of the most promising options is to use Sec4p a protein involved in SNARE-pin formation. Sec4p is a Rab-GTPase which plays an important role in polarized secretion by tethering secretory vesicles to the plasmamembrane 89, 90. Sec4p is localized at Golgi-derived vesicles with a geranylgeranyl anchor. Sec4p GEF (Sec2p) and Sec4p GAP (Msb3p and Msb4p) are described and are controlling the activity of Sec4p 91. This makes Sec4p and its partners

In the quest for alternative secretory routes the peroxicretion concept was developed 42, which shows that it is possible to redirect peroxisomes to the cell membrane where they are able to fuse and release their cargo in the extracellular medium, as shown in Figure 7. The peroxisomes can be loaded with intracellular enzymes by adding a C terminal peroxisomal

PTS1.

HTKKKRLEALRKCGLPDMVG

sequence (based on An04g09130) is depicted in blue.

aa, explaining the dramatic decrease of peroxicretion.

potential leads for improving the peroxicretion technology.

signal sequence like SKL. A combination of the peroxicretion approach with more conventional approaches to improve protein production like removal of proteases and increasing folding capacity of, in this case, the cytosol may further improve protein production. This proof of principle shows that by relocalizing the SNARE molecules to alternative compartments, it is possible to redirect vesicle trafficking in cells, which could also enable production of toxic compounds in micro organisms. This exciting possibility of constructing chimeric proteins of a specific membrane anchor with a specific SNARE functionality is an important tool to engineer membrane trafficking.

Moreover, it can also be hypothesized that due to the modified peroxisomes the complete biomass could now be used for production of enzymes, and not only the hyphal tips as is being suggested for normal secretory proteins 92. A speculative idea would be to determine the components that allow the peroxisomes to utilize all of the biomass to release their content into the medium and adjust the conventional secretory vesicles in a similar way which would enable secretion of secretory proteins using the complete biomass instead of just the hyphal tips. Using this approach we could even increase fermentation yields of secretory proteins.
