**Peroxicretion, a Novel Tool for Engineering Membrane Trafficking**

Cees M.J. Sagt *DSM Biotechnology Center, Delft The Netherlands* 

#### **1. Introduction**

136 Crosstalk and Integration of Membrane Trafficking Pathways

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The production of proteins by recombinant micro-organisms has been possible since the late 70's. Since then enormous steps have been made to improve protein titers and to expand the range of possible proteins to be produced. The development of genetic modification tools and improved ease of use of cloning strategies have played an important role in this. The time to construct a strain and develop a cost-effective bioprocess has decreased significantly. Concurrently, sequencing data, which serve as a library for donor genes to be overexpressed, have increased exponentially. Therefore, the speed and flexibility to engineer custom-made protein factories has improved tremendously during the last decades. As in any developed technology, a certain degree of standardization is desired. In order to make this ambition more tangible the concept of cell factories is often used. This concept is based on the principle that the cell factory should be able to produce any protein to a certain desired level. Based on the concept of standardized expression vectors and expression hosts the basics of this cell factory concept were built. However, in reality this concept proved to be far too simple, as a standardized input does not result in a standardized output. This is caused by the immense complexity and dynamics of cellular systems. Even when all components are described, by sequencing the genome, the interaction, compartmentalization and dynamics of these components are largely unknown. A striking example is the difference between homologous protein expression which are produced to levels up to 50 g/l in filamentous fungi whereas heterologous proteins are produced at levels which are usually 100-1000 fold less 1. So even when identical tools are used in a standardized approach the final result can vary over several orders of magnitude.

The general consensus is that this large difference is linked to cellular events and responses which are caused by the overexpression of the heterologous proteins 2, therefore the scientific community, together with biotech industries, have been studying these effects for several decades. The cellular stress responses and intracellular events which are linked to the use of cells as protein production factories are very well described 3-9. This profound insight in the molecular mechanism of the cellular stress reactions has facilitated the increase in expression and secretion levels of some heterologous proteins. However, levels comparable to homologous proteins have not been reached 1, 10. It is evident that, when even more demanding sources of biodiversity are tapped, like intracellular proteins, the engineering of a robust and versatile cell factory which is able to produce this wide range of proteins becomes a major challenge.

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking 139

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

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

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

additional tool to increase the efficiency of the protein production factory.

natural secreted proteins, most of them being hydrolases.

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 approach.

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 absolute numbers of enzyme function distribution.

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 level as well.
