Team:DTU-Denmark/Poster

RESHAPE: Tuning morphology and secretion in Aspergillus niger for improved industrial applications
Presented by DTU BioBuilders 2020

Daniel Bavnhøja, Peter Binga, Clara Drachmannb, Martí M. Gómezb, Cecilia D. V. Graaea, Bira A. Khana, Niels MKa, Lucas Levassora, Margrethe Mærsk-Møllerb, Victoria V. Nissena, Christine Pedersena, Cecilie A. N. Thystrupb, Timian Rindala

aDTU Bioengineering, bDTU Health Tech, Technical University of Denmark


Abstract: Every week, almost 6 billion people use products made with the aid of fungal cell factories. Many of these fungi stem from the Aspergillus genus. We aim to improve the production efficiency of Aspergillus niger by creating a synthetic biology toolbox that focuses on mycelial morphology and secretion. Morphology has a significant effect on productivity of certain compounds, while proper secretion is important for later recovery and purification of a compound. We have approached our goal in three ways:

  1. Characterising morphological changes of A. niger by engineering seven morphology related genes.
  2. Establishing a computational model of mycelium growth based on imaging data.
  3. Developing a library of native and synthetic signal peptides for protein secretion.

By improving the efficiency of bio-based production processes, we can improve the economic incentive to use them, and decrease our heavy reliance on oil-based substrates in the chemical industry.

Problem

Non-renewable natural resources are being depleted at an alarming rate, and the by-products of industrial production are contributing to climate change. This stands in the way of reaching the UN sustainability goals, particularly goal 12: Ensure sustainable consumption and production patterns and goal 8: Decent work and economic growth. To combat these issues, production practices must be changed.

Shifting production of industrially important compounds to be developed through enzymes, and therefore be less reliant on petrochemicals, would alleviate some of the stress which is put on the current supply chains. One way of making this shift is by using filamentous fungi as cell factories. For many compounds which could be produced by cell factories, however, the cost and nuisance of working with filamentous fungi is prohibitive to their widespread use.

We hoped to contribute to the development of more sustainable production practices by improving the filamentous fungus Aspergillus niger as a cell factory.

Solution

To make cell factory production more economically advantageous and less troublesome, we worked to RESHAPE the morphology of Aspergillus niger, which is already a highly used cell factory (Meyer et al., 2016).



We approached this task from several angles, as seen below. We both worked to alter the morphology of A. Niger by targeting relevant gene alterations and simultaneously worked to increase its ability as a protein producer. Results gathered from these alterations were further used to create modelling tools that could be used for predicting growth of mycelia.

Methods

During this project, nine different A. niger morphology knockout strains were constructed and characterized, of which five were novel.

All strains were constructed using a CRISPR-Cas9 system (Nødvig et al., 2015). The CRISPR knockout vectors were constructed using USER-cloning and were designed to knockout seven chosen genes related to morphology. Six of the mutants were successful and three additional mutants were constructed by combining the knockouts of genes from three of the most promising strains, thereby creating three double knockout strains.

To assess the effects of the mutations, the strains were tested through a variety of experiments. These included growth on different solid media, microscopy, growth in a BioLector and in 1L bioreactors. Samples from the bioreactor were analyzed through glucoamylase and BCA assays along with HPLC analysis.

Model and Software

To aid our parts characterization, we developed two models for studying the morphological features of Aspergillus niger, both based on our experimental data. The first model, Morphologizer, automatically analyzes microscopic images of mycelia. The second model, Mycemulator, is a stochastic model that simulates mycelial growth based on morphological parameters, including some from the Morphologizer.

Showcase of our first model, Morphologizer. The model analyzes microscope images of mycelia, converts them into graph objects, and extracts morphological features of interest from the graph, such as the number of branches, branching angles, and hyphal curvature.

Showcase of our second model, Mycemulator. A stochastic simulation of background strain ATCC 1015 based on parameters obtained from both experimental measurements and microscopic image analysis. The substrate gradient is shown in green, i.e. the greener the higher substrate concentration and the mycelium is shown in purple with newer hyphal elements in lighter colors.

Results

During the project we managed to make nine new morphology strains and nine mycelium growth simulations based on real life data. The results from the three most promising strains are shown here with the reference strain ATCC 1015:

The three most promising morphology strains and the reference strain ATCC 1015 plated on CYA media. They are made by knocking out gul-1 separately and in combination with either chsC or spaA. All three strains are novel work.

Microscope pictures of the three strains with the reference strain ATCC 1015. The pictures (stained version with Calcofluor White) were used by Morphologizer to estimate the parameters used in the Mycemulator simulations below.

12h Mycemulator simulations of the three strains and reference strain ATCC 1015 in the order (ATCC 1015, Δgul-1, ΔchsC_Δgul-1 and ΔspaA_Δgul-1). Parameters for the simulations are estimated by Morphlogizer and growth rate is found from the BioLector growth data.

A comparison of growth rates for the three strains and reference strain ATCC 1015 when grown in 1L bioreactors. ΔchsC_Δgul-1 showed a minor increase in growth rate whereas the two others showed minor decreases in growth rates compared to the reference strain. None of them showed a significant difference from the growth rate of the reference strain.

Protein secretion and glucoamylase activity for Δgul-1, ΔchsC_Δgul-1 and ΔspaA_Δgul-1 compared to the reference strain ATCC 1015. Samples analyzed from the last time-point of the runs. All three strains showed an increase in specific activity, with Δgul-1 having the highest increase (with the exception of the one ΔspaA_Δgul-1 replicate, but results here are not conclusive.)
(Green: Glucoamylase activity in UA/mL. Blue: Specific activity in UA/mg calculated from the activity and the protein concentration. Purple: Protein concentration in mg/mL.)

These results confirmed our original hypothesis that morphology has an impact on protein production. This is significant as increasing protein production while maintaining a similar or increased growth rate would lead to decreased production costs compared to existing systems.

Part collection

Genetic engineering of the Aspergillus niger genome can be achieved using various methods, including CRISPR-Cas9 recently adapted from Streptococcus pyogenes (Nødvig et. al, 2015 ; Nodvig et al., 2018).

Here, we present our extensive collection of CRISPR-Cas9 parts for engineering the morphology of A. niger. The presented collection comprises 40 parts for knockout of a total of 7 genes related to morphology. These parts can be combined to make a wide range of knockouts.

The CRISPR-Cas9 vector, pFC330, can also be applied to target other specific genes (e.g. glucoamylase). Find the sequence (e.g. a gene) in the A. niger genome that you want to cut out - find a PAM sequence (NGG) upstream and downstream of the sequence - design crRNA (20 bp upstream of the PAM sequence) - use pFC902 as a template to primer elongate - clone the bioblocks into the PacI/Nt.BbvCI cassette in pFC330 - and transform the plasmid into A. niger along with a 90 nt repair oligo to get a mutant of your choice!


Among our nine mutants, we constructed five novel strains of which two were single knockouts and three were double knockouts. Some of these mutants had increased growth rate, protein secretion and glucoamylase activity.

Integrated Human Practices

Consultation with external stakeholders played a crucial role in our project. We therefore contacted and held four meetings with industry stakeholders to gain insights into the problems facing industry regarding the inability to control morphology. Soon after we consulted with five leading edge experts to make sure that our engineering approach had the best chance of success. We then held two more meetings with industry to consult and learn what types of measurements that would be needed for implementation.





Finally, all of our work culminated in a project pitch for Novozymes with people attending from multiple departments. Here we presented all our results. Through continuous contact with experts from the biotech industry, we improved our project and tuned it to fit industrial purposes. With this work, we hope that our project can result in real impact for future applications of A. niger as a cell factory.

Achievements

New morphology strains
We have made 9 new morphology strains and characterized their performance as cell factories based on growth and protein secretion when grown in BioLectors and Bioreactors. We even showed that some of our new strains, the three novel strains were gul-1 was knocked-out, had up to three-fold increased protein production compared to the reference strain.

Morphological toolbox for A. niger
We have made a collection of CRISPR-Cas9 parts that can be used to engineer morphology in Aspergillus niger. This part collection consists of in total 40 parts including seven CRISPR-Cas9 vectors that can be used to knockout 7 morphology related genes in A. niger. The parts can be combined in endless ways to help engineer morphology in the future.

Analysis of fungal microscopic images
We have developed Morphologizer - a tool for analyzing microscope images of mycelia. Using Morphologizer, one can easily and automatically estimate morphological parameters for a fungal strain.

Simulation of mycelial growth
We have developed Mycemulator - a model that can simulate mycelial growth based on real-world parameters - for example the parameters estimated by our Morphologizer model.

Acknowledgements and references

This project would not have been the same without the feedback from so many dedicated and experienced people in this field - from other iGEM teams to the people from the industry who helped us shape the project. Thank you. Without all of you, we wouldn’t have made it this far.

A huge thanks to our sponsors DTU Blue Dot Project, Otto Mønsteds Fond, Novo Nordisk Fonden, Amplicon, Eurofins, Integrated DNA Technologies, Twist bioscience, and New England Biolabs who supported us throughout this project. Your support has been greatly appreciated.

Also a huge thanks to the companies Novozymes A/S, Novo Nordisk A/S and Bolt Threads, who helped us during this project. Your advice has been invaluable and helped us shape our project to what is it today.

Thank you to the other iGEM teams we collaborated with - FCB-UANL and Korea-HS - for continuous support and good work ethic. And thanks to all the other teams we made smaller collaborations with - it was a pleasure working with you.


References
  1. Meyer, V., Andersen, M. R., Brakhage, A. A., Braus, G. H., Caddick, M. X., Cairns, T. C., de Vries, R. P., Haarmann, T., Hansen, K., Hertz-Fowler, C., Krappmann, S., Mortensen, U. H., Peñalva, M. A., Ram, A. F. J., & Head, R. M. (2016). Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper. Fungal Biology and Biotechnology, 3, 6. doi: 10.1186/s40694-016-0024-8
  2. 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
  3. 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