Difference between revisions of "Part:BBa K3930003"

 
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
<p><b>The pVIOLETTE part (BBa_K3930003) enables the production of &alpha;-ionone from lycopene and is composed by:</b></p>
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<p><b>The pVIOLETTE part (BBa_K3930003) enables the production of &alpha;-ionone from lycopene and is composed of:</b></p>
<p>- the up (BBa_K3930021) and down (BBa_K3930022) integration sites in the XII-4 locus of the <i>S.cerevisiae</i> genome (based on the plasmid pCfb3040 from Easyclone Marker free kit (Jessop-Fabre et al.,2016))</p>
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<p>- the up <a href="https://parts.igem.org/Part:BBa_K3930021" class="pr-0" target="_blank">(BBa_K3930021)</a> and down <a href="https://parts.igem.org/Part:BBa_K3930022" class="pr-0" target="_blank">(BBa_K3930022)</a> integration sites in the XII-4 locus (Chr XII:830227..831248)  of the <i>S. cerevisiae</i> genome (based on the plasmid pCfB3040 from Easyclone Marker free kit (Jessop-Fabre et al.,2016)).</p>
<p>- the LcyE-ofCCD1 enzymatic fusion (BBa_K3930024) that allows the production of &alpha;-ionone  
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<p>- the <i>LcyE-ofCCD1</i> fusion <a href="https://parts.igem.org/Part:BBa_K3930024" class="pr-0" target="_blank">(BBa_K3930024)</a> codes for a carotenoid cleavage dioxygenase fused to a lycopene cyclase, which allows the production of &alpha;-ionone. The sequences were codon optimized for expression into <i>S. cerevisiae</i>.</p>
<p>- the inducible promoters Gal 1 with galactose (BBa_K3930023), driving the expression of LcyE-phCCD1</p>
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<p>- the galactose inducible promoter pGal1 <a href="https://parts.igem.org/Part:BBa_K3930023" class="pr-0" target="_blank">(BBa_K3930023)</a>, driving the expression of LcyE-phCCD1.</p>
<p>- the resistance marker NsrR (BBa_K3930025) to select yeast integrants</p>
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<p>- the resistance marker NsrR <a href="https://parts.igem.org/Part:BBa_K3930025" class="pr-0" target="_blank">(BBa_K3930025)</a> to select yeast integrants.</p>
 
<h2>Construction</h2>
 
<h2>Construction</h2>
<p>IDT and Twist Bioscience performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned with an In-Fusion Takara kit into the pCfB3040 plasmid and then transformed into E.coli Dh5&alpha; strain. Figure 1 shows the restriction map of the resulting clones. The expected restriction profile was obtained for clone 3.</p>
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<p>IDT and Twist Bioscience performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned with the In-Fusion Takara kit into the pCfB3040 plasmid and then transformed into <i>E.coli</i> Dh5&alpha; strain. Figure 1 shows the restriction map of a correct resulting clones.</p>
 
      
 
      
  
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                     <b>Figure 1: </b> <b>Figure 1: pViolette assembly:</b>
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                     <b>Figure 1: pVIOLETTE assembly</b>
                     pVIOLETTE restriction profile from clone 3 was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the right of each gel and the NEB 1 kb DNA ladder on the left (note that a different ladder is presented on the theoretical gel)
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                     <p>pVIOLETTE restriction profile from clone 3 was checked by digestion visualised on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel).</p>
 
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<p>The plasmid containing the pVIOLETTE construct was then linearized with the F and R linearization primers pVIOLETTE. Then the amplicon was integrated into the genome of our LycoYeast strain with the Takara Yeast transformation protocol. Figure 2 shows the electrophoresis gel of PCR on colony to verify clones.The expected size was obtained for clone 2.</p>
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<p>pVIOLETTE insert was then linearized with the pVIOLETTE_pCfB3040_Forward and pVIOLETTE_pCfB3040_Reverse linearization primers pVIOLETTE. Then the amplicon was integrated into the genome of our LycoYeast strain with the Takara Yeast transformation protocol. Figure 2 shows the electrophoresis gel of colony PCR to verify integrants genotype. The expected size was obtained for clone 2.</p>
 
<p><b>Primer used to clone this part in the pCfB3040:</b></p>
 
<p><b>Primer used to clone this part in the pCfB3040:</b></p>
 
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     <li>pVIOLETTE_pCfB3040_Reverse : 5' cgtacctggatggtcatttc 3'</li>
 
     <li>pVIOLETTE_pCfB3040_Reverse : 5' cgtacctggatggtcatttc 3'</li>
 
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                 <b>Figure 2: </b> <b>Figure 1:  Integration of pVIOLETTE in LycoYeast:</b>
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                 <b>Figure 2: </b> <b> Integration of pVIOLETTE insert in LycoYeast</b>
                 pVIOLETTE integration from clone 1 and 2 was checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the right of each gel and the NEB 1 kb DNA ladder on the left (note that a different ladder is presented on the theoretical gel)
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                 <p>pVIOLETTE insert integration from clone 1 and 2 was checked by PCR visualised on EtBr stained agarose electrophoresis ge. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel).</p>
 
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<p>pVIOLETTE insert at locus XII-4 was successful. The integrant strain was named LycoYeast-VIOLETTE and saved as glycerol stock.</p>
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<h2>Characterisation</h2>
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<p><b>pVIOLETTE insert integration at locus XII-4 was successful. The integrant strain was named LycoYeast-pVIOLETTE and saved as glycerol stock.</b></p>
<h3>Production of &alpha;-ionone</h3>
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<h2>Characterization</h2>
<p>After verifying the correct integration of our constructs by PCR, our engineered LycoYeast strains were placed on YPD plates containing the inducers with the aim to detect color changes due to the conversion of lycopenes (red) to carotenes (orange).</p>
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<h3>Production of &epsilon;-carotene</h3>
<p>Figure 3 shows the colors of the colonies with or without the inducer, the galactose. The LycoYeast-VIOLETTE strain plated on a YPGal Petri dish shows a yellow coloration, indicating the degradation of lycopene into &epsilon;-carotene.</p>
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<p>After verifying the correct integration of our insert by PCR, our engineered LycoYeast strains were placed on YPD plates containing the inducers with the aim to detect color changes due to the conversion of lycopenes (orange) to carotenes (yellow).</p>
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<p>Figure 3 shows the colors of the colonies with or without the inducer, the galactose. The LycoYeast-pVIOLETTE strain plated on a YPD + galactose Petri plate shows a yellow coloration, indicating the conversion of lycopene into &epsilon;-carotene.</p>
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                     <b>Figure 3: </b> <b>Color change in the modified LycoYeast strains</b>   
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                     <b>Figure 3: </b> <b>Color change in the modified LycoYeast-pVIOLETTE strain</b>   
                     The mutants seem to change from red (lycopene) to orange (carotene) when plated with the galactose activator, which was the expected result
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                     <p>The modified LycoYeast strains change from orange (lycopene) to yellow (carotene) upon galactose induction, which was the expected result.</p>
 
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<p>The carotenoids contained in the cells were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after this lysis was analyzed by RP-HPLC using a C18 column.In the LycoYeast-VIOLETTE strains, Figure 4 shows that lycopene is converted into a new product with a higher retention time upon induction. Considering the yellow color of pVIOLETTE strains, as well as the in-line following alpha ionone production results, this new peak most likely corresponds to ε-carotene, the expected precursor.</p>
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<p>The carotenoids contained in the cells were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pVIOLETTE strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 4). Considering the yellow color of pVIOLETTE strain, as well as the &alpha;-ionone production results, this new peak most likely corresponds to &epsilon;-carotene, the expected precursor.</p>
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                     <b>Figure 4: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pVIOLETTE</b>   
 
                     <b>Figure 4: </b> <b>Carotenoid analysis of the engineered strain LycoYeast-pVIOLETTE</b>   
                     tr= retention time; 3 peaks are observed in a non-modified and a modified but not induced LycoYeast while 4 peaks are present in a LycoYeast-pVIOLETTE strain.
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                     <p>tr= retention time; 3 peaks are observed in a non-modified and a modified but not induced LycoYeast, while a 4th peak is present in a LycoYeast-pVIOLETTE strain.</p>
 
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<p>Anti-His-tag antibodies revealed a band at about 45 kDa in the sample corresponding to IPTG induction in the presence of AzF (lane I+AzF). This band is not present in control samples (lane NI and I-AzF), indicating that it corresponds to the Cerberus protein (theoretical size: 41 kDa). In addition to the full length protein, we observed several extra bands which very likely correspond to proteolysis products since they are detected with the anti-His-tag antibodies. Moreover, the band at 45 kDa is clearly detected in elution samples (lanes E). The purification level of Cerberus with monomeric streptavidin in the elution samples was estimated about 62%. These data show that Cerberus was efficiently purified and can be used for subsequent assays. In addition, these results show that experimental setup to produce Cerberus also leads to the production of a protein where the amber stop codon has not been recognized by the AzF-charged orthogonal tRNA (a construction we named Orthos in our project). Although Orthos does not contain a His-tag at its C-terminus, the protein seems to be efficiently co-purified with Cerberus. The basis of this observation is unclear but this result may suggest that Orthos and Cerberus interact together.</p>
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<h3>Validation of Cerberus</h3>
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<h3>Production of &alpha;-ionone</h3>
    <h4>Validation of the AzF and CBM3a heads using FITC (Fluorescein isothiocyanate) molecules</h4>
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<p>The &alpha;-ionone is very volatile. A common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest.The most common organic solvent used is dodecane for ionones (Chen et al. 2019; López et al. 2020).Figure 5 shows the GC-MS spectrum for the LycoYeast-VIOLETTE strain. A peak can be observed at the same retention time as the &alpha;-ionone standard for the induced LycoYeast-VIOLETTE strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The &alpha;-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). The production of &alpha;-ionone, the main molecule of the violet odor, was successfully achieved with this construction.</p>
<p>To validate both the AzF and CBM3a heads of Cerberus, we challenged its potential to functionalize cellulose with fluorescence. To generate a fluorescently labelled Cerberus protein, we first performed a SPAAC reaction on 3.2 µM Cerberus protein using 31.9 µM of FITC-DBCO (Jena Bioscience). In the control experiment, 31.9 µM of FITC alone was used. These samples were then incubated with cellulose for 16 hours and after several washes with resuspension buffer (50mM Tris HCl pH 8), fluorescence levels were measured in the cellulose pellet fractions (Figure 5). We observed that the fluorescence levels in the cellulose pellet incubated with Cerberus protein clicked to FITC were about 3 times higher than in the control experiments corresponding to the cellulose pellet incubated with FITC alone. These results show that Cerberus can efficiently be conjugated with fluorescent molecules bearing a DBCO group by click chemistry and that the resulting fluorescent molecules strongly interact with cellulose.This result proves that cerberus AzF and CBM3a heads are valid and therefore, makes our construction both a practical and potent platform to functionalize cellulose.</p>
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                     <b>Figure 5: </b> <b>Fluorescence remaining in cellulose fraction after several washes  </b>
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                     <b>Figure 5: </b> <b>GC-MS analysis of the dodecane layer of the LycoYeast-pVIOLETTE</b>  
                     Experiment were performed in quadruplicate test in 50 mM Tris HCl pH 8 using 100&mu;L of 10mg/ml Regenerated Amorphous Cellulose and 100&mu;L of click reaction. Samples were incubated for 30 minutes, centrifugated, supernatant discarted and resuspended in Tris HCl four times , *Mann Whitney test p-value 0.03 computed using R 3.4.2.
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                     <p>α-ionone is produced in vivo by our strain upon galactose induction. On the right are presented the mass spectra mass spectra of the observed peak corresponding to &alpha;-ionone. First panel is the &alpha;-ionone standard. Second panel is the LycoYeast WT. Third panel is the LycoYeast-pVIOLETTE non-induced. Forth panel is the LycoYeast-pVIOLETTE induced with galactose.</p>
 
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<h4>Validation of the Streptavidin and CBM3a heads</h4>
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<p>To asses the functionality of the Streptavidin head, we used a non AzF version of Cerberus (its Orthos version). We performed SPAAC reaction to ligate <em>in vitro</em> biotin-DBCO to an azide-functionalized fluorescein (FITC), thus leading to the expected biotinylated FITC. 5.8 &mu;M of Orthos were incubated with 58.0 &mu;M of biotinylated FITC for one hour under shaking and then incubated with regenerated amorphous cellulose as described previously. Fluorescence was measured after four washes and results are presented in figure 6.</p>
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                    <b>Figure 6: </b> <b>Fluorescence remaining in cellulose fraction after several washes  </b> 
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                    Experiment were performed in quadruplicate test in 50 mM Tris HCl pH 8 using 100&mu;L of 10mg/ml Regenerated Amorphous Cellulose and 100&mu;L of binding reaction. Samples were incubated for 30 minutes, centrifugated, supernatant discarted and resuspended in Tris HCl four times , *Mann Whitney test p-value 0.1 computed using R 3.4.2.
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<p>The results indicates that fluorescence retained in cellulose pellet is higher when orthos is present which indicates that the binding between biotinylated FITC and Streptavidin was effective, proving that the activity of both streptavidin and CBM3 remains intact when fused. This validated the Streptavidine head.</p>
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<h4>Validation using paramagnetic beads</h4>
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<p> The functionality of Cerberus was further characterized using paramagnetic beads. To bind paramagnetic beads to Cerberus, we performed a click reaction using 3.2 µM of Cerberus protein and 32 µM DBCO-conjugated paramagnetic beads. In control experiment, 32 µM of paramagnetic beads were used. These samples were then incubated with cellulose, and after several washes with resuspension buffer, the magnetic capacity of cellulose using a magnet was observed and filmed (See video below). The cellulose incubated with the Cerberus protein conjugated to paramagnetic beads responded quickly and was totally collected by the magnet in contrast to the control experiment. This definitely highlights the potential of Cerberus for many applications</p>
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<video controls preload="auto" muted="true" style="width : 100%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/1/14/T--Toulouse-INSA-UPS--Collaborations--angeline--ferocell.MOV" alt="Magnetic cellulose">
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<b>Figure 7: </b> <b>Video of Cellulose functionnalised with magnetic beads using Cerberus  </b> 
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Left : Negative control using Paramagnetic beads alone, Right: Cerberus-Paramagnetic beads
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<h2>Conclusion and Perspectives</h2>
 
<h2>Conclusion and Perspectives</h2>
<p>These results show that Cerberus has the ability to interact simultaneously with cellulose and molecules with DBCO group or biotinylated compounds. This allows multiple possibilities to functionalize cellulose through its linker containing unnatural amino acid (AzF) and/or the linker presenting the engineered monomeric Streptavidin (mSA2). </p>
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<p>Our LycoYeast-pVIOLETTE strain effectively degrades degrade lycopene into &epsilon;-carotene and further transforms it into &alpha;-ionone. The quantification of &alpha;-ionone production remains to be determined under optimal conditions.</p>
<p>Fixation of various compounds that can be chemically functionalized can now be achieved envisioning endless possibilities to functionalize cellulose. We sincerely thank the future teams that will use this construction and encourage them to contact us for further details</p>
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<p>&alpha;-ionone belongs to the terpenes family and may have other uses besides perfumery, notably in medicine. We sincerely thank the future teams that will use this construction and encourage them to contact us for further details.</p>
 
<h2>References</h2>
 
<h2>References</h2>
 
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<ol>
 
     <i>
 
     <i>
     <li>Morag E, Lapidot A, Govorko D, Lamed R, Wilchek M, Bayer EA, Shoham Y: Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum. Applied and Environmental Microbiology 1995, 61:1980-1986.</li>
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     <li>Chen X, Shukal S, Zhang C. 2019. Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production. J Agric Food Chem. 67(49):13451–13459. doi:10.1021/acs.jafc.9b00860.</li>
     <li>Nogueira ES, Schleier T, Durrenberger M, Ballmer-Hofer K, Ward TR, Jaussi R: High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis. Protein Expr Purif 2014, 93:54-62. DOI: 10.1016/j.pep.2013.10.015.</li>
+
     <li>Jessop-Fabre MM, Jakočiūnas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I. 2016. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnol J. 11(8):1110–1117. doi:10.1002/biot.201600147.</li>
     <li>Young TS, Schultz PG: Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem 2010, 285:11039-11044. DOI: 10.1074/jbc.R109.091306.</li>
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     <li>López J, Bustos D, Camilo C, Arenas N, Saa PA, Agosin E. 2020. Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene. Front Bioeng Biotechnol. 8:578793. doi:10.3389/fbioe.2020.578793.</li>
 
</i>
 
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Latest revision as of 17:02, 17 October 2021


α-ionone induction system and expression in Saccharomyces cerevisiae (pViolette) Sequence and Features


Assembly Compatibility:
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Introduction

The pVIOLETTE part (BBa_K3930003) enables the production of α-ionone from lycopene and is composed of:

- the up (BBa_K3930021) and down (BBa_K3930022) integration sites in the XII-4 locus (Chr XII:830227..831248) of the S. cerevisiae genome (based on the plasmid pCfB3040 from Easyclone Marker free kit (Jessop-Fabre et al.,2016)).

- the LcyE-ofCCD1 fusion (BBa_K3930024) codes for a carotenoid cleavage dioxygenase fused to a lycopene cyclase, which allows the production of α-ionone. The sequences were codon optimized for expression into S. cerevisiae.

- the galactose inducible promoter pGal1 (BBa_K3930023), driving the expression of LcyE-phCCD1.

- the resistance marker NsrR (BBa_K3930025) to select yeast integrants.

Construction

IDT and Twist Bioscience performed the DNA synthesis and delivered the part as gBlock. The construct was cloned with the In-Fusion Takara kit into the pCfB3040 plasmid and then transformed into E.coli Dh5α strain. Figure 1 shows the restriction map of a correct resulting clones.

Figure 1: pVIOLETTE assembly

pVIOLETTE restriction profile from clone 3 was checked by digestion visualised on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel).


pVIOLETTE insert was then linearized with the pVIOLETTE_pCfB3040_Forward and pVIOLETTE_pCfB3040_Reverse linearization primers pVIOLETTE. Then the amplicon was integrated into the genome of our LycoYeast strain with the Takara Yeast transformation protocol. Figure 2 shows the electrophoresis gel of colony PCR to verify integrants genotype. The expected size was obtained for clone 2.

Primer used to clone this part in the pCfB3040:

  • pVIOLETTE_pCfB3040_Forward : 5' cgcccttattcgactctatag 3'
  • pVIOLETTE_pCfB3040_Reverse : 5' cgtacctggatggtcatttc 3'

Figure 2: Integration of pVIOLETTE insert in LycoYeast

pVIOLETTE insert integration from clone 1 and 2 was checked by PCR visualised on EtBr stained agarose electrophoresis ge. A theoretical gel is presented on the right (note that a different ladder is presented on the theoretical gel).


pVIOLETTE insert integration at locus XII-4 was successful. The integrant strain was named LycoYeast-pVIOLETTE and saved as glycerol stock.

Characterization

Production of ε-carotene

After verifying the correct integration of our insert by PCR, our engineered LycoYeast strains were placed on YPD plates containing the inducers with the aim to detect color changes due to the conversion of lycopenes (orange) to carotenes (yellow).

Figure 3 shows the colors of the colonies with or without the inducer, the galactose. The LycoYeast-pVIOLETTE strain plated on a YPD + galactose Petri plate shows a yellow coloration, indicating the conversion of lycopene into ε-carotene.


Figure 3: Color change in the modified LycoYeast-pVIOLETTE strain

The modified LycoYeast strains change from orange (lycopene) to yellow (carotene) upon galactose induction, which was the expected result.


The carotenoids contained in the cells were extracted using the method described by López et al. (2020). Yeast cells were lysed in acetone using glass beads and the supernatant obtained after lysis was analyzed by RP-HPLC on a C18 column. In the LycoYeast-pVIOLETTE strains, lycopene is converted into a new product with a higher retention time upon induction (Figure 4). Considering the yellow color of pVIOLETTE strain, as well as the α-ionone production results, this new peak most likely corresponds to ε-carotene, the expected precursor.


Figure 4: Carotenoid analysis of the engineered strain LycoYeast-pVIOLETTE

tr= retention time; 3 peaks are observed in a non-modified and a modified but not induced LycoYeast, while a 4th peak is present in a LycoYeast-pVIOLETTE strain.


Production of α-ionone

The α-ionone is very volatile. A common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest.The most common organic solvent used is dodecane for ionones (Chen et al. 2019; López et al. 2020).Figure 5 shows the GC-MS spectrum for the LycoYeast-VIOLETTE strain. A peak can be observed at the same retention time as the α-ionone standard for the induced LycoYeast-VIOLETTE strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The α-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). The production of α-ionone, the main molecule of the violet odor, was successfully achieved with this construction.


Figure 5: GC-MS analysis of the dodecane layer of the LycoYeast-pVIOLETTE

α-ionone is produced in vivo by our strain upon galactose induction. On the right are presented the mass spectra mass spectra of the observed peak corresponding to α-ionone. First panel is the α-ionone standard. Second panel is the LycoYeast WT. Third panel is the LycoYeast-pVIOLETTE non-induced. Forth panel is the LycoYeast-pVIOLETTE induced with galactose.


Conclusion and Perspectives

Our LycoYeast-pVIOLETTE strain effectively degrades degrade lycopene into ε-carotene and further transforms it into α-ionone. The quantification of α-ionone production remains to be determined under optimal conditions.

α-ionone belongs to the terpenes family and may have other uses besides perfumery, notably in medicine. We sincerely thank the future teams that will use this construction and encourage them to contact us for further details.

References

  1. Chen X, Shukal S, Zhang C. 2019. Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production. J Agric Food Chem. 67(49):13451–13459. doi:10.1021/acs.jafc.9b00860.
  2. Jessop-Fabre MM, Jakočiūnas T, Stovicek V, Dai Z, Jensen MK, Keasling JD, Borodina I. 2016. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Biotechnol J. 11(8):1110–1117. doi:10.1002/biot.201600147.
  3. López J, Bustos D, Camilo C, Arenas N, Saa PA, Agosin E. 2020. Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene. Front Bioeng Biotechnol. 8:578793. doi:10.3389/fbioe.2020.578793.