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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 the fungal secondary metabolites that have the potential to prolong the human lifespan.

The production of lovastatin, a drug used in lowering LDL cholesterol for patients suffering from cardiovascular disease, has been chosen as the Herculean task for our team. Currently, this secondary metabolite is produced in the fungus Aspergillus terreus, which also produces less desirable toxins. We aim to transfer the entire lovastatin metabolic pathway from A. terreus into the GRAS organism Aspergillus niger. A. niger is amenable to genetic engineering which already works efficively for citric acid.

Modular Structure of DNA Sequence

The production of secondary metabolites in most cases involves very big genes that code for large backbone enzymes which contain multiple catalytic domains. One of our goals is to establish a modular system of domain shuffling to generate a plethora of novel enzymes with new functionalities. 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 Fugure 1.

Figure 1: Modularity approach of lovB.

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 bioreports such as secondary selection markers. These tools allow one to obtain insight in cellular economy, physiology and architecture, which can be used to optimize the production processes in A. niger.