Difference between revisions of "Team:DTU-Denmark/Integrated Human Practices"

Kenneth Bruno and Grayson Wawrzyn from Zymergen visited DTU in May 2019. On this occasion, we met with them and discussed our project. As Zymergen works with i.a. filamentous fungi and know what other biotechnology companies look for in promoters, they were able to provide us with a lot of valuable advice on how to approach our project. Among other things, they advised us to use the Aspergillus niger strain ATCC 1015 since this strain is commonly used in industry and contains an auxotrophic selection marker.
Zymergen specialises in genetically engineering a broad range of non-standard hosts, and this directed us to open the software to be able to parse any given organism genome. Additionally, Zymergen is in the business of gene expression regulation/control, and a stable promoter ladder is therefore vital to their setup. Accordingly, we directed our experimental setup to also test the scalability of our synthetic promoters. Additionally, we learned that the industry is interested in scarless assemblies between the promoter region and the start codon, as well as promoters that are active in the stationary phase. We took all of this into consideration when designing the software to create the promoters and when assembling the plasmids.

We talked to Peter Richard, a principal scientist at VTT in Finland. He is an expert in using synthetic biology with regards to Aspergillus spp. and provided feedback on our approach, in addition to advice on making our results as applicable as possible. He advised us to grow the fungi under comparable conditions, i.e. that we should keep things such as growth phase, temperature, media, and reporter gene consistent and in mind when testing the promoters. We implemented this throughout the project, most notably in the characterization phase where a standardized media was used. RFP was used as a reporter and the growth was measured over time, enabling evaluation of the promoters during both the lag phase, exponential phase, stationary phase, and eventually the death phase. Furthermore, he recommended keeping the plasmid copy number in mind while running the experiments as this would enable us to correlate gene expression to each promoter. We, therefore, decided to integrate the constructs in the Aspergillus genome, but unfortunately, we did not have time to test our promoters further in this construct, as described in the results.

In June, the team visited the Novozymes facilities in Lyngby to learn about their approach to working with filamentious fungi in the industry. Apart from a guided tour around the facilities, the team also had an inspiring talk with Mikako Sasa, Science Manager and fungi specialist, where we discussed different fungal species and their potential applications.

From this initial meeting, we learned that promoters are valuable assets for a company such as Novozymes, and that our promoters would be even more valuable if they were consistent across different scales and conditions. This allows the user to worry less about the implementation of the promoters and enables them to focus on optimizing pathways and fermentation conditions.

In August, we came back and talked with Mary Ann Stringer and Kirk Matthew Schnorr, both scientists at Novozymes. Here, we learned more about what Novozymes looks for in a desirable promoter. As they are in the business of producing enzymes, they are usually looking for strong promoters, however, ideally they would want a promoter that was inactive in the exponential phase and highly active in the stationary phase. This would limit the stress on the organism while growing and building biomass, and maintain the high production rates when the amount of biomass is sufficiently large. This is consistent with what we had previously heard from Zymergen, and we decided to prioritize promoters that fulfilled these criteria.

At this meeting, we also learned about their concept of “Tank Years”. This refers to the number of times a tank can be run in a year and takes into consideration cleaning, sterilization, inoculating, running, and extraction of the product. Novozymes is interested in producing as much as possible, as quickly as possible, and this means that the production time in the tanks should be minimal, the cleaning easy, and the enzyme titers maximized.
We have taken this into account when designing our bioreactor experiments, where a conscious effort was taken to minimize downtime between runs.

We have been in contact with the bio-materials company Bolt Threads which among other things work with filamentous fungi. We were in contact with Edyta Szewczyk, senior scientist at Bolt Threads, who gave us a lot of feedback and ideas. Especially three key points were emphasized: Filamentous fungi takes a long time to grow, which means we need to plan ahead; good reporter systems are essential for detecting transformants and expression, and; targeted genomic integration is a good solution to control for copy-number and genomic context. We have tackled these point by, 1) transforming our fungi as soon as possible, 2) using fluorescence (both GFP and RFP) to measure the expression, and 3) testing genomic integration methods which can be seen on Design page.

We reached out to one of the leading research scientists in filamentous fungi, Jens Christian Frisvad. Among other things, he told us to look at a several different fungal species, and not only the standard ones, as these might be able to expand the range of products that are possible to produce industrially. By basing our software on genomes from many different Aspergillus spp. our promoters are ideally applicable in several different species. Additionally, our software allows for the application of any list of genomes and the creation of promoters for any given species, enabling the creation of promoter libraries for non-standard organisms.

Following the discussion with Zymergen, we decided to contact Jakob Blæsbjerg Hoof, associate professor at the Department of Biotechnology and Biomedicine. He told us that using a fluorescence marker was the easiest approach to select for transformants, and that we should add a secretion tag to the fluorescent protein in order to prevent accumulation inside the cell. This was later backed up by Bolt Threads. Additionally, Blæsbjerg provided us with the ATCC 1015 Aspergillus niger strain, in addition to a protease-deficient variation (prtT KO), which were used in the experiment as well as protocols for protoplasting and transformation.