Team:DTU-Denmark/Design
Awards
Introduction
The non-conventional yeast Komagataella phaffii (K. phaffii) is a popular production chassis for the production of recombinant proteins. However, the genetic engineering of non-conventional yeasts entails several hurdles. Therefore the aim of this project was to expand the repertoire of available genetic engineering tools for the industrially relevant production host, K. phaffii.
By utilising the tools of synthetic biology we developed an easy to use system for integration and gene deletion in the genome of K. phaffii GS115. By doing so, we provide a versatile platform for genetic engineering in this non-conventional yeast. Further, this toolbox contributes to the publically available knowledge and tools for the scientific community and future iGEM teams.
With an established toolbox for engineering K. phaffii, our final aim was to introduce methane utilisation in the methylotrophic yeast by genomic integration of prokaryotic genes. The necessary genes encode a particular methane monooxygenase complex, pMMO, consisting of three subunits: PmoA, PmoB and PmoC [1]. To test the functionality of recombinant methane utilisation in K. phaffii, we included a validation step in our experimental design to account for the efficiency of methane conversion of into methanol. This was done by knocking out genes involved in a native methanol assimilation pathway in K. phaffii. In summary, our experimental plan consisted of developing a toolbox and use it to engineering K. phaffii for methane utilisation.
Within our lab work, the three main goals were:
From our design, we managed to create several different CRISPR-plasmids for directed genetic engineering of K. phaffii’s genome in its different chromosomes. With these plasmids we are able to either perform a knockout or knock-in of genes. With our contributions, future iGEM teams and scientists have access to this standalone platform for efficient and easy engineering of K. phaffii GS115 for stable genomic integration and scarless deletion of genes. The toolbox has been tested extensively by this year's DTU BioBuilders team and is ready to be used. In depth description of all the considerations that went into the design of our project in the tabs below.
Why Komagataella phaffii?
When designing a methane utilising cell factory, we considered our choice of organism carefully. Through literature review, discussions with supervisors and interviews with stakeholders in our Human Practices work, we kept circling back to the potential of non-conventional yeasts due to their metabolic properties and relevance to the biobased industry [2].
As we were interested in designing a cell factory for methane valorisation, the non-conventional methylotrophic yeast Komagataella phaffii (K. phaffi) spiked our interest. K. phaffii is a popular chassis due to its versatile range of recombinant proteins and narrow range of endogenous secretory proteins [2]. Further, one of the cornerstones of K. phaffii’s popularity as an industrial production host is it’s strong native system for methanol induced gene expression [3]. K. phaffii can not utilise methane as a substrate since it lacks an enzyme catalysing the conversion of methane to methanol (methane monooxygenase). However, this enzyme is found In prokaryotic methanotrophs such as Methylococcus capsulatus. This led us to devising a plan to introduce the necessary parts for K. phaffii to be able to oxidize methane to methanol, which would then strongly induce native methanol regulated promoters.
We quickly realized that there was a lack of established and publicly available tools for genomic integration and expression of heterologous proteins for K. phaffii.
In order to make engineering of K. phaffii readily available, we decided to develop a genetic toolbox based on CRISPR/Cas9 and describe the workflow with attached protocols, which can also be found on Contribution.
The reference strain we used is Komagataella phaffii GS115, in which we have performed all our genetic engineering studies. We used GS115 ku70. This strain lacks the ku70 gene which makes it unable to do Non-Homologous End Joining (NHEJ). This forces the strain to use Homologous Direct Repair (HDR) to repair double-stranded breaks in the DNA caused by either natural events or by genetic engineering tools such as CRISPR based systems. This facilitates easier integration of necessary parts into K. phaffii, for instance by CRISPR based systems where integrations happen at a much higher frequency compared to the wild type GSS115 strain, as a ku70 mutant HDR will predominate over NHEJ [4].
References
[1] Chan, S. I., Nguyen, H. H. T., Chen, K. H. C., & Yu, S. S. F. (2011). Overexpression and purification of the particulate methane Monooxygenase from Methylococcus capsulatus (Bath). Methods in Enzymology, 495, 177–193. https://doi.org/10.1016/B978-0-12-386905-0.00012-7
[2] Karbalaei, M., Rezaee, S. A., & Farsiani, H. (2020). Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology, 235(9), 5867–5881. https://doi.org/10.1002/jcp.29583
[3] 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
[4] Mistlberger, B., Ruth, C., Hajek, T., S. Hartner, F., & Glieder, A. (2016). Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology.
Toolbox for Chromosomal integration and deletion in K. phaffii
To modify the genome of our K. phaffii, we decided to use USER cloning for plasmid construction, and CRISPR-Cas9 for genome engineering.
USER Cloning and Fusion
A USER cloning cassette within the USER compatible vector can be digested either with the restriction enzymes PacI and Nt.BbvCI or AsiSI and Nb.BsmI to create overhangs that match the insert’s USER overhangs. PacI or AsiSI creates a small overhang whereafter Nt.BbvCI or Nb.BsmI makes a single-strand nick, which creates a 5’ overhang. When mixed, the insert and vector will assemble and create the designed vector. The vector can then be cloned into competent DH5α E. coli cells. Not providing the inserts to the reaction will prevent the linearized plasmid from annealing, which will in consequence disable subsequent cloning of the plasmid into competent cells. Cells without a plasmid will not gain antibiotic resistance, therefore reaction samples that lack inserts can be used as a negative control.
USER is an abbreviation for Uracil-Specific Excision Reagent. It is a method to directionally clone inserts into a USER compatible vector by the use of a USER nicking enzyme. The sequence for insert is created through primers, with one uracil and a tail that is compatible with a uracil cloning site in the vector, amplified through PCR. This product is treated with Uracil-Specific Excision Reagent, removing the U and the attached strand from the double-stranded DNA, creating an overhang ready for cloning conceptualized Fig 1.
CRISPR-Cas9
To design a robust K. phaffii cell-factory utilising the concepts of synthetic biology, we opted for chromosomal integration of our parts for increased stability and to relieve the need of maintaining selection pressure. The main tool we used to facilitate genomic integration or gene knockouts in K. phaffii was a CRISPR-Cas9 based system designed by the Diversify group at DTU Bioengineering, provided by Tomas Strucko [Nødvig references x2]. We applied this platform to design a state of the art CRISPR based toolbox for easy and fast genetic engineering of K. phaffii GS115.
Design of sgRNAs for Knockout and Insertion
Before we began designing sgRNAs for our toolbox we had to identify neutral integration sites in K. phaffii GS115. We mined the genome for integration sites of approximately 500-2000 base pairs of non-coding regions (in neutral intergenic regions) flanked by household genes or highly expressed genes that (divergently transcribed) (Fig. 2). Another criterion was that the intergenetic region was easily accessible for Cas9 and not located near chromosomal ends, to avoid the presence of telomeres. We identified neutral integration sites on each of the four chromosomes that we named IS1, IS2, IS3, and IS4.
How to design sgRNAs
To design sgRNAs targeting the neutral integration sites, we uploaded the intragenetic region between two divergent genes according to Fig. 3.150 basepairs upstream of each of the divergent genes, to avoid that the sgRNAs might cut in the promoter region of the genes, onto CRISPRdirect and CHOPCHOP.
Our criteria for choosing sgRNAs are summarized in the table below. Based on these criteria we selected four sgRNAs for each of the four chromosomes in K. phaffii GS115.
How to integrate them into our CRISPR-Cas9 plasmid
Primer extension PCR was used to integrate the site specific sgRNA targeting the neutral integration sites or genes for deletion into a USER casetttee. The casettee was subsequently cloned into the pDIV151 plasmid backbone as shown in Fig. 4.
From our design above we constructed CRISPR-Cas9 based plasmids for both genomic integration and targeted gene deletion by USER cloning. The assembled CRISPR-Cas9 based plasmids each contain an variable crRNA sequence within their sgRNA, targeting a protospacer in the genome of K. phaffii GS115. For a full overview of the build process go to our Experiments page → USER CLONING protocol. We designed sgRNAs for cloning into CRISPR/Cas9 plasmids to target either neutral integration sites for integration of genes of interest, or the deletion of genes of interest in K. phaffii.
Test Of sgRNAs
To choose which integration sites we wanted to use for gene insertion in K. phaffii we started with identifying the efficiency of the different protospacers. The cutting efficiency of the designed sgRNAs for gene insertion were tested using the TAPE method []. We relied on a K. phaffii GS115 Δku70 strain and the wild type GS115 strain for comparison of the cell viability as a result of transformation with the different CRISPR/Cas9 plasmids (see Fig. 5). This strain was chosen to increase our success in genomic integration since it depends solely on Homology Directed Repair for mending double stranded breaks in its DNA. For more details on the results visit Results.
Knockout of genes using CRISPR
A typical repair template for knockouts of genes can be seen in Fig. 6. The oligo is made in vitro by fusing the 45 basepairs directly upstream with the 45 basepairs directly downstream of the gene you want to knock out. Co-transformation with a CRISPR-Cas9 plasmid and a repair oligo like the one below will mediate homology directed repair (HDR) (Fig. 7).
Knock-in of genes using CRISPR
In Fig. 8 the elemental design of a repair template for gene integration is shown. To have a conceptual overview of how a gene of interest is inserted into the host cell genome as a result of a double-stranded break caused by the Cas9 complex can be seen in Fig. 9.
References
[1] Chan, S. I., Nguyen, H. H. T., Chen, K. H. C., & Yu, S. S. F. (2011). Overexpression and purification of the particulate methane Monooxygenase from Methylococcus capsulatus (Bath). Methods in Enzymology, 495, 177–193. https://doi.org/10.1016/B978-0-12-386905-0.00012-7
[2] Garcia Vanegas, K., Lehka, B. J., & Mortensen, U. H. (2017). SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae. Microbial Cell Factories, 16(25), 25. https://doi.org/10.1186/s12934-017-0632-x
Methanotrophic yeast toolbox
With an established genetic toolbox for engineering K. phaffi, the next step was to test our neutral integration sites to introduce methane utilisation in our strain and knocking out genes involved in methanol metabolism to test the efficiency of the conversion of methane into methanol. To integrate the necessary parts for the conversion of methane to methanol in K. phaffii GS115, we designed repair templates encoding the particular methane monooxygenase complex (pMMO) subunits: PmoA, PmoB and PmoC [1]. For optimal expression of the prokaryotic genes in K. phaffii we did codon-optimization.
Genes For Methane Consumption
In order to make K. phaffii methanotrophic we had an ambitious plan for integrating several (pMMO) subunits from the bactetrium Methylococcus capsulatus. When the complex assembles in the cell membrane methane can be oxidized to methanol and transported across the cell membrane as shown in Fig. 1 [1].
All three subunits need to be transported to the plasma membrane to assemble into a nonamer (3α3β3γ). We designed a repertoire of composite parts that can be viewed in the tables below.
The complexity of integration repair templates varied with regards to various versions of the pMMO subunits such as with and without signal peptides and/or reporter fusions.
Please see the Registry.
Choice of promoters
We wanted the pMMO subunits to be expressed constitutively so that methane would be converted into methanol as soon as it is present in the media. We designed the repair templates for pMMO subunit integration with the strong constitutive promoters GAP1 and TEF1.
Signal Peptides
Signal peptides (SPs) typically consist of short amino acid sequences of 16 to 30 amino acids, aiding in the localization of proteins to the functioning location in the cell. The SPs can be found in either end of the protein sequence or in the middle, as Jens Preben Morth explained in our interview. This means that it can be hard to predict exaclty where in the gene a native SP is located, however SPs are typically found in the N-terminal of a protein sequence.
In order to increase the likelihood of the subunits of pMMO ending up in the plasmamembrane, a signal peptide was taken from K. phaffii’s native glycosyltransferase, which is a membrane bound protein. By using the protein localization tool, DeepLoc, we predicted where in the cell a protein fused to the given SP would end up, the result of this prediction can be seen in Fig. 2.
Knock-ins
The repair templates for co-transformation with our constructed CRISPR-Cas9 plasmids were built using Golden Gate Assembly (gene insertion) and USER cloning (gene deletion). Since the repair templates for gene insertion consist of more parts compared to gene deletion, we chose to use Golden Gate Assembly for simultaneous modular assembly of five biobricks in a predetermined manner. For both gene insertion and gene deletion, the repair templates were PCR amplified from respective plasmids.
For gene insertion, we tried to integrate the pMMO repair templates into the genome of K. phaffii. We prepared competent K. phaffii GS115 Δku70 cells for electroporation with a constructed CRISPR-Cas9 plasmid together with a respective repair template. Obtained transformant colonies were screened prior to confirmation of genomic integration by sequencing.
Knock-outs
To test the functionality of recombinant methane utilisation in K. phaffii, we included a validation step in our experimental design to account for the efficiency of methane conversion into methanol. This was done by knocking out non-essential genes involved in a native methanol metabolism pathway inK. phaffii. This has been done before in literature yielding a K. phaffii mutant that is unable to metabolize methanol [2]. A similar approach to the gene integration was used. (Fig. 4). The only difference resides in the repair template. To knock out a gene, one should provide DNA oligo that is complementary to the 45 base pairs upstream and the 45 base pairs downstream of the gene one wants to knock out.
References
[1]Chan, S. I., Nguyen, H. H. T., Chen, K. H. C., & Yu, S. S. F. (2011). Overexpression and purification of the particulate methane Monooxygenase from Methylococcus capsulatus (Bath). Methods in Enzymology, 495, 177–193. https://doi.org/10.1016/B978-0-12-386905-0.00012-7
[2] Zavec, D., Troyer, C., Maresch, D., Altmann, F., Hann, S., Gasser, B., & Mattanovich, D. (2021). Beyond alcohol oxidase: The methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2. Fems Yeast Research, 21(2), foab009. https://doi.org/10.1093/femsyr/foab009
Reporter Library
For stable expression of recombinant proteins in K. phaffii, we relied on the strong native methanol induced promoters AOX1, FLD1. One of the cornerstones of K. phaffii’s popularity as an industrial production host of recombinant proteins is its strong native system for methanol induced gene expression, regulated by promoters such as the AOX1 and FLD1 promoters. We wanted to see whether we could provide the iGEM community with an improved version of the AOX1 promoter for methanol induced recombinant protein expression in K. phaffii GS115. Therefore we investigated two other versions of the AOX1 promoter from related species, namely an AOX1 promoter with 99% and 89% similarity (AOX1 99% and AOX1 89%, respectively) to the GSS115 AOX1 promoter. The FLD1 promoter was investigated for comparison to the AOX1 promoters, for us to find the strongest promoter for our recombinant production pathway (Table 1) (AOX1 and FLD1 Reference: https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-019-1194-x).
We designed reporter protein constructs to investigate the efficiency of each of our chosen promoters, as well as to provide experimental data or the iGEM registry on some of their fluorescent reporters. This resulted in the following two different libraries of reporter constructs:
- Promoter investigation, using different promoters on our codon optimized Venus (BBa_K3841006) and our soy leghemoglobin (BBa_K3841014).
- Fluorescent reporter library, here using the same promoter, AOX1, and 3 different fluorescent reporters from the iGEM distribution kit, namely GFP (BBa_K895006), mOrange (BBa_E2050) and Venus optimized for Saccharomyces cerevisiae (BBa_K165005)
Promoter Investigation
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841022 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter AOX1 |
| BBa_K3841024 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter |
| BBa_K3841025 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter |
| BBa_K3841026 | Reporter | Expression cassette of yeast optimized Venus from regulated FLD1 promoter |
| BBa_K3841088 | Reporter | Expression cassette of soy leghemoglobin from methanol-dependent promoter |
| BBa_K3841089 | Reporter | Expression cassette of soy leghemoglobin from methanol-dependent promoter |
| BBa_K3841090 | Reporter | Expression cassette of soy leghemoglobin from methanol-dependent promoter |
| BBa_K3841091 | Reporter | Expression cassette of soy leghemoglobin from regulated promoter |
This resulted in a total of 8 different combinations of promoter and gene to be expressed, an overview of which can be seen in Fig. X and Fig. Y below.
Figure x: y
Figure x: y
For the reporter gene investigation, we had a total of 4 constructs to compare. All constructs include AOX1 as their promoter. This was designed to provide the iGEM registry with additional data on the expression of said fluorescent proteins in K. phaffii (Table 2 and Fig Z).
Reporter Investigation
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841022 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter AOX1 |
| BBa_K3841092 | Reporter | Expression cassette of GFP from methanol-dependent promoter |
| BBa_K3841093 | Reporter | Expression cassette of mOrange from methanol-dependent promoter |
| BBa_K3841094 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter |
In order to integrate the above mentioned constructs into the genome of K. phaffii, we designed primers for amplification of parts by primer extension PCR to be compatible for the Golden Gate assembly. A conceptual overview of the repair template assembly can be seen in Fig. XYZ.
From the GGA, we PCR amplify our repair templates in order to transform the resulting linear DNA oligos into our K. phaffii strain along with our previously designed sgRNA plasmids. With this we have designed our protein expression platform. A conceptual animation of our gene integration in K. phaffii can be seen in THE GIF of gene insertion (Fig. X).
References
[1] Chan, S. I., Nguyen, H. H. T., Chen, K. H. C., & Yu, S. S. F. (2011). Overexpression and purification of the particulate methane Monooxygenase from Methylococcus capsulatus (Bath). Methods in Enzymology, 495, 177–193. https://doi.org/10.1016/B978-0-12-386905-0.00012-7
