Team:DTU-Denmark/Proof Of Concept

This year our team wanted to approach the need for reducing the harmful greenhouse gas emissions on the climate from a biotechnological point of view. Methane is a potent greenhouse gas which is 28-36 times more harmful than carbon dioxide [1]. In nature, methane is being used as a valuable raw material by some organisms. However domesticating these microbes is troublesome due to their codependent lifestyles [2]. Our project, PHEAST, started with the ambitious goal of engineering the methylotrophic yeast, Komagataella phaffii, (previously known as Pichia pastoris)to become a methane-consuming cell factory that can produce recombinant proteins, such as leghemoglobin, from methane.

However, we quickly realized there was a lack of established and publicly available tools for genomic integration and expression of heterologous proteins for K. phaffii in the iGEM Registry. In fact, after developing a search on the iGEM Registry, we found out that K. phaffii (and also Pichia pastoris) parts represented less than 1% of all the available parts (Fig. 1). This shocked us considering the great relevance K. phaffii has currently on industry. And that is what made us entail to provide a better knowledge for K. phaffii engineering.

Figure 1 : Percentage presence of Komagataella phaffii in relation to the whole iGEM Registry. For this results, the total amount of 242 parts for K. phaffii after PHEAST contribution are compared to the rough estimate of 20000 parts found on the main iGEM Registry page.

Figure 2 : Barchart showing PHEAST contribution to the iGEM Registry for K. phaffii parts.

Additionally, as it can be seen in Fig. 2, PHEAST contribution implied an increase of 60% for the amount of parts available for K. phaffii in the iGEM Registry, going from 151 to 242 parts.

Therefore, we had to start by building a toolbox for genomic integration in K. phaffii. Here we present a proof of concept state of the art CRISPR based toolbox for easy and fast genetic engineering of K. phaffii GS115 Part Collection.

We chose to use a CRISPR based system with type II Cas9 endonucleases for site-directed genome editing in K. phaffii. The main reasons for choosing a RNA-to-DNA based targeting method over protein-to-DNA based methods were lower technical complexity and time-efficient design and employment of the system [3]. Currently, there is a big incentive for developing CRISPR-Cas9 based tools for engineering K. phaffii [4,5].

To get started on the CRISPR/Cas9 based toolbox, single guide RNAs (sgRNAs) were designed to target neutral insertion sites in K. phaffii GS115 [6,7]. We created several sgRNAs, and homology arms. The homology arms can be used as modular parts when constructing repair templates. These parts have been described in-depth in our Part Collection.

Figure 3: Repair Template for Generic Gene Insertion

Figure 4: Repair Template for Generic Gene Knockout

Figure 5: Animated Gene Insertion in the Genome of a Cell Factory

Figure 6: Animated Gene Knockout in the Genome of a Cell Factory

This provides the user with a baseline for marker-free integration into respective neutral integration sites in the four chromosomes of K. phaffii. Further, we designed and experimentally validated a sgRNA and corresponding homology arm fusion for efficient and scarless knockout of the adh2 gene. The same methodology can be applied for the knockout of other genes of interest.

Figure 7: Colonies Guide RNAs

Figure 8: Gels Guide RNAs

We constructed 16 CRISPR/Cas9 plasmids targeting neutral integration sites for integration of genes of interest in K. phaffii. For our proof of concept, a subset consisting of 9 plasmids was chosen for sequencing. Our results show that all 9 plasmids were successfully constructed. From the subset of sequenced plasmids, 6 were experimentally shown to efficiently target neutral integration sites in K. phaffii using the TAPE method (Technique to Assess Protospacer Efficiency) [8].

Figure 9: Sequencing Results gRNAs

As an addition to our toolbox and for the validation of pMMO functionality in K. phaffii, another set of 6 CRISPR/Cas9 plasmids were successfully constructed to knockout three different genes, aox1, aox2 and adh2. Colony PCR results indicated successful deletion of each gene, respectively. However due to time constraint and trouble with sequencing, only one scarless gene knockout was validated by sequencining K. phaffii GS115 Δku70 for one of the CRISPR/Cas9 plasmids, namely a crRNA targeting adh2.

Figure 10: Colony PCRs performed on Δaox1, Δaox2, and Δaox2. Each band corresponds to a unique colony picked from a plate of transformed K. phaffii GS115 Δku70. 16 colony PCRs were loaded to the gel on the left, eight aox1 and eight aox2 knockout colony PCRs, respectively. Eight adh2 knockout colonies were loaded the gel on the right. The gel pictures have been cut to fit both in one figure. One band was observed for aox1 and aox2 while two bands were observed for adh2

Figure 11: Transformed K. phaffii GS115 Δku70 with respective gRNAs and repair templates, if provided. Each gRNA had two candidates (C1 & C2). C1 seems to more efficient than C2. The negative control consists of K. phaffii GS115 Δku70 electroporated with water to check for contamination. The other control was K. phaffii GS115 Δku70 strain transformed without a repair template (RT)(BBa K3841033). The Triple knockout refers to an experiment were all three knockout plasmids were co-electroporated into K. phaffii GS115 Δku70 with respective repair templates. Since it only relies on taking up one plasmid to obtain resistance to NTC, a different approach is recommended to do multiple knockouts.

Figure 12: Sanger Sequencing confirmation of knockout of adh2.

Figure 13: BioLector growth curves of aox1 and adh2 double knockout mutant and the K. phaffii GS115 Δku70 mutant strain. It is evident that the increasing methanol concentrations affects the growth of the double mutant more than the K. phaffii GS115 Δku70 mutant strain, proving that the double knockout mutant is more susceptible to methanol.

The results above confirm that our CRISPR based toolbox works. With our contributions, future iGEM teams and scientists will have access to this standalone platform for efficient and easy engineering of K. phaffii GS115 for stable genomic integrations and deletions. The toolbox has been tested extensively by this year's DTU BioBuilders team and is ready to be used. Extensive documentation of our efforts during this project can be accessed in the results section of our wiki Results.

References

[1] https://2021.igem.org/Team:DTU-Denmark/Proof_Of_Concept
[2] Chen, Y., Li, Y. L., Zhou, G. T., Li, H., Lin, Y. T., Xiao, X., & Wang, F. P. (2014). Biomineralization mediated by anaerobic methane-consuming cell consortia. Scientific Reports, 4(1), 5696. https://doi.org/10.1038/srep05696
[3] Gaj, T., Sirk, S. J., Shui, S. L., & Liu, J. (2016). Genome-editing technologies: Principles and applications. Cold Spring Harbor Perspectives in Biology, 8(12), a023754. https://doi.org/10.1101/cshperspect.a023754
[4] Cai, P., Duan, X., Wu, X., Gao, L., Ye, M., & Zhou, Y. J. (2021). Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Research, 49(13), 7791–7805. https://doi.org/10.1093/nar/gkab535
[5] Liu, Q., Shi, X., Song, L., Liu, H., Zhou, X., Wang, Q., … Cai, M. (2019). CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris. Microbial Cell Factories, 18(1), 144. https://doi.org/10.1186/s12934-019-1194-x
[6] Brady, J. R., Tan, M. C., Whittaker, C. A., Colant, N. A., Dalvie, N. C., Love, K. R., & Love, J. C. (2020). Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq. Acs Synthetic Biology, 9(9), 2515–2524. https://doi.org/10.1021/acssynbio.0c00299
[7] De Schutter, Kristof, et al. “Genome Sequence of the Recombinant Protein Production Host Pichia Pastoris.” Nature Biotechnology, vol. 27, no. 6, NATURE PUBLISHING GROUP, 2009, pp. 561–66, doi:10.1038/nbt.1544.
[8] Garcia Vanegas, Katherina, et al. “SWITCH: a Dynamic CRISPR Tool for Genome Engineering and Metabolic Pathway Control for Cell Factory Construction in Saccharomyces Cerevisiae.” Microbial Cell Factories, vol. 16, no. 25, BioMed Central Ltd., 2017, p. 25, doi:10.1186/s12934-017-0632-x.