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Molecular toolbox
In the substrate section we established that Yarrowia Lipolytica constitutes a great platform utilizing waste streams. In order to unlock the potential of Y. Lipolytica, we developed a molecular toolbox allowing us to efficiently engineering Y. Lipolytica. In this section we present the theory and results of the development of a BioBrick backbone and CRISPR tools for Y. Lipolytica

Man is a tool-using animal. Without tools he is nothing, with tools he is all.
Thomas Carlyle Signs of the Times (1829)

BioBrick plasmid

Introduction

A key part of synthetic biology is to streamline the process of engineering biological systems, by standardizing parts and methods¹. Perhaps the most versatile standards available is the BioBrick standard, in part due to the contributions made during the annual iGEM competition. The BioBrick registry currently has over 20,000², and by creating BioBrick plasmid backbones compatible with a new organism, one is effectively unlocking the entire BioBrick registry available for that specific organism. Realizing this, it was decided to develop a plasmid that supports the BioBrick standard and replicates in Y. Lipolytica. Due to the convenience of manipulating Escherichia coli it was determined to develop a shuttle vector that allows for cloning and confirmation of the construct in E. coli, before the construct is transformed in Y. Lipolytica. Additionally, as the only replicative plasmids currently available for Y. Lipolytica is low copy yeast chromosomal plasmids (YCp)³ this allows for high amounts of DNA to easily be propagated in E. coli, before the the plasmid is purified and transformed into Y. Lipolytica. Figure 1 shows a suggested workflow for the proposed BioBrick plasmid.

DESCRIPTION — **Figure 1:** The suggested workflow using pSB1A8YL described above. The workflow is broken down to three distinct steps: cloning, confirmation and expression. Cloning is the assembly of the construct, which is conducted in *E. coli* using typical cloning methods. Confirmation is where the identity of the construct is confirmed, using analytical methods such as PCR, analytical restriction enzyme digestion and/or sequencing. Expression includes the work done in the final host organism, *Y. lipolytica*, and includes inserting the construct and optimization of the expression and cultivation conditions.

Design

For the design of the plasmid, we decided to incorporate a high copy E. coli part for cloning and propagation DNA. The design was based on the pUC19 vector as it fulfils the criteria of being high copy⁴, while perhaps being one of the most widely used cloning vectors for E. coli. To support the BioBrick standard, we only used the ampicillin resistance and replication origin elements of the plasmid. It was found that the sequence in and between these elements did not contain any restriction sites of any current BioBrick assembly standard, thus no further modification of the sequence was needed.

For the Y. Lipolytica part of the plasmid we decided to base the design on the pSL16-CEN1-1(227), as it has found to exhibit high transformation efficiency compared to similar plasmids⁵, and perhaps for this reason this plasmid and its derivatives are utilized in many recent studies.^6,7,8 Again only the sequence of the replicative and selective elements were chosen. Although, the original sequence was not BioBrick compatible, and thus it was decided to order the sequence as a gBlock. This also introduced the added benefit of being able to incorporate the BioBrick prefix, suffix and a 5’ terminator in the gBlock and exchange the original leucine autotrophy marker with a uracil autotrophy marker allowing for negative selection of the plasmid with 5-Fluoroorotic Acid (5-FOA)⁹. In order to comply with the iGEM plasmid nomenclature¹⁰, the plasmid was dubbed “pSB1A8YL”, YL was added in the end to emphasize that the plasmid is used for Y. Lipolytica. Figure 2 shows a graphical representation of the sequence map of pSB1A8YL.

Cloning

The pUC19 part and the gBlock fragments was amplified using primers with USER tails, and fused using USER cloning (See Figure 3).

The USER reactions was transformed into chemically competent E. coli DH5alpha cells, and purified. The identity of the product was checked using PCR, restriction analysis (see Figure 4) and sequencing (data not shown).

Testing

After having confirmed the identity of the plasmid, we set out to test its functionality. This was done in three steps: 1. Testing the plasmids replicability and selectivity in Y. Lipolytica, 2. Testing the plasmids cloning capabilities in E. coli and finally 3. Combining the two first tests by cloning a construct in E. coli and which is expressed when transformed into Y. Lipolytica.

1. Replicability and selectability in Yarrowia lipolytica

The pSB1A8YL plasmid was purified from E. coli DH5alpha, and transformed in Y. Lipolytica PO1f cells. The transformants was selected on selective dropout media not containing uracil, thus only yielding uracil autotroph transformants. A negative control was included substituting the plasmid for MQ water. The transformations only yielded colonies on the plated containing the cells which were transformed with the plasmid. To ensure that these results indeed meant that our plasmid was stably replicating in the Y. Lipolytica cells, a few colonies were subjected to colony PCR (see Figure 5).

These results confirm that pSB1A8YL replicates in Y. Lipolytica, and the chosen uracil selection marker allows for selection of transformants. To further assess the functionality of pSB1A8YL, the possibility of counter selection was investigated. This was done by growing colonies containing pSB1A8YL on plates containing 5-FOA. Colonies appearing on these plates were then transferred to selective dropout media not containing uracil. As no growth was observed on the latter plate, this proved that pSB1A8YL supports counter selection.

2. Cloning capabilities in E. coli

In order to test this, we decided to produce a device using BioBricks from the distribution kit which would allow us to easily assess whether the cloning were successful. When deciding BioBricks that would allow this, we received inputs from the SDU iGEM team. We ended up choosing the strong Andersson promoter/strong RBS BioBrick (BBa_K880005) and pair this with three chromoproteins: amilCP (BBa_K592009), amilGFP (BBa_K592010) and mRFP (E1010), which would allow us to easily pick transformants and visually inspect if the cloning was successful. The cloning flow is shown in Figure 6.

The BioBricks were retrieved from the distribution kit, and assembled with our plasmid as carrier backbone using standard 3A assembly and transformed into chemically competent E. coli DH5alpha cells. The transformants yielded colored colonies (See Figure 7), and the identity of the constructs were confirmed using restriction analysis and PCR (data not shown). These results confirm that pSB1A8YL can be used for cloning in E. coli and supports the BioBrick standard.

3. Combining construct cloning in E. coli and expression in Y. Lipolytica

To test if our plasmid would support cloning of a construct in E. coli, which would be expressed, and ultimately prove that our plasmid works as intended, we chose to develop our own BioBricks. Namely a constitutive TEF promoter (BBa_K2117000) and a hrGFP gene (BBa_K2117003) previously used successfully in Y. lipolytica⁷. By combining these two parts in a device (BBa_K2117004), the expression should, in theory, be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 8.

The parts were ordered as gBlocks and assembled in E. coli using A3 assembly. The assembly was confirmed using PCR, restriction analysis and sequencing. Afterwards the construct was transformed into Y. lipolytica PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy and measured in a fluorometer. Unfortunately we did not observe any fluorescence signal, despite the sequencing confirmed the identity of the construct. We hope that a future team will debug the construct, and unlock the true potential of pSB1A8YL.

Conclusion

We successfully developed a shuttle vector that allows the user to harvest the efficiency and accessibility of cloning in E. coli, while still allowing for replication in Y. lipolytica. We extensively tested the plasmid, and showed that it allows for replication, selection and expression in E. coli and replication, selection and counterselection Y. lipolytica. The plasmid was shown to be compatible with BioBricks which, to our knowledge, makes it the first tool that allows for the use BioBricks in Y. lipolytica. Unfortunately, we were not able to detect any expression from the constructs we were able to clone. The plasmid was submitted to the registry, and we hope that future teams will debug the construct, facilitating the use of Y. lipolytica to a larger extend in iGEM and general research context. It is our hope that other actors in iGEM community will use this tool in order harvest the great potentials of Y. lipolytica as a chassis for biorefineries of the future.

CRISPR

Introduction

By creating a modular plasmid that is compatible with the BioBrick standards and that is able to replicate in both E. coli and Y. lipolytica, the entire biobrick registry has been made available for use in Y. lipolytica. However, one important aspect of industrial-scale protein expression is the stable and homogenous expression of relevant pathway genes. In production organisms with multi-enzyme pathways there is often a need for accurate control of the expression levels of the associated genes, both in terms of promoter strength and copy numbers¹¹. Plasmid based systems for the expression of heterologous genes are widely used, but offer limited control of copy number and in many cases show notable segregational instability even during growth in selective media. Therefore, it is often preferred to integrate the genetic pathway of interest into the genome of the production organism of choice to ensure long-term strain stability and homogenous expression of pathway genes¹¹.

The CRISPR-Cas9 has revolutionized the way in which model organisms can be engineered. In addition, it has made genetic engineering applicable to a wide range of organisms that were previously considered to be uninteresting for cell factory purposes, largely because the genetic engineering tools for these organisms were under characterized. The CRISPR-Cas9 system is ideally suited for the integration of heterologous genes in production organisms, as it relies on Double Strand Break (DSB) induction and its subsequent repair by homologous recombination (HR). In addition, almost any sequence of interest can be targeted with the most commonly used Cas9 from Streptococcus pyogenes¹². The basic mechanism of CRISPR-Cas9 is illustrated in Figure 9, for a more detailed overview the reader is referred to Nødvig et al.¹²

We adapt the CRISPR-Cas9 system for use also in Y. lipolytica in order to obtain a proof-of-concept of the construction of a stable production strain. We do so by a proof-of-concept integration of a gene in Y. lipolytica. In addition, we obtain a proof-of-concept of the deletion of a native Y. lipolytica gene. With these proof-of-concepts, we envision that the metabolic flux towards a possible compound of interest could be increased, leading to tailored Y. lipolytica strains for cell factory engineering purposes.

Experimental design

The CRISPR-Cas9 system has previously been successfully applied to Y. lipolytica by Schwartz et al.⁸ As this was a proof-of-concept study, the native Y. lipolytica PEX10 gene was targeted for disruption, which allowed for an easily selectable phenotype. This is because PEX10 codes for Peroxisome biogenesis factor 10, a protein that is involved in peroxisome biogenesis. Consequently, PEX10 disruption results in an inability to catabolize long-chain fatty acids. We set out to confirm the observed PEX10 deletion. In addition, we set out to integrate URA3, a commonly used yeast auxotrophic marker gene which codes for Orotidine 5’-phosphate decarboxylase, an enzyme involved in pyrimidine biosynthesis.

We had obtained both a commonly used Y. lipolytica laboratory strain (PO1f, MATA URA3-302 LEU2-270 XPR2-322 AXP2 ΔNU49 XPR2::SUC2; obtained from Schwartz et al.⁸) and also this same strain with a knockout of the Non-Homologous End-Joining (NHEJ) DNA repair pathway gene Ku70 (PO1fΔKu70, MATA URA3-302 LEU2-270 XPR2-322 AXP2 ΔNU49 ΔKu70 XPR2::SUC2; obtained from Schwartz et al.⁸). The NHEJ pathway for DNA repair is commonly disrupted in laboratory strains to improve the efficiency of CRISPR-Cas9 induced HR integration of heterologous DNA. However, an intact NHEJ pathway can be utilized to disrupt genes without the need for an DNA repair template, as this pathway is intrinsically more error-prone than HR. Thus, in order to disrupt the native Y. lipolytica PEX10 gene, we used a CRISPR-Cas9 plasmid (pCRISPRyl) in the PO1f strain. For the URA3 insertion, we co-transformed the same CRISPR-Cas9 plasmid and a linearized HR donor plasmid (pIW501) in the PO1fΔKu70 strain. An overview of our workflow is provided in Figure 10. Overviews of the pCRISPRyl and pIW501 are provided in Figures 11 and 12, respectively.

Results & Discussion

1. URA3 insertion

In order to integrate the URA3 gene into the genome of the Y. lipolytica PO1fΔKu70 strain, we first constructed a pCRISPRyl-derived plasmid which contained a protospacer that targets the PEX10 locus. This protospacer was used previously for successful disruption of PEX10 (cory). The pCRISPRyl-derived plasmid (pIW357) was constructed through the Gibson assembly of a 60 bp fragment containing the protospacer of interest into the AvrII linearized pCRISPRyl. Subsequently, pIW357 was purified from selected E. coli DH5ɑ transformants and a double restriction analysis was performed with PstI and AvrII (Figure 13). As the purified pIW357 was no longer digested with AvrII, the protospacer had been correctly inserted. In addition, the protospacer insertion was confirmed through Sanger sequencing (data not shown).

Next, we tested pIW357 in the Y. lipolytica PO1fΔKu70 strain. In order to account for Cas9 activity of pIW357, we also transformed the pCRISPRyl as a positive control. The results of these transformations are depicted in Figure 14. With an intact NHEJ repair pathway and/or a template for HR, the induced DSB should easily be repaired. However, as this is the PO1fΔKu70 strain and as there is no HR donor template available, cells die, indicating that the pIW357 is functioning properly. The observed residual growth can be attributed to either mutations in pIW357 (for example in Cas9 or in the the sgRNA/tRNA promoter) or possible repair by the Microhomology-Mediated End-Joining (MMEJ) pathway ¹⁴.

With a functioning pIW357, we then co-transformed pIW357 and pIW501 (linearized) into the Y. lipolytica PO1fΔKu70 strain (Figure 15). The observation of growth on SC-ura media indicates that the URA3 gene had been successfully integrated. Growth was also observed when solely the linearized pIW501 was transformed. In addition, transformants from the co-transformation of pIW357 and pIW501 were screened for URA3 integration through gap-check colony PCR (Figure 16). The observed shift in band size between non-pIW501 transformants and pIW501 transformants corresponds to the 1700 bp size of the URA3 promoter+CDS+terminator sequence that is present on pIW501.

The pIW501 is linearized prior to transformation in order to increase HR efficiency and in order to reduce the amount of false positive transformants (a gene cannot be expressed from a linear DNA fragment). However, growth was also observed when solely the pIW501 is transformed, indicating that CRISPR-Cas9 DSB induction is not necessarily required for the integration of an auxotrophic marker gene. We did not have time to compare the efficiencies of co-transformation of pIW357 and pIW501 as compared to solely pIW501 transformation. However, it has been shown previously that co-transformation with a CRISPR-Cas9 plasmid increases the HR efficiency¹². We thus hypothesize that the integration of the URA3 gene was facilitated by pIW357. We also did not have time to test the integration of a non-marker gene (which would still allow for an easily selectable phenotype such as GFP or lacZ). This might have been a better option, as the efficiency of non-CRISPR-Cas9 induced HR is lower for a non-marker gene. This would have allowed us to more easily distinguish between CRISPR-Cas9-mediated and non-CRISPR-Cas9-mediated gene integration.

2. PEX10 deletion

1

Conclusion

As a proof-of-concept, we have shown that we can integrate a gene of interest into Y. lipolytica and also disrupt a native gene of interest. Although in our project we have used a plasmid-based protein expression system this proof-of-concept validates the use of Y. lipolytica for future cell factory engineering purposes.

Here, we have focused on the integration of a commonly used auxotrophic marker gene. However, the CRISPR-Cas9 system can be applied for markerless gene integrations ¹³. Markerless genetic modifications are advantageous as they do not affect cell physiology and allow for iterative genetic modification cycles of the production organism of interest. In addition, this iterative process can be sped up as the modular nature of the CRISPR-Cas9 system makes it ideally suited for simultaneous multiplex genome editing^13,15.

References

Shetty, R. P., Endy, D., & Knight, T. F. (2008). Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering, 2(1), 5. article. http://doi.org/10.1186/1754-1611-2-5
iGEM collections
Liu, L., Otoupal, P., Pan, A., & Alper, H. S. (2014). Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. Fems Yeast Research, 14(7), 1124–1127. doi:10.1111/1567-1364.12201
Yanisch-Perron, C., Vieira, J., & Messing, J. (1984). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 33(33), 103–119. doi:10.1016/0378-1119(85)90120-9
Yamane, T., Sakai, H., Nagahama, K., Ogawa, T., & Matsuoka, M. (2008). Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission. Journal of Bioscience and Bioengineering, 105(6), 571–578. doi:10.1263/jbb.105.571
Liu, L., Otoupal, P., Pan, A., & Alper, H. S. (2014). Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. Fems Yeast Research, 14(7), 1124–1127. doi:10.1111/1567-1364.12201
Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. Applied and Environmental Microbiology, 77(22), 7905–7914. doi:10.1128/AEM.05763-11
Schwartz, C. M., Hussain, M. S., Blenner, M., & Wheeldon, I. (2016). Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica. Acs Synthetic Biology, 5(4), 356–359. doi:10.1021/acssynbio.5b00162
Sakaguchi, T., Nakajima, K., & Matsuda, Y. (2011). Identification of the UMP Synthase Gene by Establishment of Uracil Auxotrophic Mutants and the Phenotypic Complementation System in the Marine Diatom Phaeodactylum tricornutum. Plant Physiol, 156(1), 78–89.
iGEM nomenclature
Ronda, C., Maury, J., Jakočiu̅nas, T., Baallal Jacobsen, S. A., Germann, S. M., Harrison, S. J., Borodina, I., Keasling, J. D., Jensen, M. K., & Nielsen, A. T. (2015). CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae. Microbial Cell Factories, 14(1), 97. http://doi.org/10.1186/s12934-015-0288-3
Nødvig, C. S., Nielsen, J. B., Kogle, M. E., & Mortensen, U. H. (2015). A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS ONE, 10(7), 1–18. http://doi.org/10.1371/journal.pone.0133085
Jessop-Fabre, M. M., Jakočiūnas, T., Stovicek, V., Dai, Z., Jensen, M. K., Keasling, J. D. & Borodina, I. (2016), EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnology Journal, 11: 1110–1117. doi:10.1002/biot.201600147
Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K.-I. T., & Yamamoto, T. (2015). MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nature Protocols, 11(1), 118–133. http://doi.org/10.1038/nprot.2015.140
Gao, S., Tong, Y., Wen, Z., Zhu, L., Ge, M., Chen, D., Jiang, S., & Yang, S. (2016). Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system. Journal of Industrial Microbiology & Biotechnology, 43(8), 1085–1093. http://doi.org/10.1007/s10295-016-1789-8

Team:DTU-Denmark/molecular toolbox

BioBrick plasmid

Introduction

Design

Cloning

Testing

1. Replicability and selectability in Yarrowia lipolytica

2. Cloning capabilities in E. coli

3. Combining construct cloning in E. coli and expression in Y. Lipolytica

Conclusion

CRISPR

Introduction

Experimental design

Results & Discussion

1. URA3 insertion

2. PEX10 deletion

Conclusion

References