**2. Conventional engineering strategies for improved protein production**

The conventional strategy to improve protein production is to increase the expression levels of the gene of interest by using multiple gene copies and strong promotors 11, 12. As a consequence, the cell is faced with very high levels of mRNA which should rapidly be translated into active and correctly folded proteins. To handle this increased demand for folding capacity the cell often reacts with the so called Unfolded Protein Response (UPR). This UPR is a well studied mechanism 13-17 which is also applied to increase folding capacity. By upregulating the transcription factors needed, or the resulting chaperones and folding enzymes, the folding capacity of the cell is enhanced 18-21.

The production of intracellular proteins in a secreted form, to avoid complex and costly downstream processing, has hardly been touched upon. Several strategies have been developed to increase protein titers, however, these efforts have all been focused on secreted proteins. The functional diversity of intracellular proteins is very broad, while extracellular enzymes are mostly hydrolases, as shown in Figure 1. If the cell would be able to secrete these intracellular enzymes in an active form a wide range of applications could be within reach. Current strategies do not suffice but when these are combined with membrane engineering to redirect vesicle trafficking this could become a very promising

> isomerases ligases lyases hydrolases transferases oxidoreductases

Fig. 1. Distribution of enzyme functionalities in *Aspergillus niger*. The genome sequence of *Aspergillus niger* was mined for annotated functionalities based on EC classifications. The extracellular (extra) or intracellular (intra) protein distribution is based on this annotated genome. The left panel indicates relative function distribution, the right panel shows

This chapter will discuss the different strategies to improve the productivity of the cellular protein factory. Especially the possibility of engineering membrane trafficking will be addressed. This technology is not only useful for developing a versatile and robust cell factory but can be very valuable to study intracellular membrane trafficking on an academic

**2. Conventional engineering strategies for improved protein production** 

folding enzymes, the folding capacity of the cell is enhanced 18-21.

The conventional strategy to improve protein production is to increase the expression levels of the gene of interest by using multiple gene copies and strong promotors 11, 12. As a consequence, the cell is faced with very high levels of mRNA which should rapidly be translated into active and correctly folded proteins. To handle this increased demand for folding capacity the cell often reacts with the so called Unfolded Protein Response (UPR). This UPR is a well studied mechanism 13-17 which is also applied to increase folding capacity. By upregulating the transcription factors needed, or the resulting chaperones and

intra extra

**Distribution** *A. niger*  **functionalities**

approach.

0% 20% 40% 60% 80% 100%

level as well.

intra extra

absolute numbers of enzyme function distribution.

**Relative distribution** *A. niger*  **functionalities**

For heterologous proteins the fusion peptide concept has been proven to be successful 22. This concept is used for heterologous proteins which are secreted by their native host but are overexpressed in an alternative system to increase production titers. By fusing the protein of interest to a homologous protein which is naturally secreted by the host, the desired protein is also transported outside the cell, for example, *Acremonium murorum* phenol oxidase expressed in *Aspergillus awamori* 23. This carrier protein is often an N – terminal part of a very well secreted homologous protein which is fused to the protein of interest. Often a cleavage site is engineered, like Kex2 with the aim to produce only the full length protein. In reality often a mixture of unprocessed protein, partially processed protein and fully processed protein is produced, which can have consequences for downstream processing in order to meet the required product specifications. On the other hand, there are also reports which describe secretion of heterologous proteins by using only signal sequences without any additional fusion proteins like the production of llama variable heavy-chain antibody fragments (VHHs) 24 and the production of *Arthromyces ramosus* peroxidase 25 both in *Aspergillus awamori*. However, the titers obtained by these systems are in the mg/l range which is generally not sufficient for an economically feasible process.

The next generation of tools to increase protein titers are based on technology developments within bio-IT combined with the possibility of designing custom-made genes. Recently, the concept of codon adaptation was demonstrated. This concept is based on the use of favorable codons so that translation of the mRNA encoding the protein of interest is not a bottleneck. An excellent *in silico* study has recently been published 26 describing that translational speed and, concomitantly, ribosome density are determined by the combination of coding sequences and the tRNA pool. The first dozens of codons are translated at lower rates and create an area where ribosomes are very densely packed, especially on transcripts with a high mRNA level. This strategy could prevent ribosome jamming in later stages of the translational process and therefore be of physiological advantage of the cell. This elevation of translational speed towards the end of transcripts is often seen in fungi (which are biotechnological work-horses) and provides the cell with an effective tool against late abortions of protein synthesis which is an energy-consuming process. As suggested by the authors the fine-tuning of this ramping principle could be an additional tool to increase the efficiency of the protein production factory.

As optimizing codon usage is aimed at improving the translation of mRNA into protein, the folding capacity of the cell can become limiting, especially when high copy numbers and strong promoters are combined with codon optimization. The folding and refolding of proteins is tightly linked to membrane trafficking, a very stringent quality control system existing in the endoplasmic reticulum acts as a gatekeeper for proteins to be transported further into the secretory pathway. This quality control system has been reviewed recently 2. Also the removal of proteolytic activity has been applied to improve protein production 27-33 this approach has shown to be very effective in specific cases. The success of the different approaches is hard to predict and highly dependent on the protein of interest being overproduced. In addition, all of the strategies above are focused to improve secretion of natural secreted proteins, most of them being hydrolases.

An overview on conventional strategies to improve the productivity of the cellular protein factory is shown in Figure 2. As shown, the modification of DNA, which includes cloning strong promotors in front of the gene of interest and constructing multiple copies of this

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking 141

The conventional strategies described above are focused on incremental improvement, overproducing proteins which are naturally secreted products. Therefore, the large diversity of intracellular proteins remains untapped. In addition, these strategies can be seen as generic, at least in part, but very large differences in secretion efficiency exist between different proteins; changes of a few amino acids can often have a detrimental effect on protein secretion 7, 36. This is probably linked to different interactions with chaperones and folding enzymes. In the case of cutinase secretion in yeast, the bottleneck was circumvented by engineering an N-glycosylation site at the amino terminus of cutinase 8. The restrictions of conventional strategies are not only illustrated by the order of magnitude of differences in protein secretion but also by the limited class of proteins which are secreted. As shown in Figure 1, in *A. niger* most of the secreted proteins are hydrolases whereas the enzyme variation of intracellular enzymes is much larger. This enormous potential of enzymes is hardly being touched upon. There are strategies to overproduce intracellular proteins inside the cell but this is always followed by a complex downstream processing step to liberate the proteins and to purify them to an acceptable level 37, 38. In addition, toxic proteins or compounds cannot be produced by these methods at economical levels because they will very likely damage the host cell 39-41. In order to get access to the large variety of intracellular functionalities and to be able to produce toxic compounds into a cell compartment which does not impact the viability of the host cell, an additional strategy

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

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

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

**3. Limitations of conventional strategies** 

based on membrane engineering would be very valuable.

without damaging the host cell.

molecules.

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

expression cassette is a generic strategy which can lead to improvement of protein production. Also the optimization of codon(pair) usage and mRNA stabilizing elements can be applied in a generic way. The adaptation of the folding capacity of the host cell is not a generic approach. The folding and modification of proteins is intertwined and very protein specific. Improvement of regular transport of secretory vesicles is a strategy which is less well-known, but some possible routes have been described 34. In addition the overexpression of SEC4, a Rab protein associated with vesicles, doubled protein production in *Pichia pastoris* 35, which is a direct proof that modification of vesicle flow can result in enhanced protein production. However all these approaches are focused on enhancing the efficiency of existing protein production and secretory processes in the host cell.

Fig. 2. Conventional improvement options for protein secretion in biological processes. Indicated in blue are the major steps in biological (eukaryotic) systems which are crucial for protein secretion, indicated in orange are improvement strategies which can be applied at the different stages of the protein secretion process.

#### **3. Limitations of conventional strategies**

140 Crosstalk and Integration of Membrane Trafficking Pathways

expression cassette is a generic strategy which can lead to improvement of protein production. Also the optimization of codon(pair) usage and mRNA stabilizing elements can be applied in a generic way. The adaptation of the folding capacity of the host cell is not a generic approach. The folding and modification of proteins is intertwined and very protein specific. Improvement of regular transport of secretory vesicles is a strategy which is less well-known, but some possible routes have been described 34. In addition the overexpression of SEC4, a Rab protein associated with vesicles, doubled protein production in *Pichia pastoris* 35, which is a direct proof that modification of vesicle flow can result in enhanced protein production. However all these approaches are focused on enhancing the

efficiency of existing protein production and secretory processes in the host cell.

Fig. 2. Conventional improvement options for protein secretion in biological processes. Indicated in blue are the major steps in biological (eukaryotic) systems which are crucial for protein secretion, indicated in orange are improvement strategies which can be applied at

the different stages of the protein secretion process.

The conventional strategies described above are focused on incremental improvement, overproducing proteins which are naturally secreted products. Therefore, the large diversity of intracellular proteins remains untapped. In addition, these strategies can be seen as generic, at least in part, but very large differences in secretion efficiency exist between different proteins; changes of a few amino acids can often have a detrimental effect on protein secretion 7, 36. This is probably linked to different interactions with chaperones and folding enzymes. In the case of cutinase secretion in yeast, the bottleneck was circumvented by engineering an N-glycosylation site at the amino terminus of cutinase 8. The restrictions of conventional strategies are not only illustrated by the order of magnitude of differences in protein secretion but also by the limited class of proteins which are secreted. As shown in Figure 1, in *A. niger* most of the secreted proteins are hydrolases whereas the enzyme variation of intracellular enzymes is much larger. This enormous potential of enzymes is hardly being touched upon. There are strategies to overproduce intracellular proteins inside the cell but this is always followed by a complex downstream processing step to liberate the proteins and to purify them to an acceptable level 37, 38. In addition, toxic proteins or compounds cannot be produced by these methods at economical levels because they will very likely damage the host cell 39-41. In order to get access to the large variety of intracellular functionalities and to be able to produce toxic compounds into a cell compartment which does not impact the viability of the host cell, an additional strategy based on membrane engineering would be very valuable.
