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A Versatile and Powerful Synbio Host

Biotechnology has brought about a revolution in drug manufacturing since its inception. Many life-saving drugs have spun out of biotech companies over the past few decades. However, there is still a vast body of unexplored compounds, such as fungal secondary metabolites, that have the potential to prolong human lifespan and increase the quality of life.

The production of lovastatin, a drug used in lowering LDL cholesterol for patients suffering from cardiovascular diseases, has been chosen as the Herculean task for our team. Currently, this secondary metabolite is produced in the fungus Aspergillus terreus, which also produces some less undesirable toxins. We aim to transfer the entire lovastatin metabolic pathway from A. terreus into the Generally Regarded as Safe organism Aspergillus niger. A. niger is much more amenable to genetic engineering, which already had profound effects in citric acid production.

Modular Structure of DNA Sequence

The production of secondary metabolites in most cases involves very big genes that code for large backbone enzymes that contain multiple, apparently independent, catalytic domains. One of our goals is to establish a modular system of domain shuffling to be able to generate a plethora of novel enzymes with new functions. The possibilities are endless as there are various different domains from fungi that can be added, removed, reordered, or even customized by this modularity approach, which is shown in Figure 1.

Figure 1. Modularity approach of lovB. Since secondary metabolite producing enzymes consist of large modular proteins, it could be possible to brick the individual modules and use our new frame-shift-less assembly strategy to generate multidomain proteins in all kinds of configurations.

For instance, one of the main enzymes involved in the production of lovastatin is the 277kDa LovB enzyme, which contains 7 different catalytic domains. Prior studies on this pathway provide a good insight into the catalytic mechanisms of these individual domains. The individual domains have been mapped by homology modeling and are found to be well defined as well, which makes the modularity approach more feasible.

A Toolkit for A. niger

In addition to the modularity approach, a toolkit for A. niger is also being created, consisting of an ATP and a pH biosensor that can be targeted to specific compartments with the use of signal peptides, actin and septa GFP fusions to visualize the cytoskeleton and the junctions between adjacent cells, and chromoproteins that can serve as simple bioreporters such as secondary or neutral selection markers. These tools allow one to obtain insight in the cellular economy, physiology and architecture and logistics, which can be used to optimize production processes in A. niger.

Host Engineering

A. niger is a ‘workhorse’ commonly used in industry. However in this multicellular host only the hyphal tips are actively secreting whereas most of the biomass is vegetative. This is why we chose to expand the scope of our project by including host engineering. The goal is to create a single cell from the mycelium of A. niger that represents the active hyphal tip. Two different approaches are taken into consideration to do so.