Team:DTU-Denmark/Engineering


To RESHAPE Aspergillus niger we have used the tools of synthetic biology. We have designed, built and tested a total of eight parts for gene knockout in the A. niger genome. Additionally, we have designed and built a new parts collection to add novel signal peptides to our protein products, containing 15 parts. In total we added 76 parts to the iGEM registry benefitting further teams choosing to work with A. niger.


Engineering morphology:

We wanted to ease the work with Aspergillus niger and thereby improve it as a cell factory. Quickly, we figured out that morphology has a huge impact on A. niger's performance as a cell factory. Its way of growing in filamentous structures can be difficult in large fermentation tanks as the filaments can tangle around stirrers and probes. Furthermore, morphology is closely linked to the production, and depending on the product of interest different types of morphology are preferred. We chose to focus on protein production, thereby changing the morphology to ease the work with A. niger without hurting its ability for protein production.

To do this, we went into the literature to find genes affecting morphology. We found seven genes that were proven to alter the morphology, either when down/up regulated or completely knocked-out. An overview of the genes is found in the design page.We decided for the initial work to only do knockouts of the seven genes as this would also be the first step in making up/down regulated strains.


To make our new morphology strains we used CRISPR-Cas9 to meditate gene knockouts in the A. niger genome. The CRISPR-Cas9 system we used was developed and optimized for Aspergilli by Uffe Mortensens lab (Nødvig et. al., 2015; Nødvig et al., 2018). The system is described in detail in our design page.

To use the system a SingleGuideRNA (sgRNA) with the sequence matching the gene to be knocked-out needs to be designed. sgRNA consists of gRNA, that attaches to Cas9, and crRNA, that is the sequence matching the cutting site of the gene of interest. By identifying two crRNA regions for each of the genes, we managed to design seven CRISPR-Cas9 plasmids.

The design of the seven CRISPR-Cas9 plasmids for knockout of morphology genes are found in the iGEM registry with the following part numbers:


From our design we constructed the seven CRISPR-Cas9 plasmids. The sgRNAs are made using primer extending PCR amplification with primers containing the crRNA sequence. The plasmids were assembled using USER cloning. For a complete overview of the build process visit our experiments page.


The plasmids were transformed into A. niger, meditating knockouts of the genes. Six of the seven gene knockouts resulted in viable strains. From these initial six strains we again used the CRISPR-Cas9 plasmids resulting in three double knockout strains. In total we managed to make nine new morphology strains.


To characterize the new strains, we observed their macroscopic morphology on different solid medias as well as through the microscope. We measured their growth in a BioLector and 1L bioreactors. The strains protein production was also assessed from bioreactor samples. Read the details of all the measurements in the measurement page.

We learned how the gene knockouts affected the strains behavior. You will find all the data we obtained for the new morphology strains and our conclusions in our results page.


While some of the strains showed increased protein production and good growth data, other gene knockouts did not benefit the strains on these parameters. This was expected from our initial research. The original design for up and down regulating the genes was also made but never built. You can read about this design on our design page.



Engineering protein secretion:

We wanted to improve protein secretion in A. niger. To do this we wanted to research the rather abundant area of signal peptides. In literature we found the top secreted proteins in A. niger and identified their signal peptides. Furthermore, our drylab team developed a tool that predicts signal peptides, SignalPrepper.From this prediction we got five synthetic signal peptides that we also wanted to test.


We were able to characterize our signal peptides by their ability to facilitate export of glucoamylase enzyme (encoded by glaA) as its activity can easily be measured by an assay. We designed an expression cassette for integration in A. niger. This cassette was based on an integration platform designed by the DTU-Denmark 2019 team. We also used one of their promoters. The initial construct is seen in the figure to the right. The signal peptide can easily be replaced with primer extending PCR amplification and USER cloning as illustrated in the figure below.


The design of the 15 expression constructs for testing the signal peptides are found in the iGEM registry with the following part numbers:
Additionally, a CRISPR-Cas9 plasmid, as the ones designed for the morphology gene knockouts, was designed to knockout the gene glaA:


From our design we built the 15 expression constructs and the CRISPR-Cas9 plasmid. We first knocked-out the glaA gene in A. niger. Thereafter, we successfully inserted 11 of the 15 expression constructs into the genome of A. niger.


Unfortunately, we did only reach the initial part of this step. Due to time constrictions we did not obtain conclusive data for the eleven constructed strains but we hope to be able to continue this work soon!


References
  1. Nødvig, C., Nielsen, J., Kogle, M., & Mortensen, U. (2015). A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLOS ONE, 10(7). doi: 10.1371/journal. Pone.0133085
  2. Nødvig, C., Hoof, J., Kogle, M., Jarczynska, Z., Lehmbeck, J., Klitgaard, D., & Mortensen, U. (2018). Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genetics And Biology, 115, 78-79. doi: 10.1016/j.fgb.2018.01.004