iGEM 2022 — Team wwu-muenster


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[Engineering success of *Saccharomyces cerevisiae*](#section-1)
[Fusion of afraGFP with PTS1 for localization in
peroxisomes](#section-2)
[Engineering success of DNPH assay](#section-3)

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To engineer *Saccharomyces cerevisiae* for α-pinene production, we applied the concept of Modular Cloning (MoClo)
targeting the cytosol and peroxisomes. Following strain engineering, we characterized the production of our product of
interest, α-pinene, by using gas chromatography-mass spectrometry (GC-MS) analysis. We identified several genes which
have a greater impact on α-pinene production than others, leading to new potential gene combinations. Furthermore, we
developed a colorimetric assay based on 2,4 dinitrophenylhydrazine (DNPH) to detect and quantify verbenone produced by
our *in vitro* system. After we completed the design-build-test-learn cycles three times, we applied the assay
for standardized verbenone detection. Additionally, this can be used as a mutation screening for protein engineering
in the future.
We designed, built, and tested several new parts including a C-terminal SKL peroxisomal targeting signal-1 (PTS1) to
relocate the mevalonate pathway inside the peroxisome. Our microscopy results verify that the C-terminal SKL (PTS1)
works as expected.

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## Engineering success of *Saccharomyces cerevisiae*

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/engineering-success-yeast-modifications-hevk.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/engineering-success-yeast-modifications-hevk.png)

**Figure 1: Graphical abstract of the design-build-test-learn (DBTL) cycle applied to yeast metabolic
engineering.**

In the implementation and experimental design of MonChassis, we were driven by the general principle of synthetic
biology - design, build, test, and learn (DBTL).

### Design

We engineered the yeast’s mevalonate pathway to overcome its rate-limiting steps. By introducing additional
heterologous genes and silencing endogenous genes, we [modified the pathway to suit our needs](https://2022.igem.wiki/wwu-muenster/results#sccytosol). Our goal was to increase geranyl diphosphate (GPP) production in *Saccharomyces cerevisiae*, as it acts as an essential precursor for monoterpenoid synthesis such as α-pinene. Hence, we
engineered the cytosolic pathway, but also targeted another cell compartment. Since physiological conditions in
peroxisomes are favorable for GPP production, we hypothesize that an increased α-pinene concentration is observed if
the corresponding synthesis pathway is relocated there.

### Build

Since we decided to clone all genes using the RFC1000 modular cloning system (MoClo), we designed specific overhangs
for our promoter, gene of interest and terminator. We added them to our genes via Polymerase Chain Reaction (PCR) to generate our level 0 plasmids.
Those form the basis on which we build multiple parts and vectors that can be easily assembled in the correct order. To
verify our design, we cloned *in silico* using SnapGene software to simulate composition models according to the
actual process in the laboratory.

### Test

GC-MS analysis was chosen as a suitable assay to evaluate our metabolically modified yeast strains. Corresponding
yeasts were cultured to achieve high cell densities, followed by extraction of α-pinene to obtain a high
concentration. Using previously assigned internal standards for GC-MS measurements, we quantified final α-pinene
levels and thus tested our strains.

### Learn

After obtaining our GC-MS results, we compared our yeast strains equipped with different heterologous genes for
α-pinene production. When testing our previous hypothesis, we realized that this was not consistently met. To draw
actual conclusions from strain comparison, we need to ensure uniform conditions in their generation. At the metabolic
level, this implies that α-pinene synthesis proceeds via the same precursors. Based on this, we can use our
self-developed software in future approaches to specifically identify additional metabolic targets for further
optimization [[model](https://2022.igem.wiki/wwu-muenster/model)].

## Fusion of afraGFP with PTS1 for localization in peroxisomes

For verification of the general functionality of the C-terminal SKL peroxisomal targeting signal-1 (PTS1) described
by Dusséaux et al., (2020), *Agaricia fragilis*-derived afra green fluorescent protein (afraGFP) originated from
the iGEM distribution (BBF10K\_003348) was fused with a C-terminal PTS1 tag according to our [general cloning strategy](https://2022.igem.wiki/wwu-muenster/results#cloning_strategy). The *ScpTEF* promoter was selected for strong gene expression. As a
control, an afraGFP shuttle construct lacking a C-terminal PTS1 tag was designed to accumulate in the cytosol.
Subsequent yeast transformation was performed utilizing the shuttle vectors that resulted from our partnership with
the iGEM Team TU Dresden. The fluorescence microscopy of transformed yeast cells was carried out in cooperation with
the research group of Dr. Maria Bohnert at the Institute of Cell Dynamics and Imaging.

Yeast cultures were inoculated with overnight cultures incubated shaking at 30 °C for two hours prior to microscopy.
To adhere the cells, the wells of an 8-well glass bottom were primed with concanavalin A. 50 µl were added to each
well and discarded after 10 minutes of incubation. Subsequently, 100 µl of cell suspension were added and removed after
a 10-minute adhesion time. Cells were covered with 600 µl of fresh Sc-all medium. Imaging was performed using a Nikon
N-SIM microscope with a 100x objective and a CMOS camera. A wavelength of 488 nm was used for excitation and emission
spectra was captured at 510 nm with a bandwidth of 15 nm. Exposure time was set to 100 ms and laser intensity to 2%.

Control cells expressing the afraGFP without the PTS1 tag showed bright, diffuse fluorescence throughout the cell,
indicating cytosolic GFP localization (Fig. 2, A). In contrast, the introduction of [*afraGFP\_PTS1*](http://parts.igem.org/Part%3ABBa_K4188006) resulted in localization of fluorescence
signal in distinct bright spots, indicating functional targeting to peroxisomes (Fig. 1, B), consistent with Dusséaux
et al., (2020). Moreover, microscopy of *S. cerevisiae* wild-type CEN.PK2-1C as a negative control displayed no
fluorescence signal at all. Hence, we could demonstrate proper function of our shuttle vectors and the PST1 tag
resulting in strong expression and targeted translocation. Therefore, we included the PTS1 tag accordingly into our
constructs for targeted monoterpenoid production in yeast peroxisomes.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/partnership-4-fig-2.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/partnership-4-fig-2.png)

**Figure 2: Fluorescence microscopy of *S. cerevisiae* cells exhibiting strong afraGFP signal in (A)
cytosol and (B) peroxisomes.**  100 µl of a 1:5 diluted yeast suspension derived from cultures incubated for two
hours were added to a well plate primed with concanavalin A. After 10 min of incubation, the yeast suspension was
replaced with Sc-all medium and the cells were imaged using a Nikon N-SIM microscope with an exposure time of 100 ms
at 2% laser intensity. AfraGFP fluorescence was excited at 488 nm and emission was detected at 510 ± 15 nm.
Subsequent image editing in **(B)** allowed for better visibility of the cell background. Scale bar 5 µm.

## Engineering success of DNPH assay

We developed a colorimetric assay as a part of mutational screening for protein engineering of a cytochrome P450
monooxygenase (BM3). This rapid mutation screening method for the detection of verbenone is based on the reaction of
2,4-dinitrophenylhydrazine (DNPH) with enones like verbenone that leads to a red complex formation of a hydrazone
(Fig. 3). These can be examined visually or quantified by photometric measurements in a 96-well plate (Liu et al.,
2016). To avoid extraction of verbenone from the cells by lysis, we used the autodisplay system (Jose et al., 2012).
As the BM3 is thus presented on the surface of *Escherichia coli* cells, catalysis of α-pinene to verbenone takes
place in the medium. During the process, we encountered several challenges and constantly improved our assay to
properly quantify our produced verbenone. Among others, we adapted the composition of the DNPH reagent to form a
soluble mixture and avoid red precipitates that interfere with the measurement. In total, we went through three
Design-build-test-learn (DBTL) cycles (Fig. 4) and further development should tackle the interference of nicotinamide adenine dinucleotide phosphate (NADPH) in
photometric detection. In summary, we successfully showed that the assay is suitable for verbenone quantification and
could be used as a tool for improving the BM3 through mutational screening if the conversion ratio of verbenone is
exceeding 10%.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp6-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp6-fh.png)

**Figure 3: Schematic representation of the chemical reaction of 2,4-dinitrophenylhydrazine (DNPH) with
verbenone.** The two substances react in an acidic environment to form a reddish colored phenylhydrazone
precipitate.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/engineering-success-dnph-assay-fig-3-ai-ml.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/engineering-success-dnph-assay-fig-3-ai-ml.png)

**Figure 4: Design-build-test-learn cycles for the establishment of the 2,4-dinitrophenylhydrazine
(DNPH) assay.**

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[Experiments](https://2022.igem.wiki/wwu-muenster/experiments)
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## References

Dusséaux, S., Wajn, W. T., Liu, Y., Ignea, C., & Kampranis, S. C. (2020). Transforming yeast peroxisomes into
microfactories for the efficient production of high-value isoprenoids. Proceedings of the National Academy of
Sciences of the United States of America, 117(50), 31789–31799. https://doi.org/10.1073/pnas.2013968117

Jose, J., Maas, R. M. and Teese, M. G. (2012) ‘Autodisplay of enzymes--molecular basis and perspectives’, Journal
of biotechnology, vol. 161, no. 2, pp. 92–103.

Liu, Y., Cao, F., Xiong, H., Shen, Y. and Wang, M. (2016) ‘Application of 2,4-Dinitrophenylhydrazine (DNPH) in
High-Throughput Screening for Microorganism Mutants Accumulating 9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD)’,
PloS one, vol. 11, no. 10, e0163836.

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[##### {{ self.title() }}](#summary)

[Cloning Strategy](#cloning_strategy)
[Microbial α-pinene production](#sccytosol)
[in *Saccharomyces cerevisiae* cytosol](#sccytosol)
[in *Saccharomyces cerevisiae* peroxisome](#section-4)
[in *Yarrowia lipolytica* cytosol](#section-5)
[in *Yarrowia lipolytica* peroxisome](#section-6)
[Proposed MoClo compatible vectors for faciliated engineering of *Yarrowia lipolytica*](#section-7)
[E-driven CYP](#section-8)
[Conclusion & Outlook](#section-9)

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In the wet lab, we divided into several subgroups to achieve intermediate objectives in yeast metabolic engineering
and our *in vitro* system in parallel. Cytosol-targeted *Saccharomyces cerevisiae* strain generation aimed
at an α-pinene production of about 4.1 mg/l. In addition, metabolic engineering of yeast peroxisomes
yielded about 3.6 mg/l α-pinene and 12.8 mg/l verbenone. Despite the successful production in *S. cerevisiae*, the metabolic engineering of the less established *Yarrowia lipolytica*, and the targeted synthesis of
precursors was not successful. We have developed the BioReactor for Enzymatic ElectroSynthesis (BREES) for the
enzymatic electrocatalysis of yeast-derived α-pinene which enables efficient and cost-effective
verbenone synthesis that can be used as a platform system to produce numerous monoterpenoids. For further improvement,
we have also developed a colorimetric assay for high throughput screening of enzyme variants. Our overall concept and
key results pave the way for a possible industrial scale-up for pioneering steps towards bioeconomy.

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## Cloning Strategy

Given our metabolic engineering approach in yeast, a well-designed cloning strategy was indispensable. Following the official iGEM rules, general cloning was performed according to the RFC1000 modular cloning system (MoClo) (Fig. G1). For all required gene constructs and plasmids, this was accomplished in *Escherichia coli* Turbo. Initial genes, as well as plasmids, originated from internal research groups of our institute, from this year’s iGEM teams from Dresden and Bochum or have been provided by Integrated DNA Technologies IDT. In addition, some were obtained from the official iGEM distribution and the Free Genes Project.

The MoClo system relies on IIS-type restriction enzymes to specifically cleave DNA beyond its recognition site, resulting in 5′ or 3′ overhangs. Generation of diverse overhangs allows efficient assembly of multiple DNA fragments in one simple ligation. A suitable overhang design ensures fragments only to be assembled in a predefined order (Engler et al., 2008). This gave us the great advantage of designing standardized overhangs to establish individual building blocks. In this way, we have established a library of genetic parts that can be easily arranged as desired, making them accessible to future iGEM teams.

All genes have been amplified by initial Polymerase Chain Reaction (PCR) using specific primers with the appropriate overhangs attached. For promoter sequences, the forward primer (fw) contained TACAGCTCTTCATCG and fusion site GGAG. The reverse primer (rv) included the 3′ fusion site AATG and the overhang CGAGTGAAGAGCCGAT leading to a final construct without 5′ UTRs. All coding sequences were equipped with the 5′ overhang GACGGCTCTTCATCG and the fusion site AATG, and the 3′ fusion site GCTT and the overhang CGAGTGAAGAGCATAC. Finally, all terminator sequences featured 5′ overhang TGCAGCTCTTCATCG and fusion site GCTT and 3′ fusion site GGCT and the overhang CGAGTGAAGAGCATAA.

Since metabolic engineering of certain metabolic pathways targeted the peroxisome, proper localization within cells had to be considered when cloning the corresponding genes. To achieve this, we added a tag comprising nine specific base pairs to the overhang of the matching rv primer. According to Dusséaux et al., 2020, the C-terminal SKL peroxisomal targeting signal-1 (PTS1) tag consisted of TCCAAGCTG encoding serine, lysine, and leucine, leading to reliable peroxisome localization.

After attaching the specific overhangs to all genes, Golden Gate Assembly into iGEM-derived pSB1C00 backbone resulted in the level 0 parts. These served to design the level 1 constructs where a promoter, coding sequence, and terminator formed single transcription units (TU). We achieved varying levels of gene expression by using different promoters. It was important to choose between *pSB1K01*-04 as a backbone in view of the later level 2 composition. Up to four level 1 constructs can be assembled into the *pSB3C01* backbone as a final level 2 construct. For each position, a spacer was designed in case less than four TUs are to be assembled. All plasmid backbones carried a constitutively monomeric red fluorescent protein (*mRFP1*) to allow selection of transformed colonies ensuring successful integration of the desired gene construct.

Yeast transformation was to be accomplished either by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-assotiated protein 9 (CRISPR-Cas9)-mediated stable genome integration or by using extrachromosomal shuttle vectors. In addition, four different level 2 backbones *pSB3C01* were each equipped with His, Leu, Trp, or Ura auxotrophies (*pSB3CY-TRP1/LEU2/URA3/HIS3*) to allow for subsequent selection. In addition, *pSB1KY-TRP1/LEU2/URA3/HIS3* were used as level 1 shuttle vectors. We developed an alternative backbone using cyan fluorescent protein (CFP), *pSB3CY-CFP* as a second selection marker instead of auxotrophy. For uniform gene expression, all shuttle vectors contained the yeast 2µ ori, which in contrast to the single copy integration, leads to a high-copy number.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/results-g1-lm.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/results-g1-lm.png)

**Figure G1: Schematic summary of our RFC1000 MoClo strategy.** Since we cloned over 50 individual gene constructs, we aimed to keep the process as simple and convenient as possible. As part of the iGEM regulations, we used the MoClo system to build a library of gene constructs with uniform overhangs allowing them to be easily assembled in a single ligation step. Individual CDSs were assembled into level 0 backbones, followed by the assembly of level 1 TUs with corresponding promoters and terminators. In accordance with the level 2 design, one of each of four different level 1 backbones was selected. Subsequent yeast transformation was performed either by genome insertion with the CRISPR-Cas9 system or with level 2 shuttle vectors. **Abbreviations:** CDS = coding sequence; PCR = Polymerase Chain Reaction; HF = homology flanks; MTUs = multiple transcription units.

---

## Cytosolic production of α-pinene in *Saccharomyces cerevisiae*

#### Summary

To create a chassis organism that produces the monoterpenoid α-pinene, we engineered the
mevalonate pathway of the model organism *Saccharomyces cerevisiae*. We used the MoClo
strategy for efficient engineering. In total we cloned 27 different constructs using Golden Gate Assembly.
Thereby, we inserted heterologous genes like *PptAPS* and upregulated endogenous genes like *ScERG13* or
*SctHMGR* in *S. cerevisiae* with a strong promoter - *ScpTEF*. Further, we tried to knock out two genes which are the bottleneck of
the mevalonate pathway (MVA) using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated
protein 9 (CRISPR-Cas9). Overall, we created two yeast strains that produce the monoterpenoid α-pinene
 in concentrations up to 4.1 mg/l.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/pathway-cytosol-scerevisiae-ks.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/pathway-cytosol-scerevisiae-ks.png)

**Figure Scc 1: Engineered mevalonate pathway in the cytosol of *S. cerevisiae*.** The MVA was engineered at different positions. *ScERG13*, *SctHMGR*, *AgtGPPS*2,
*GgmFPS144*, *SltNPPS*1 and *PptAPS* were upregulated by *ScpTEF*. Simultaneously,
*ScROX1* and *ScERG20* were either knocked out, or downregulated by *ScpCTR3*.

#### Brief project description

The lab group *S. cerevisiae* cytosol engineered the MVA that takes place in the cytosol of the baker’s yeast. The integration of all genes was plasmid based. For genomic knockouts we used a CRISPR-Cas9 system. Thereby, we engineered a synthetic pathway with a final product: α-pinene. By silencing *ScROX1* and upregulating *ScERG13* as well as *SctHMGR* with a strong promoter, we planned to increase the mevalonate concentration and thus the concentration of subsequent intermediate substrates of the pathway. The knockout of *ScROX1* was favourable for increasing the mevalonate concentration since it inhibits enzymes in the MVA (Bröker et al., 2018). Further, we introduced the heterologous genes *GgmFPS144* from *Gallus gallus* and *AgtGPPS*2 from *Abies grandis* to increase the concentration of geranyl diphosphate (GPP), a precursor molecule of all monoterpenoids. By knocking out *ScERG20* and simultaneously inserting *GgmFPS144* we tried to inhibit but not completely imped the vital reaction of GPP to farnesyl diphosphate (FPP) (Brown et al., 2015). Lastly, we introduced the heterologous α-pinene synthase (*PptAPS*) from *Pinus ponderosa* to facilitate the production of α-pinene within the cytosol of *S. cerevisiae* (Fig. Scc 1). A schematic representation of the transformed level 2 constructs is displayed in Figure Scc 2.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/level2-module-scerevisiae-cytosol.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/level2-module-scerevisiae-cytosol.png)

**Figure Scc 2: Schematic abstract of level 2 constructs that were ligated into shuttle
vectors and thus transformed into *S. cerevisiae*.** **(A)**
level 2 construct consisting of three TUs and one spacer. TUs include a
promoter (*ScpTEF*), a gene of interest and a terminator (*ScCYC1t*). **(B)**
level 2 construct consisting of two transcription units and two spacers. **Abbreviations**: P =
promoter; T = terminator; S = spacer.

#### Results

As a strategy for introducing our genes of interest into *S. cerevisiae*, we used modular cloning. We successfully cloned all level 0, level 1 and level 2 plasmids that were needed, as well as three level 2 shuttle vectors with [Golden Gate](#cloning_strategy) cloning. We used a mutated *ScpTEF* as a strong promoter. Since the promoter naturally contains a *Bsa*I restriction site and we were using *Bsa*I restriction enzymes for restriction of our level 1 constructs, we introduced a mutation. Hereinafter the mutated promoter is called *ScpTEF*. The correct cloning of level 0 and level 1 constructs was confirmed by transforming them into *Escherichia coli* using antibiotic selection, colony Polymerase Chain Reaction (cPCR) (Fig. Scc 3) and sequencing. The finished level 1 plasmids were used to clone level 2 constructs. Additionally, successful transformation of our level 2 constructs into *E. coli* was confirmed by selective media.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/gelbildlevel1.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/gelbildlevel1.png)

**Figure Scc 3: Successfully cloned level 1 constructs.** The level 1
constructs for the genes *SltNPPS*1 (1), *GgmFPS144* (2), *AgtGPPS*2 (3), *ScERG13* (4),
*SctHMGR* (5), *ApL3H* (6) and *PptAPS* (7) were cloned successfully using Golden Gate Assembly
and can be seen in the agarose gel. While the band for *PptAPS* looks to be smaller than the expected 1767
bp, *SltNPPS* (1404 bp), *GgmFPS144* (1724 bp), *ScERG13* (2097 bp), *SctHMGR* (2130 bp),
*ApL3H* (2206 bp) and *PptAPS* (2364 bp) show bands with the correct sizes.

The genomic knockout of *ScROX1* was carried out using CRISPR-Cas9. We tried to replace *ScROX1* with
*ScERG13* and *SctHMGR* to create *S. cerevisiae*\_MS\_1. Thus, the upregulated transcription of *ScERG13* and *SctHMGR* led to an increase of
subsequent metabolites which further results in an increased GPP concentration (Bröker et al., 2018). We planned
two different strains to further optimize the metabolic flux towards GPP and finally towards α-pinene
.

In the first approach, we used *S. cerevisiae*\_MS\_1 and tried to knock out *ScERG20* by using CRISPR-Cas9. Simultaneously, we wanted to replace this
gene with two heterologous genes: *AgtGPPS*2 and *GgmFPS144*. Both catalyze the reaction to GPP. The
latter, also catalyzes the reaction of GPP to FPP and therefore ergosterol synthesis would still occur, enabling
the yeast to endure. In a second approach, we planned to replace *ScpERG20* with a copper dependent promoter
(*pCTR3*) and insert *SltNPPS*1. This would have resulted in a downregulated FPP synthesis as well as in
an upregulated NPP production (Brown et al., 2015; Ignea et al., 2019).

The genomic targets within *ScROX1*, *ScERG20* and *ScpERG20* were predicted using online tool [CRISPOR](http://crispor.tefor.net/). Like Laughery et al., (2015) we used the
**pML104** plasmid containing Cas9, AmpR and URA3. We restricted and ligated the sgRNA for
*ScROX1*, *ScERG20* and *ScpERG20* into **pML104** and designed homology flanks for these
genes to fuse them to the level 2 constructs which held the transcription units we wanted to insert
(Fig. Scc 4).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/homology-flanks-sccytosol-he.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/homology-flanks-sccytosol-he.png)

**Figure Scc 4: Schematic abstract of transcription units with homology flanks to serve as repair
templates during CRISPR-Cas9.** **(A)** Template to be inserted into the genomic DNA for *ScROX1*.
**(B)**  Template to be inserted into the genomic DNA for *ScERG20*. **(C)**  Template to be inserted
into the genomic DNA for *ScpERG20*. **Abbreviations:** HF = homology flank; P = promoter; T =
terminator; S = spacer.

However, we were not able to amplify these flanked constructs. While the reason for this hindrance remains
unknown, we decided to introduce a new strategy to stay within our schedule. Instead of inserting our constructs
into the genomic DNA, we cloned the constructs into [shuttle vectors with different auxotrophies](https://2022.igem.wiki/wwu-muenster/contribution#section-1). However, this strategy is not
applicable for the genomic integration of the promoter *ScpCTR3* upstream of *ScERG20*. Due to time
limitation, we had to relinquish the plan to produce NPP.

Another part of our project that had to be left out is the introduction of the limonene-3-hydroxylase
(*ApL3H*), an enzyme which catalyzes the reaction of α-pinene to verbenone. Again, the fusion
of homology flanks to the level 2 construct failed. As we had to change our transformation strategy,
we were limited by the number of auxotrophy markers. Since there was no auxotrophy marker left to select for the
*ApL3H*, this idea had to be abandoned.

Nonetheless, we used CRISPR-Cas9 to insert a disruption into *ScROX1* and *ScERG20*. Simultaneously, we
transformed shuttle vectors that held transcription units of our genes of interest. The transformation process
resulted in three different yeast strains (Tab. Scc 1). However, due to time limitation it was not possible to
verify the transformation success with cPCR. Further, verification of a successful frameshift was unachievable. We
therefore solely relied on the yeast’s growth on selective SD media as well as on SD medium containing
5-Fluoroorotic acid.

**Table Scc 1: Engineered yeast strains.**Three different yeast strains were generated. In *S. cerevisiae*\_MS\_1 we tried to knockout *ScROX1* while *ScERG13*, *SctHMGR* and *PptAPS* were
inserted. *S. cerevisiae*\_MS\_2 and *S. cerevisiae*\_MS\_3 lack *ScROX1* and *ScERG20* while *AgtGPPS*2, *mFPS144*, *ScERG13*,
*SctHMGR* and *PptAPS.*

![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/tabelle-strains.svg)

For quantification of α-pinene GC-MS was performed. The engineered strains were inoculated for 48
hours at 30 °C in liquid minimal medium, followed by a subsequent lysis and extraction with ethyl acetate for [analysis](https://2022.igem.wiki/wwu-muenster/experiments). A standard curve of α-pinene covering a concentration range of 0.2 mg/l to 200 mg/l containing eugenol and para-xylene
(p-xylene) as internal standards was recorded. The chromatogram exhibit elution time of α-pinene
at 6.5 minutes, para-xylene at 5.3 minutes, and eugenol at 12.0 minutes.

We expected α-pinene production for *S. cerevisiae*\_MS\_1 since we inserted *PptAPS*. However, GC-MS measurement resulted in a chromatogram, in which α-pinene peak was detectable. The internal standards eugenol and p-xylene eluted at expected times.
We assume the extraction of α-pinene did not work properly since we used an extraction protocol that included cell
lysis with one bigger metal bead instead of a mix of many small glass beads.

Nonetheless, Figure Scc 5 shows the chromatograms and mass spectra of *S. cerevisiae*\_MS\_2 and *S. cerevisiae*\_MS\_3. In the chromatograms, the retention time in minutes is plotted against the absolute intensity. The mass
spectra show relative intensity against m/z. Both gas chromatograms display a clear peak at a retention time of
6.5 minutes. These were verified by a distinct α-pinene pattern within the mass spectra.
Quantification of α-pinene resulted in concentrations of 4.1 mg/l produced from *S. cerevisiae*\_MS\_2 and 3.5 mg/l produced from *S. cerevisiae*\_MS\_3. Further, *S. cerevisiae* CEN.PK2-1C was included as a negative control. No peak can be observed in the chromatogram at 6.5 minutes.
Therefore, we conclude no α-pinene production in this strain. Based on the GC-MS results, we
verified the functionality of the heterologous α-pinene synthase. Furthermore, we verified the
functionality of our self-designed and build [shuttle vectors](https://2022.igem.wiki/wwu-muenster/contribution#section-1).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/gc-ms-chromatogram-massspectrum-jpg.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-cytosol/gc-ms-chromatogram-massspectrum-jpg.png)

**Figure Scc 5: Results of GC-MS measurements.** Eugenol and para-xylene were used as universal
internal standards and elute at 12 minutes and 5.3 minutes. **(A)** Chromatogram of extracted sample gained
from strain *S. cerevisiae*\_MS\_2. α-pinene elutes at 6.5 minutes (marked with orange arrow). **(B)** Chromatogram of
extracted sample gained from strain *S. cerevisiae*\_MS\_3. α-pinene elutes at 6.5 minutes (marked with orange arrow). **(C)** Chromatogram
with comparative representation of α-pinene producing strain *S. cerevisiae*\_MS\_2 and *S. cerevisiae* CEN.PK2-1C. **(D)** Chromatogram with comparative representation of α-pinene producing
strain *S. cerevisiae*\_MS\_3 and *S. cerevisiae* CEN.PK2-1C. **(E)** Exemplary mass spectrum of α-pinene. **Abbreviations:** GC = gas
chromatography; MS = mass spectrometry.

---

## Peroxisomal production of α-pinene in *Saccharomyces cerevisiae*

#### Summary

This part of MonChassis comprised metabolic engineering to target the mevalonate metabolism of *S. cerevisiae* and the monoterpene synthesis to the yeast peroxisomes. Our main objective aimed to microbially produce the monoterpenoid precursor α-pinene in yeast. To achieve this goal, we cloned more than 25 level 0, 1, and 2 constructs and designed multiple yeast expression vectors using Golden Gate Assembly. Yeast expression vectors contained heterologous genes and upregulated endogenous genes equipped with a C-terminal SKL PTS1. Final GC-MS analysis revealed that our generated yeast strain *S. cerevisiae*\_MS\_8 produced about 3.6 mg/l α-pinene and *S. cerevisiae*\_MS\_9 with additional ApL3H 12.8 mg/l verbenone.

#### Brief project description

The budding yeast *Saccharomyces cerevisiae* is undoubtedly one of the most established model organisms in
applied biotechnology (Ostergaard et al., 2000). Extensive knowledge of cultivation, genomic modification
techniques, and downstream processing enable a convenient experimental design. In addition to the cytosol, the
peroxisome became a promising target for metabolic engineering, as its physiological properties, such as substrate
availability, were thought to significantly enhance product synthesis (Dusséaux et al., 2020). Thus, metabolic
engineering targeting the peroxisome comprised cloning of plasmids to drive heterologous gene expression. We used
yeast expression vectors to introduce numerous genes into *S. cerevisiae*. For targeted peroxisome localization in yeast cells, we equipped all our genes of interest with the
C-terminal SKL PTS1 previously established by Dusséaux et al., 2020. For the verification of functional
peroxisomal tagging, we additionally fused a green fluorescent protein (GFP) to PTS1 and performed [fluorescence microscopy](https://2022.igem.wiki/wwu-muenster/engineering#section-2) of
corresponding yeast cells.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/pathway-peroxisome-scerevisiae-ks.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/pathway-peroxisome-scerevisiae-ks.png)

**Figure Scp 1: Schematic overview of the heterologous metabolic pathway introduced into the
peroxisome along the genes involved in α-pinene synthesis.** *ScERG13*, *SctHMGR*,
as well as *SItNPPS1*, *AgtGPPS2*, *GgmFPS144*, and *PptAPS* were upregulated by the strong
constitutive promoter *ScpTEF*.

Since mevalonate metabolism does not occur naturally in peroxisomes (Zhang et al., 2020), we incorporated the
required PTS1-fused genes *ScERG13*, *SctHMGR*, *ScERG12*, *ScERG8*, and *ScERG19* into
each yeast strain under the control of the constitutive *pADH* promoter for uniform gene expression (Fig. Scp
1). In a first approach, we additionally introduced the synthase SltNPPS1 from tomato which resulted in the
production of the monoterpenoid precursor neryl diphosphate (NPP). In a second approach, we further inserted the
heterologous genes *GgmFPS144* and *AgtGPPS2* from *Gallus* *gallus* and *Abies*
*grandis*, respectively, which led to the generation of the NPP structural isomer geranyl diphosphate (GPP).
For both approaches, we finally introduced *PptAPS* which catalyzes the reaction of NPP or GPP to α-pinene. Two specific genes are involved in the bottleneck phase of the mevalonate pathway,
*ScERG13*, and *SctHMGR*, that are considered rate-determining enzymes in synthesis. Therefore, we
upregulated gene expression using *ScpTEF* instead of *ScpADH*, which was expected to result in
increased product yields due to stronger promoter activity of *ScpTEF*. The same promoter sequences were used
in the [other yeast part](#sccytosol) of the project. *ScpTEF* promoter has also
been used to control expression of *PptAPS*, *SltNPPS1*, *GgmFPS144*, and *AgtGPPS2*. For the
comparison of the cell-free production of verbenone in our *in vitro* approach, we also generated a yeast
strain containing cytochrome P450 monooxygenase (CYP) limonene-3-hydroxylase (*L3H*) in addition to GPP and
NPP production. Thereby, we were able to obtain microbially-derived verbenone in addition to the
electrically-driven CYP product. The heterologously produced monoterpenoids were quantified by gas
chromatography-mass spectrometry (GC-MS).

Within the partnership of MonChassis and the [iGEM Team TU Dresden](https://2022.igem.wiki/tu-dresden/), we attempted to produce therapeutical thymol
for the treatment of chronic wounds (Costa et al., 2018). This project required the insertion of additional genes
including *TvtGTPS1*, *TvCYP71D179*, and *TvSDR1* to synthesize [thymol](https://2022.igem.wiki/wwu-muenster/partnership#section-2). This monoterpenoid is also obtained by
three-step catalysis of GPP (Krause et al., 2021).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/level2-module-scerevisiae-peroxisom.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/level2-module-scerevisiae-peroxisom.png)

**Figure Scp 2: Schematic abstract of level 2 constructs that were transformed into *S. cerevisiae***. level 2 constructs consist of two to four transcription units (TU) and spacer (grey).
The TUs include a promoter (*ScpADH* = dark green, *ScpTEF* = light green), the gene of interest
(yellow), and a terminator (*CYC1t* = dark blue). **(A)** shows the mevalonate module, **(B)** the
downstream module, and **(C)** NPP module. **Abbreviations:** P = promoter; T = terminator; TU = targeting units S = spacer.

#### Results

For targeted metabolic engineering of yeast peroxisomes, the corresponding gene constructs were initially [cloned](#cloning_strategy) up to level 2. Each construct was transformed into *E. coli* and confirmed by antibiotic selection, cPCR, and all parts of levels 0 and 1 were additionally sequenced. Final level 2 shuttle vectors were used for transformation to achieve a high plasmid copy number in the resulting yeast. The arrangement was designed to functionally combine multiple genes into a single module (Fig. Scp 2). First, *ScERG10*, *ScERG13*, and *SctHMGR* composed the mevalonate module. Second, *ScERG8*, *ScERG12*, *ScERG19*, and *ScIDI1* formed the downstream module, and finally, *PptAPS* and *SItNPPS1* built the NPP module (Tab. Scp1). In planning our experiments, we considered using the genome editing system CRISPR-Cas9 to integrate the required genes into the *S. cerevisiae* genome. Compared to plasmid-based systems, genomic integration is usually more reliable in terms of gene copy number and stability (Reider Apel et al., 2017). Due to time limitations, the stable integration was not implemented and is pending for future projects.

**Table Scp 1: Successfully generated yeast strains of *S. cerevisiae* with metabolically engineered peroxisomes.** Listed yeast strains were modified by gene insertion via
shuttle vectors to produce NPP and verbenone. **Abbreviations:** afraGFP = *Agaricia fragilis*-derived
green fluorescent protein;
NPP = neryl diphosphate; PTS1 = peroxisomal targeting signal-1; *ApL3H* = limonene-3-hydroxylase.

![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/table-scp1-vk.svg)

Some of the yeast strains that we planned to deliver in MonChassis could not be implemented due to difficulties encountered during the cloning process and limited time. Although the establishment of the yeast strain designed to produce GPP-derived α-pinene is still pending, we succeeded in cloning the level 0 construct *AgtGPPS2* including PTS1. For the thymol-producing yeast strain a correct level 2 shuttle construct was obtained (Fig. Scp 3).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/lv0gpps-lv2thymol-vk.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/lv0gpps-lv2thymol-vk.png)

**Figure Scp 3: cPCR of (A) level 0 *AgtGPPS2* and (B) level 2 shuttle construct of thymol module.** Amplified gene constructs were examined by gel electrophoresis to ensure correct construct assembly. **(A)** Samples from colonies 1-5 were used as a template for cPCR and loaded onto the gel, expecting amplified fragments of 901 bp. Amplification was achieved by GoTaq polymerase. **(B)** amplified level 2 thymol module 1 = in *pSB3C01* and 2 = in *pSB3CY-URA3* both expected at 6047 bp. Ladder: Thermo Scientific GeneRuler 1kb. **Abbreviations:** cPCR = colony polymerase chain reaction; kb = kilobase;
bp = base pair.

In all five samples, we succeeded in performing a correct level 0 assembly of *AgtGPPS2* into *pSB1C00* including the C-terminal PTS1 fusion, which consists of approximately 900 bp. Additional fragments of varying sizes that were apparent in the Midori Green-stained agarose gels were probably synthesized due to nonspecific primer binding or represent the template fragments. cPCR of the level 2 thymol module targeted 6 kb which was partially confirmed by gel electrophoresis. However, the corresponding bands showed rather low intensity, whereas off-target fragments were more prominent. Consequently, successfully cloned vectors can be used in further experiments after the iGEM period to obtain transformed yeasts for α-pinene production. All generated yeast strains from Table Scp 1 were analyzed by cPCR (Fig. Scp 4) by amplifying a region within the inserted construct using specific primer combinations.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/yeastcpcr-vk.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/yeastcpcr-vk.png)

**Figure Scp 4: Yeast cPCR of positively transformed *S. cerevisiae* equipped with corresponding shuttle vectors.** **(A)** Yeasts transformed with single downstream
modules (1-3) and NPP modules (4-6) and **(B)** yeasts with all three mevalonate, downstream (1-3), and NPP
(4-6) modules were analyzed for successful insertion. Amplification of 1107 bp was expected for the downstream
module and 828 bp for the NPP module. Ladder: Thermo Scientific GeneRuler 1kb. **Abbreviations:** cPCR =
colony polymerase chain reaction; kb = kilobase; bp = base pair, NPP = neryl diphosphate.

Suitable primer combinations were selected to confirm both plasmid and gene insertion. Together with the
auxotrophy selection markers, we assumed that we had successfully introduced these constructs into the yeast
strains. In contrast, we could not successfully verify the mevalonate module because it was not reliably
amplified. Further experiments would require confirmation of the construct by repeated cPCR. Since the generated
yeast strain *S. cerevisiae*\_MS\_9 possessed an additional ApL3H, confirmation of the latter was also necessary. However, this could not be
done due to time constraints within the iGEM regulations.

The final evaluation of our genetically modified yeast strains was performed by quantification using GC-MS
analysis (Fig. Scp 5). For this, α-pinene and verbenone have been considered important
monoterpenoids to infer synthesis efficiency. Yeast strains *S. cerevisiae*\_MS\_8 and *S. cerevisiae*\_MS\_9 were grown in liquid culture for 48 hours and then extracted with ethyl acetate and small glass beads
for [analysis](https://2022.igem.wiki/wwu-muenster/experiments). P-xylene for α-pinene and eugenol for verbenone and verbenol were chosen as internal standards. For comparison, the
*S. cerevisiae* CEN.PK2-1C wild type was also included.

The GC chromatogram shows the absolute intensities of detected peaks along their specific retention time. The
mass spectra show relative intensity against m/z. The internal standards p-xylene and eugenol displayed peaks at
approximately 5 and 12.0 minutes, respectively (Fig. Scp 5 A). Obtained results of *S. cerevisiae*\_MS\_8 demonstrated the requested α-pinene expected to eluate at 6.5 minutes. A review of the MS
spectrum reveals a distinct pattern that indeed confirmed α-pinene, subsequently quantified at 3.6
mg/l. GC output of *S. cerevisiae* wildtype did not show a targeted α-pinene peak, indicating that no production occurred (Fig.
Scp 5 B). Secondly, *S. cerevisiae*\_MS\_9 yielded in 12.8 mg/l verbenone eluting at 10.8 minutes indicating proper function of inserted ApL3H
catalyzing α-pinene-derived verbenone that is additionally confirmed by the MS spectrum (Fig. Scp 5 D). In addition to catalytic conversion, autooxidation of α-pinene to verbenone was also likely in
line with previous studies (Moore et al., 1956). The peaks for α-pinene and verbenone showed identical retention
and fragmentation patterns as the previously measured authentic standards (Fig. Scp 5 C). Consistent with our
prediction, both strains indeed appear to exhibit targeted synthesis, supporting our original approach of
microbially produced high-value precursors.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/gc-ms-data-scp-vk.svg)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/sc-peroxisome/gc-ms-data-scp-vk.svg)

**Figure Scp 5: GC-MS measurements of yeast strains comprising peroxisome-targeted metabolic
engineering aimed for NPP-derived α-pinene synthesis.** **(A)** GC output of *S. cerevisiae*\_MS\_8 including mevalonate, downstream, and NPP module (Tab. Scp1) verifying α-pinene
synthesis which eluted at 6.5 minutes (marked with red arrow). **(B)** Comparison of yeast strain *S. cerevisiae*\_MS\_8 with *S. cerevisiae* CEN.PK2-1C wildtype in α-pinene production. **(C)** GC output of *S. cerevisiae*\_MS\_9 including additional ApL3H to mevalonate, downstream, and NPP module (Tab. Scp 1) displaying
α-pinene and verbenone which eluted at 10.8 minutes (marked with red arrow). **(D)** MS output
displayed a distinct pattern for α-pinene (top) and verbenone (bottom) which was in line with
prior expectations. P-xylene and eugenol were used as universal internal standards. **Abbreviations:** GC =
gas chromatography; MS = mass spectrometry; NPP = neryl diphosphate; L3H = limonene-3-hydroxylase.

## Cytosolic production of α-pinene in *Yarrowia lipolytica*

#### Summary

*Yarrowia lipolytica* is a promising organism for monoterpenoid synthesis, since it contains a high concentration of acetyl-CoA, is able to efficiently accumulate lipids, and shows high tolerance toward monoterpenoids (Ji et al., 2020, Wei et al., 2021). We aimed to introduce the *Saccharomyces cerevisiae* genes *SctHMGR* and *ScERG13* to eradicate the bottleneck of the mevalonate pathway. Additionally, we tried to introduce the *AgtGPPS2* and *GgMFPS144* genes into the *YlERG20* locus of *Y. lipolytica* to reduce the amount of produced farnesyl pyrophosphate (FPP) and increase the amount of geranyl pyrophosphate (GPP). In a second approach, we sought to introduce the *SltNPPS1* to produce neryl pyrophosphate (NPP), and insert the copper repressible promoter *YlpCTR1* upstream of the *YlERG20* gene to reduce the amount of FPP produced, decreasing competition for the reactants isopentenyl pyrophosphate and dimethylallyl pyrophosphate (IPP and DMAPP). Lastly, we wanted to introduce the *PptAPS* to produce α-pinene from NPP or GPP (Fig. Ylc 1).

To demonstrate the versatility of the NPP and GPP producing strains we also [planned on introducing genes responsible for the synthesis of thymol](https://2022.igem.wiki/wwu-muenster/partnership#section-2) (*TvGTPS1, TvCYP71D179, TvSDR1*)s an alternative to the α-pinene producing *PptAPS*.
We were able to assemble all (multi -) transcription units on level 1 and level 2 plasmids. The creation of linear fragments containing the (multi -) transcription units with fitting homology flanks up and downstream caused some difficulties. Therefore, the CRISPR-Cas9 mediated gene insertion or gene knockout in *Y. lipolytica* could not be achieved in the timeframe of MonChassis.
We are confident to have found some of the potential key problems concerning the gene insertion that can be tackled in the future.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-1.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-1.png)

**Figure Ylc 1: Proposed pathway engineering for optimized α-pinene production within *Y. lipolytica*** Green marks represent heterologous genes, which we aimed to introduce, orange marks represent the gene repression mediated by the *YlpCTR1* promoter, and red marks represent a gene knockout.

#### MoClo-based assembly of transcription units

We could create all designed level 1 plasmids. The correct assembly was confirmed via colony-PCR
(cPCR) (Fig. Ylc 2). Although white colonies, [that suggest successful gene
insertion](#cloning_strategy), were found on plates for the level 2 plasmid assemblies, cPCR appeared to be
negative. Later, we were able to amplify the entire multi-transcription unit (MTU) from the
*pSB3C01-Yl-SctHMGR-ScERG13* and *pSB3C01-Yl-AgtGPPS2-GgmFPS144* plasmids using primers binding to the
promoter and terminator, confirming the correct assembly of these two level 2 plasmids (Fig. Ylc 3).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-2.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-2.png)

**Figure Ylc 2: cPCR of level 1 plasmids.**
The agarose gels show PCRs to confirm the successful cloning of different level 1 plasmids. The
size after the name of the respective coding sequence refers to the expected size of the amplified fragment.
Thermo Scientific GeneRuler 1kb DNA-ladder was used. Kilobase pair (kb).
A: cPCR of level 1 plasmids 1: *PptAPS* (2339 bp), 2: *SltNPPS1* (1379 bp), 3:
*ApL3H* (2181 bp), 4: *AgtCS* (2303 bp)
B: cPCR of level 1 plasmids 1: *SctHMGR* (2105 bp) 2: *ScERG13* (2072 bp) 3:
*AgtGPPS2* (1742 bp) 4: *GgmFPS144* (1700 bp)
C: cPCR of level 1 plasmids 1 and 2: *TvCYP71D179* (2230 bp), 3 and 4: *TvGTPS1* (2368
bp), 5 and 6: *AgtCS* (2446 bp)
D: cPCR of level 1 plasmids 4 and 5: *TvSDR1* (1534 bp)

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-3.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-3.png)

**Figure Ylc 3: Amplification of level 1 and 2 (multi-)transcription units.**
The size after the name of the respective coding sequence refers to the expected size of the amplified fragment.
Thermo Scientific GeneRuler 1kb DNA-ladder was used. Kilobase pair (kb). 1: *ScERG13* (2072 bp) 2:
*ScERG13-SctHMGR* (4177 bp) 3: *AgtGPPS2* (1487) 5: *AgtGPPS2-GgmFPS144* (3187 bp)

#### CRISPR-Cas9-mediated gene insertion or knockout

While completing the cloning of our regular level 0, 1, and 2 plasmids, we started cloning our CRISPR plasmids. We decided on using the *pJME4472* plasmid (Larroude et al., 2020) since it contains the *URA3* gene as a selection marker. When positive transformants are plated on media containing 5-fluoroorotic acid (5-FOA), the yeasts eliminate the plasmid, since 5’FOA is converted into a toxic compound when the uracil synthase coded by *URA3* gene is present. This way, we can later introduce a different *pJME4472* plasmid for subsequent gene insertions or knockouts.

We introduced crisprRNAs (crRNAs), hybridized from oligonucleotides, into the *pJME4472* via Golden Gate Assembly using *BsmB*I, which excises a red fluorescent protein gene (*mRFP1*) from *pJME4472*. Thus, positive transformants are white, while *E. coli* colonies with the original plasmid turn red.

The genomic loci for the insertions were chosen based on Schwartz et al., 2011. *YlERG20* was also chosen as a locus since the *YlERG20* gene was a target for deletion or downregulation.

To improve the efficiency of homologous recombination, we aimed to knockout the *KU70* gene. The KU70 protein is part of the non-homologous end joining complex, which is the preferred repair method for DNA strand breaks in *Y. lipolytica* (Ji et al., 2020, Larroude et al., 2020). The genomic target sequences were chosen to be close to the starting sequence of the gene, and with high efficiency, specificity, and no off-targets based on [CRISPOR web software](http://crispor.tefor.net/).

Three different target sequences were selected for the *YlKU70* knockout, which displayed a high probability of a frameshift (Fig. Ylc 4). Successful cloning was confirmed via cPCR.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-4.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-4.png)

**Figure Ylc 4: Location of CRSPR-Cas9 targets within the *YlKU70 gene*.**  For the CRISPR-Cas9
mediated *YlKU70* knockout, three targets with high probability of a frameshift were chosen. After
transformation with the CRISPR-Cas9 vectors *pJME4472-YlKU70sgRNA1*, *pJME4472-YlKU70sgRNA2* and
*pJME4472-YlKU70sgRNA3*, the fragment between the oligonucleotides “ku70\_seq\_fwd” and “ku70\_seq\_rev”, was
amplified and sequenced.

After overcoming initial problems with the transformation method of *Y. lipolytica*, we were able to transform all three of the plasmids using an electroporation-based approach. A sequence of
777 bp was amplified from the genome, purified and sequenced, but no mutation was found.

#### Overlap-Extension PCR to obtain fragments for CRISPR-Cas9 mediated gene insertion

Gene insertion required the addition of 1 kb homology flanks (HFs) complementary to the desired gene locus
upstream and downstream of our transcription units (Fig. Ylc 5). The genomic loci were selected based on the work of
Schwartz et al., 2016.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-modules.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-modules.png)

**Figure Ylc 5: Schematic abstract of (multi-) transcription units with homology flanks for gene
insertion into *Y. lipolytica*.** Multi transcription units consists of two transcription units (TU). The TUs include the promoter
*YlpTEF* (dark red), the gene of interest (yellow), and a terminator (*CYC1t* = dark blue). The copper
repressible promoter YlpCTR1 is marked in light red. Homology flanks to the respectable *Y. lipolytica* gene are shown in dark grey. **(A)** The transcription units for *SctHMGR* and *ScERG13* with
HFs for the *YlAXP* gene. **(B)** The transcription units for *AgtGPPS1* and *GgmFPS144* with
HFs for the *YlERG20* gene, for gene insertion and simultaneous knockout of *YlERG20* **(C)** The
transcription unit for *SltNPPS1* with HFs for the *YlXPR2* gene. **(D)** The transcription unit
for *PptAPS* with HFs for the *YlA08* gene. **(E)** The transcription unit for *ApL3H* with
HFs for the YlD17 gene. **(F)** The copper repressible promoter *YlpCTR1* with HFs for insertion into
the promoter region of the *YlERG20* gene, to replace the endogenous *YlERG20* promoter.
Abbreviations: P = promoter; T = terminator.

We amplified the HF from *Y. lipolytica* gDNA using primers with a 20 bp overhang to the transcription units (Fig. Ylc 6). For further information on
our strategy, [see above](#cloning_strategy).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-5.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-5.png)

**Figure Ylc 6: Example of homology flank amplification from *Y. lipolytica* gDNA.**
All homology flanks had a size of around 1 kb. Thermo Scientific GeneRuler 1kb DNA-ladder was used. Kilobase pair (kb).
1: *AXP\_HF1* 2: *AXP\_HF2* 3: *XPR2\_HF1* 4: *XPR2\_HF2*

Simultaneously we amplified the genes and transcription units from different level 0, level 1 and, level 2 plasmids. Amplification of the *YlpCTR1*1 from the level 0 plasmid and of the transcription units from level 1 plasmids was achieved using primers with a 20 bp overhang to the HF (Fig. Ylc 7).

For amplification of MTUs from level 2 plasmids primers that bound to the promoter or terminator as well as part of the backbone were chosen, to achieve a specific binding site. This method proved to be difficult and inconsistent, and the following overlap-extension PCRs to add the HF were not successful, although we tried several different approaches to enable specific amplification, such as gradient PCR or adding dimethylsulfoxid (DMSO) to prevent steric inhibition.
Finally, we settled on amplifying the single transcription units composing the MTUs from level 1 plasmids and linking the individual transcription units with overlap-extension PCR. We focused on the *SctHMGR-ScERG13* and *AgtGPPS2-GgmFPS144* MTUs.

For this purpose, we designed specific oligonucleotides targeting only the promoter and terminator sequences. Using these primers, the transcription units from the level 1 plasmids could be amplified, and even amplification of MTUs from level 2 was successful. The amplified MTUs were not used for overlap-extension PCR but this could confirm the correct assembly of the level 2 plasmids (Fig. Ylc3).
The amplification and subsequent overlap-extension PCR to obtain the MTUs were successful for both the
*SctHMGR-ScERG13* construct as well as the *AgtGPPS2-GgmFPS144* construct (Fig. Ylc 8).

The addition of the overhangs to the single transcription units, as well as the MTUs, was also successful, except
for the *ERG20\_HF1-AgtGPPS2-GgmFPS144-ERG20\_HF2* construct. (Fig. Ylc 8).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-6.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-6.png)

**Figure Ylc 7: Gel of overlap-extension PCR of single transcription units and homology flanks.**
The size after the name of the respective coding sequence refers to the expected size of the amplified fragment.
Thermo Scientific GeneRuler 1kb DNA-ladder was used. Kilobase pair (kb).
1:- 2: *A08\_HF1-PptAPS-A08\_HF2* (4342 bp) 3: *XPR2\_HF1-SltNPPS1-XPR2\_HF2* (3379 bp) 4:- 5:
*pERG20\_HF1-YlpCTR1-pERG20\_HF2* (2857 bp) 6: *D17\_HF1-ApL3H-D17\_HF2* (4242 bp).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-7.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-7.png)

**Figure Ylc 8: Gel of overlap-Extension PCR of *AXP\_HF1-SctHMGR-ScERG13-AXP\_HF2* and
*AgtGPPS2-GgmFPS144* constructs.**
The size after the name of the respective coding sequence refers to the expected size of the amplified fragment.
Thermo Scientific GeneRuler 1kb DNA-ladder was used. Kilobase pair (kb).
1: *AXP\_HF1-SctHMGR-ScERG13-AXP\_HF2* (6180 bp) 2: *AgtGPPS2-GgmFPS144* (3556 bp)

#### Strain generation

Subsequent transformation of *Y. lipolytica* with the fully assembled, gel-excised fragments and the complementary *pJME4472* did yield colonies,
that appeared to replicate the plasmid. cPCR never showed a successful gene insertion, but rather fragments of the
length that would be expected from the original genomic sequence.Due to time constraints, we were unable to
continue with our work on the generation of *Y. lipolytica* strains with an optimized cytosolic GPP/NPP pathway.
To improve the gene insertion efficiency, repeating the *YlKU70* knockout should be considered. It is also
possible that the electroporation-based transformation method does not allow for optimal uptake of linear
fragments (Wang et al., 2011). A lithium-acetate based transformation method, or the introduction of plasmids
containing the MTUs and fitting HFs could be a solution.

---

## Peroxisomal production of α-pinene in *Yarrowia lipolytica*

In addition to the large cytosolic acetyl-CoA pool of *Y. lipolytica* (Wei et al., 2021), its extensive fatty-acid metabolism makes *Y. lipolytica* an interesting chassis organism for the peroxisomal production of monoterpenoids. The peroxisomes of *Y. lipolytica* have already been utilized to produce the sesquiterpene α-humulene and this strategy achieved a higher humulene titer than cytosolic expressions (Farhi et al., 2011). Here we tried to implement the designed metabolic pathways that utilize either GPP or NPP as the substrate for the monoterpene synthase PptAPS in the peroxisomes of *Y. lipolytica* (Fig. Ylp 1). Within MonChassis the respective level 1 parts of the mevalonate pathway with the PTS1 tag were cloned. Genes that were supposed to be strongly expressed were under the regulation of the *YlpTEF* promoter, while genes that required weaker expression were assembled with the *YlpGAP* promoter.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/pathway-peroxisome-ylipolytica-ks.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/pathway-peroxisome-ylipolytica-ks.png)

**Figure Ylp 1: Metabolic pathway designs for α-pinene production in the peroxisomes of *Y. lipolytica*.**

All genes of the NPP utilizing pathway were successfully assembled into level 1 parts (Fig. Ylp 2).
For the GPP using pathway, however, it was not possible to successfully assemble the *pSB1K04-Yl-AgGPPS\_PTS1*
part. Moreover, even though assembled level 2 constructs were transformed, and white colonies were
observed, verification of the correct assembly proved to be difficult because of the large size of the constructs.
We did not perform analytical cPCRs of level 2 constructs, but tried to amplify the respective
multi-transcription units for the subsequent overlap-PCR to assemble multi-transcription units with homology
flanks (Fig. G1). However, no bands were visible after the PCR of level 2 constructs to add the
overlaps for the planned overlap-PCRs (data not shown). Thus, the test of our metabolic designs for peroxisomal
monoterpenoid production is pending. To overcome the limitations of MoClo-based genetic engineering of *Y. lipolytica*, we propose a new vector design for MoClo-compatible level 2 vectors for cytosolic and
peroxisomal-tagged expression strategies. More information on the proposed design of the vector can be found here.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylpresults-1-lm.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylpresults-1-lm.png)

**Figure Ylp 2: Analytical cPCR of level 1 constructs for the peroxisomal production of monoterpenoids in *Y. lipolytica*.**  All necessary transcription units for the peroxisomal producction of α-pinene utilizing NPP as the precursor for monoterpene synthase were successfully cloned. Ladder: Thermo scientific GeneRuler 1 kb; 1: *pSB1K01-Yl-ScERG10\_PTS1* (2377 bp); 2: *pSB1K02-Yl-ScERG13\_PTS1* (2081); 3: *pSB1K03-Yl-SctHMGR\_PTS1* (2114 bp); 4: *pSB1K01-Yl-ScERG8\_PTS1* (2536 bp); 5: *pSB1K02-Yl-ScERG12\_PTS1* (2512 bp); 6: *pSB1K03-Yl-ScERG19\_PTS1* (2371 bp); 7: *pSB1K04-Yl-ScIDI1\_PTS1* (2047 bp); 8: *pSB1K02-Yl-GgmFPS144\_PTS1* (1709 bp); 9: *pSB1K02-Yl-SlNPPS1\_PTS1* (1577 bp); 10: *pSB1K01-Yl-PptAPS\_PTS1* (2342 bp).

---

## Proposed MoClo compatible vectors for faciliated engineering of *Yarrowia lipolytica*

The establishment of MoClo-compatible level 1 and level 2 plasmids containing the HF up and downstream of the (multi-) transcription unit could be a feasible alternative to linear fragments. This could be achieved by Gibson Assembly, similar to the creation of our level 2 shuttle plasmids. The design of the *pSB3C01* vector containing HFs for the *AXP* gene is shown below (Fig. Yl 1). If necessary, the transcription unit, including the HF, can be excised with *Pme*I.

This improved strategy would utilize most of the already cloned plasmids, maintain the modular and versatile MoClo approach, while increasing gene insertion efficacy to generate a highly efficient monoterpenoid-producing *Yarrowia lipolytica* strain.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-8.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/ylcresults-fig-8.png)

**Figure Yl 1: Design of the *pSB3C01* plasmid for improved gene insertion into *Yarrowia
lipolytica*.**
Based on the *pSB3C01* plasmid, a plasmid for facilitated gene insertion into *Y. lipolytica* was designed. This plasmid contains the exemplary homology flanks (HF1 and 2) for insertion into the
*YlAXP* gene. The Plasmid also contains the chloramphenicol acetyltransferase gene, which confers
chloramphenicol resistance (CmR) and the bacterial origin of replication p15A. The red fluorescent protein gene
*mRFP1*, a monomeric derivative of DsRed, can be replaced by Golden Gate Assembly using *Sap*I. The
entire MTU with the HFs can be excised using *Pem*I.

---

## E-driven CYP

Our platform for the production of various monoterpenoids, MonChassis, consists of two components; in the *in vivo* part, beneficial precursors are produced by the optimal yeast strain. The *in vitro* part aims to catalyze these precursors cost-efficiently to profitable monoterpenoids. To this end, MonChassis uses a self-designed BioReactor for Enzymatic ElectroSynthesis (BREES) and a modified cytochrome P450 monooxygenase BM3. As a proof-of-concept, we aimed to catalyze the reaction from α-pinene to the bark-beetle repellent verbenone electrically-driven. Thus, we also developed a colorimetric assay for the rapid screening of verbenone-producing BM3 mutants.

### BREES

Aiming to synthesize monoterpenoids electrically-driven, we mainly built our platform on the research by Zernia
et al., 2018. They demonstrated that the recombinant fusion of an indium tin oxide (ITO) binding peptide and the
BM3 can be used for electrically driven catalysis. Thus, we aimed to reconstruct this set-up to produce verbenone
as our proof-of-concept. We managed to clone, express, and purify a recombinant BM3 and built BREES with which we
conducted initial tests.

#### Cloning, Expression & Purification of the enzyme BM3

First, we attached an N-terminal 6x HisTag for purification and a C-terminal ITO binding peptide with the amino acid sequence (RTRHK)4 to a BM3 variant. We received a plasmid including *BM3* with three mutations (A74G, F87V, L188Q) showing high activity on several fluorescence assays (Zernia et al., 2016) from the working group of Sarah Zernia from Leipzig University. Using self-designed primers with the ITO oligonucleotide sequence attached, we amplified the *BM3* via PCR. After this, we inserted the gene into the vector *pET28b* which contains a 6x HisTag at the N-terminal end of the multiple cloning site. The successful insertion was verified by sequencing using only primers for the border regions of the multiple cloning site (Fig. E-CYP 1). We transformed the expression strain *Escherichia coli* BL21 DE3with the completed plasmid.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp9-fh-png.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp9-fh-png.png)

**Figure E-CYP 1: Scheme of modified BM3 for purification.** The BM3 consists of the cytochrome
P450 (BMP) and the corresponding reductase (BMR) and was cloned into the multiple cloning site of vector
*pET28b* with N-terminal 6x HisTag, thrombin site, and T7 tag for further transformation of expression
strain *E. coli* BL21 DE3 resulting in a recombinant protein with a total molecular weight of 124
kiloDalton.

To obtain large and clean amounts of enzyme for application in BREES, we expressed and purified the desired
protein BM3 with the ITO binding peptide. For the purification, a column with Nickel-NTA was used, which binds the
N-terminal 6x HisTag of the expressed BM3. We performed a Bradford assay to determine the protein concentration of
the eluate. Therefore, we created a standard curve of protein with bovine serum albumin (BSA) in known
concentrations. The eluate containing BM3 had a total protein concentration of 4.25 mg/ml. Subsequently, we
performed an SDS-PAGE to verify the presence of the protein and check the quality. Thus, we checked samples from
the pellet and supernatant of disrupted cells, flow through, washing step, and the final eluate.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp2-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp2-fh.png)

**Figure E-CYP 2: SDS-PAGE of different steps from the purification of recombinant BM3 (124
kiloDalton).** The expression strain BL21 DE3 was disrupted, centrifuged, and the supernatant purified via
binding of 6x HisTag to the Ni-NTA column. 1: supernatant after disruption; 2: pellet after disruption; 3:
flowthrough (5 mM imidazole); 4: washing step I (24.5 mM imidazole); 5: diluted eluate (2 µg); 6: diluted eluate
(5 µg). Ladder: ThermoScientific PageRuler.

The SDS-PAGE confirms the occurrence of a protein with ~125 kDa in the pellet, supernatant, and the flow through,
as well as in both dilutions of the eluate (Fig. E-CYP 2). This is most probably the protein of interest, the
recombinant BM3 with 124 kDa. In the pellet (2), a relatively high amount of a protein with ~35 kDa is present,
indicating the presence of the in *E. coli* BL21 DE3 overexpressed membrane protein OmpT with 33 kDa.
Additionally, a protein of ~55 kDa is detected in both dilutions of the eluate (5 & 6), which can be due to
binding of amino acids like histidine to the Ni-NTA. Thus, we have a high amount of BM3 although there is also
another protein in our eluate. A second washing step with an even higher concentration of imidazole could lead to
higher purity of the protein BM3.

#### Activity tests of BM3

We wanted to test the general enzyme activity as well as the immobilization on the ITO-electrode of the purified
BM3. Since the variant of BM3 we use in MonChassis shows high activity in the deethoxylation of 7 ethoxycoumarin
to 7 hydroxycoumarin (Zernia et al., 2016) due to the three mutations, we chose a fluorometric assay to examine
the general enzyme activity. We tested the purified BM3 with the ITO binding peptide in two set-ups. In set-up I,
we diluted BM3 in assay buffer and added substrate as well as nicotinamide adenine dinucleotide phosphate (NADPH)
to start the reaction. For set-up II, BM3 was incubated with the electrode for 1 h to attach to the ITO surface
before the assay (Fig. E-CYP 3). After that, we discarded the supernatant and added substrate and NADPH to the
set-up. In both set-ups, we extracted 7-hydroxycoumarin from the reaction solutions with ethyl acetate and
measured the fluorescence with excitation wavelengths of 340 nm and 360 nm and an emission wavelength of 465 nm
using a plate reader.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp3-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp3-fh.png)

**Figure E-CYP 3: General enzyme activity assay.** We tested the activity of purified BM3 by the
conversion of 7-Ethoxycoumarin to 7-Hydroxycoumarin **(A)** in set-up I in solution and **(B)**  set-up II
attached on the ITO-electrode. The quantity of 7-Hydroxacoumarin was determined fluorometrically.

Plotting the data against a standard series of known amounts of 7-hydroxycoumarin, we saw no activity of purified
BM3 either in solution (set-up I) or on the ITO-electrode (set-up II). This could be due to mutations in the BM3
leading to a defective folding of the native protein since the protein sequence was not entirely analyzed before
transformation. Additionally, no activity in set-up I can be due to low affinity of the ITO binding peptide to the
electrode resulting in the discard of the BM3 before the substrate and NADPH were added. For this, the binding
affinity should be tested again.

#### Testing the BioReactor for Enzymatic ElectroSynthesis

MonChassis aims to use enzymatic electrosynthesis for the conversion of α-pinene to verbenone.
Therefore, we produced BREES for an inexpensive and easy way to use immobilized enzymes for electrically-driven
catalysis. The detailed design as well as further information about the bioreactor can be seen [here](https://2022.igem.wiki/wwu-muenster/contribution#section-3). We conducted different
experiments to test BREES.

At first, we examined the binding affinity of the ITO binding peptide onto the electrode. Therefore, we measured
the UV-Vis spectra of purified BM3 with the NanoDrop to determine the wavelength of its absorbance (Fig. E-CYP 4).
Several peaks at different wavelengths were detected. Since the SDS-PAGE (Fig. E-CYP 2) revealed the presence of
two proteins in the eluate, we decided to choose the peak at 426 nm as indicator for the purified BM3 which may
result from the absorbance of its oxidized heme-group. Based on the cross section of BM3 published in literature
(Zernia et al., 2018), we determined that the maximal possible amount of BM3 to be immobilized onto the
ITO-electrode is 20.83 pmol. Thus, we diluted the eluate gained from purification 1:1000 and added 280 pmol of BM3
onto the electrode to ensure its complete coverage. To determine if the binding peptide attaches to the
ITO-surface, we measured the absorbance at 426 nm before and after applying purified BM3 on the electrode using
the sample before application as standard. We assessed a difference in absorbance of 9 mAU. This could verify the
immobilization of BM3 on the ITO-electrode. But since the small number of maximal immobilized BM3 is very near to
the detection limit of the NanoDrop, these data are not significant. The binding affinity of the ITO binding
peptide must be examined with other methods, such like antibody adhesion.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp4-fhneu.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp4-fhneu.png)

**Figure E-CYP 4: UV-Vis Spectrum of purified BM3.** We measured the absorbance spectra of
**(A)** the purified BM3 and **(B)** detected several peaks. We chose the peak at 426 nm (indicated with
arrow) to determine the affinity of the ITO binding peptide to the electrode.

Although we measured no enzyme activity of the purified BM3 in the deethoxylation of 7 ethoxycoumarin, we decided
to test the enzyme’s ability to convert α-pinene to verbenone in BREES anyways. Therefore, we aimed
to immobilize the BM3 on the ITO-electrode in two set-ups with either NADPH or with electricity for reduction
(Fig. E-CYP 5). The ITO-electrodes were incubated with the purified BM3 for 30 min and then washed with PBS
buffer. Then, we added the substrate α-pinene. We started the reaction by adding NADPH in the
set-up with no electricity and by connecting the ITO-electrode with electricity in the other set-up. In both
batches the supernatants were then extracted with ethyl acetate and measured with GC-MS.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp5-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp5-fh.png)

**Figure E-CYP 5: Conversion of α-pinene to verbenone by immobilized BM3 in BREES.**
We tested the ability of immobilized BM3 to convert α-pinene to verbenone in a BioReactor for
Enzymatic ElectroSynthesis under two conditions. **(A)** Set-up I use NADPH as a reductant; **(B)** in
set-up II a voltage of -0.6 V provides the BM3 with electrons.

Unfortunately, we saw no occurrence of either verbenol or verbenone. This confirms that the purified BM3 had no
activity in the conversion of α-pinene to verbenone in our set-ups. Same as in the general activity
assay, the reasons can be mutations in the BM3 or the immobilization of the enzyme on the ITO-electrode does not
work properly. Nevertheless, we were able to do the first tests of our self-designed BioReactor for Enzymatic
Electrosynthesis (BREES) and gained experience with its application. We saw that the air pressure sealing allowed
no loss of reaction liquid and led to visible stirring and ventilation.

### Colorimetric screening of favorable BM3 mutants

MonChassis wants to produce verbenone as a proof-of-concept for electrically-driven monoterpenoid production. To
optimize the conversion from the precursor α-pinene to verbenone, [we developed a colorimetric assay](https://2022.igem.wiki/wwu-muenster/engineering) for
the rapid screening of favorable mutants of BM3. We used 2,4-Dinitrophenylhydrazin (DNPH) that reacts with
unsaturated ketones (enones) like verbenone and converts them to a colored phenylhydrazone precipitate (Fig. CYP
6). The amount of verbenone that is thereby formed, is proportional to the visible red coloration. Saturated
ketones and aldehydes do also react with DNPH, but do not form a colored complex and thus remain yellow-colored
(Liu et al., 2016).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp6-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp6-fh.png)

**Figure E-CYP 6: Schematic representation of the chemical reaction of DNPH with verbenone.** The
two substances react in an acidic environment to form a reddish colored phenylhydrazone precipitate.

The problem with an intracellular enzyme reaction is that α-pinene is toxic in higher
concentration and verbenone cannot diffuse through the membrane. To avoid a laborious extraction of verbenone from
the *E. coli* cells, we used the method of autodisplay. In this method, a passenger protein is fused to a
signal peptide and a membrane protein, called the autotransporter system. This ensures the passenger proteins
display on the surface of *E. coli* (Jose et al., 2012; Schumacher et al., 2012). In our colorimetric assay,
we used a wildtype BM3 as a passenger protein. Due to the simple insertion of different mutants in the
autotransporter system, our colorimetric assay allows rapid screening of favorable BM3 mutants for verbenone
production.

#### α-pinene toxicity test

Monoterpenoids including α-pinene are known to be toxic to bacteria such as *E. coli* (Dunlop
et al., 2011; Sarria et al., 2014; Trombetta et al., 2005). Therefore, we aimed to determine an appropriate *E.
coli* strain for autodisplay and examined the highest possible concentration of α-pinene it
can be exposed to. In total, we incubated four different *E. coli* strains with eight different α-pinene concentrations and a negative control. We measured the course of the optical density at 600 nm
over 16 h (Fig. E-CYP 7).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp7-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp7-fh.png)

**Figure E-CYP 7: Growth curves of *E. coli* exposed to different α-pinene
concentrations.** All different strains were treated with either 0.0, 0.1, 0.3, 0.6, 1.25, 2.5, 5.0, 10.0,
20.0% (v/v) of α-pinene and the optical density was measured at 600 nm for 16 h. **(A)** BL21.
**(B)** BL21 (DE3). **(C)** BL21 (DE3) plys.s. **(D)** BL21 (DE3) codonPlus. n=3, the standard
deviation is indicated through error bars.

As seen in Figure E-CYP 7, most approaches showed slow and steady growth after starting at an OD600 of ~0.7. The highest optical density was detected for an α-pinene concentration of 2.5% (v/v) for all strains. Lower concentrations showed a similar course to the control whereas higher concentrations impaired cell growth. Some data rows of BL21 (DE3) codonPlus were irregular and had a high standard deviation, indicating that the growth of the strain was negatively influenced by α-pinene. For the other strains, the results were similar with a reliable standard deviation and the highest possible α-pinene concentration of 2.5% (v/v). Thus, we decided to use *E. coli* BL21 for the colorimetric assay, since this strain lacks the T7 promoter system as well as the outer membrane protease OmpT (Jose et al., 2012). This makes the strain suitable for autodisplay, and thus, for our colorimetric assay.

#### Colorimetric assay with DNPH

To ensure that only verbenone reacts with DNPH even in the presence of other substances and/or cells, we have
performed several tests. Therefore, we grew *E. coli* BL21 in LB medium to an OD600 of 1.0 and
incubated them with known amounts of verbenone. As negative control and for comparison of coloration, we used the
same amount of verbenone diluted in water (Fig. E-CYP 8). After incubation, we added DNPH. We also examined the
ketone acetone as well as other monoterpenoids such as α-pinene and verbenol on their interaction
with DNPH and *E. coli* BL21.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp8-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp8-fh.png)

**Figure E-CYP 8: Colorimetric assay with DNPH.** Verbenone standard series with 1, 0.5, 0.25,
0.125, 0.063, 0.031, 0.015, and 0% (v/v) (1-8), as well as 1% (v/v) α-pinene (9), 1% (v/v) acetone (10), and 1% (v/v)
verbenol (11) were diluted **(A)** in H2O and **(B)** in LB media with *E. coli* BL21. Each well was
incubated for 40 min. After that, DNPH reagent was added which led to the staining after a few hours.

The results show a rapid determination of verbenone occurrence in samples with or without *E. coli* BL21
cells is possible by red staining with DNPH (Fig. E-CYP 8). A higher amount of verbenone leads to more intensive
red staining, enabling a comparison if known standards are present. Other compounds such as α-pinene
, acetone, and verbenol show no red staining and thus, do not interact with cells or DNPH. This supports
that a colorimetric assay using DNPH and autodisplay can be used as a rapid screening for favorable BM3 mutants.

These findings rely solely on a subjective determination of verbenone occurrence by visual red staining. Since an
objective assessment based on measured data is more reliable, we also wanted to determine the absorbance using
UV-Vis spectroscopy.

Since the absorption maximum of the hydrazone formed by verbenone with DNPH was not known, we measured the
spectrum against DNPH without verbenone as control (Fig. E-CYP 9). Verbenone-dinitrophenylhydrazone has an
absorption maximum at 390 nm as shown in the figure. The data was validated by measuring the DNPH absorption
maximum at 352 nm as previously described (Roberts and Green, 1946). Since the absorbance at 450 nm was so high
that it was outside the linear measurement range, both approaches were diluted in acetonitrile before the
measurements.

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp9-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp9-fh.png)

**Figure E-CYP 9: UV/Vis spectrum of verbenone-dinitrophenylhydrazone and a negative control.**
Both samples were diluted in acetonitrile. n=3.

To avoid interference with other substances and unreacted DNPH in our further experiments, we chose a higher
wavelength for the measurements. At 450 nm, an accurate standard curve could be measured for the
verbenone-dinitrophenylhydrazone (Fig. E-CYP 10).

[![](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp10-fh.png)](https://static.igem.wiki/teams/4188/wiki/wiki-images/labstuff/edrivencyp/results-edrivencyp-fig-e-cyp10-fh.png)

**Figure E-CYP 10: UV-Vis spectroscopy of absorbance of Verbenone-DNPH for a standard series.** We
incubated the autodisplay for 2 h with different amounts of verbenone. The DNPH reagent was added and after 40
min, we measured the absorbance at 450 nm. The regression line can be used to determine the concentration of
verbenone. n=3, the standard deviation is indicated through error bars.

As shown in Figure E-CYP 10 the standard series with verbenone was successful, since the R2-factor is
close to 1, confirming the possibility of protein engineering by an objective color assay as the regression line
can be used to determine the concentration of verbenone. We also incubated the autodisplay cells with verbenone
using the same method. For this purpose, the *E. coli* BL21 cells must be removed by centrifugation, as they
interfere with the absorption (Mesquita et al., 2014). The supernatant was transferred and DNPH was added. After
incubation, we measured the spectrum of the samples in the plate reader.

Problematic was that even controls with no verbenone, but NADPH showed high absorbance at 450 nm. This is unusual
since the absorption maximum of NADPH is actually around 340 nm and the maximum of NADP+ is even lower (Sugishima
et al., 2020). To avoid this problem, we decided to work with an NADPH regeneration system, since in this case
overall less NADPH/NADP+ is present, so interference is diminished. These samples showed indeed a lower
absorption, but it still influenced the results.

Therefore, mutation screening for autodisplay by UV-Vis spectroscopy still needs to be revised by possible
extraction with, for example, ethyl acetate. Nevertheless, finding a BM3 with increased verbenone production is
definitely easier with the rapid screening of our developed colorimetric assay. Thousands of variants can be
screened within days, which would take weeks for measurements by gas chromatography with mass spectrometry
(GC-MS).

---

## Conclusion and Outlook

Through extensive cloning of numerous gene constructs and yeast transformation, we have clearly demonstrated that the synthesis of α-pinene and verbenone is possible by introducing a synthetic pathway into *Saccharomyces cerevisiae* in both the cytosol and peroxisomes.

The results we obtained were indeed promising and offered insights into more advanced possibilities, as we were able to confirm the synthesis of monoterpenoid precursors in yeast. This sets the basis for future experiments regarding advanced strain optimization and exploring the promising organism *Yarrowia lipolytica*. We considered high throughput screening to substantiate the quantification and functional characterization of individual pathway members of interest. However, quantification is not reliable due to insufficient replicates and lack of improvements in experimental design. Despite our tremendous success in creating different chassis organisms, we had to adjust our expectations in finding the most efficient α-pinene producing yeast strain. Because of the limited time we were unable to exploit the full potential of MonChassis.

To unlock the full potential of metabolic engineering, we developed a powerful software tool for metabolic modeling to reveal further targets for improvement and to identify the optimal carbon source for monoterpenoid production. Accounting for all aspects mentioned the most efficient chassis organism can be determined and utilized for large-scale biotechnological applications. Concerning the *in vivo* approach, which was to produce monoterpenoids, we were able to demonstrate the functionality of MonChassis.

Finally, MonChassis also aimed at developing an *in vitro* system for downstream enzymatic electrosynthesis that converts yeast-produced precursors into any desired monoterpenoid. The use of such a cell-free platform eliminates the need for costly cofactors, like NADPH, extends toxicity-related yield limits, and provides a modular method of monoterpenoid production. To demonstrate this aspect of MonChassis, we focused on the conversion from α-pinene to verbenone. We cloned, expressed, and purified a genetically engineered cytochrome P450 monooxygenase BM3 variant, successfully immobilized it on an indium tin oxide (ITO)-electrode, and tested its activity in various experimental setups. We also designed, built, and tested a modular, [easy-to-copy bioreactor](https://2022.igem.wiki/wwu-muenster/contribution#section-3) that can be used by future iGEM teams. This platform also includes a [colorimetric assay system](https://2022.igem.wiki/wwu-muenster/engineering#section-3) for rapid mutation screening.

For industrial application of MonChassis, the next logical step will be to investigate direct purification of BM3
using the ITO-binding peptide. This could vastly decrease the costs since no complex purification methods are
necessary and the cell extract can be applied immediately onto the ITO-electrode. The yield of monoterpenoids such
as verbenone can be further increased by enzyme engineering. Promising amino acids for increased α-pinene
 specificity of BM3 have already been mentioned (Lehmann, 2016), for which the colorimetric assay
developed by MonChassis can be employed. For industrial scale-up, bioreactors with larger electrode surfaces and
multiple reaction chambers need to be designed. For all future steps, our key findings paved the way for the
enzymatic electrosynthesis of monoterpenoids. MonChassis thus provides a step towards a sustainable bioeconomy.

{% endblock %}
{% block buttonForwarding %}
[Implementation](https://2022.igem.wiki/wwu-muenster/implementation)
{% endblock %}
{% block references %}

## References

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