Part:BBa_K3570002

Provitamin A synthesis from GGPP in S. cerevisiae

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1297
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
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• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2012
  Illegal NheI site found at 5006
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  INCOMPATIBLE WITH RFC[21]

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  Illegal BglII site found at 3364
  Illegal BglII site found at 5489
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  Illegal BamHI site found at 3157
  Illegal XhoI site found at 4943
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• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1297
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
  Illegal PstI site found at 3605
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1297
  Illegal PstI site found at 2357
  Illegal PstI site found at 2627
  Illegal PstI site found at 3605
  Illegal NgoMIV site found at 2018
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

This biobrick shall be used to boost the production of produce provitamin A (𝛽-carotene) in S. cerevisiae . 𝛽-carotene is one of the carotenoids produced in yeast. The metabolic pathway comprises multiple intermediate as well as side-products before reaching 𝛽-carotene (fig. 1). It starts with geranylgeranyl diphosphate (GGPP), which is a derivative from Mevalonate pathway. GGPP is importantly used in yeast since it is a precursor to carotenoids[1], tocopherols[2], and to geranylgeranylated proteins[3]. Therefore, for the best production yield of 𝛽-carotene production using this biobrick, it is best to use it in synergy with the "GGPP production enhancement in S. cerevisiae" biobrick (BBa_K3570000).

Fig. 1: From Rabeharindranto et al. 2019. Presumed biosynthetic pathway for the synthesis of β-carotene in X. dendrorhous[5]. The bifunctional lycopene cyclase/phytoene synthase (CrtYB) enzyme is depicted in orange, the phytoene desaturase enzyme (CrtI) is depicted in red. Orange or red boxes represent the localization of the modifications introduced by the CrtYB or CrtI enzymes respectively.

Design

Biosynthesis of β-carotene in X. dendrorhous begins with the production of phytoene from GGPP by the domain B of bifunctional lycopene cyclase/phytoene synthase (CrtYB). Phytoene desaturase (CrtI) then catalyzes four successive desaturation reactions to form lycopene. In the end, the domain Y of CrtYB performs the cyclization on both sides of lycopene to produce β-carotene (fig.1).

Fig. 2: CrtYB(ek)I-pUC19. The integrative locus used in yeast is HO. The selective locus used is URA3. The genes coding for CrtYB and CrtI come from X. dendrorhous and is fused using the artificial peptide linker ek. We use the TDH1 promoter and the tCYC1 terminator.

Eukaryotic CrtY enzymes are predicted to be transmembrane proteins. On the contrary, CrtB and possibly CrtI are supposedly cytosolic[4]. Additionally, in some fungi, the phytoene CrtY and CrtB catalytic activities are fused within a multidomain protein named CrtYB in Xanthophyllomyces dendrorhous[5]. The complete biosynthetic pathway of X. dendrorhous, with a multidomain CrtYB, was efficiently expressed in S. cerevisiae[6]. Even though this heterologous system is the most productive to date, the accumulation of β-carotene precursors in the pathway, such as phytoene, was remarked. Nevertheless, the overexpression of CrtI to overcome the metabolic bottleneck turned out to be only partially successful[6].

Recently, the natural tridomain fusion (CrtIBY) from Schizotrium sp. was expressed in Yarrowia lipolytica[7] but unfortunately, the β-carotene production yields were considerably lower than those obtained with the yeast-expressed X. dendrorhous configuration [6], [8]. We then searched to design an enzyme(s), that would have at the same time high production yields of β-carotene precursors (as in X. dendrorhous) with the spatial proximity of both cytosolic enzymes (CrtB and CrtI).

A strategy for creating an enzyme that would have crtYB et crtI activities and every listed property above was adapted from Rabeharindranto, H et al., 2019. This way, a tridomain fusion protein CrtYBekI was expressed in S. cerevisiae, with ‘‘ek’’ being a peptidic linker. This spatial reorganization of the carotenoid enzymes should reduce the accumulation of intermediates and reorient the metabolic fluxes towards β-carotene production.

TDH1 promoter proved itself to be quite versatile under different carbon sources for yeast[9]. TDH1 promoter is a gene-specific promoter from the yeast TDH1 gene[10]. The sequence was extracted from TDH1 gene in SGD[10]. CYC1 terminator was chosen because of its large usage in yeast biotechnological manipulations[11]. The sequence was identified from the personal communication with Dr. Anthony Henras.

HO upstream and downstream homology arms (BBa_K3570009 and BBa_K3570010) are used to target a functional yeast integration locus. This will result in homologous recombination within the Diacylglycerol pyrophosphate phosphatase 1 (DPP1) gene and thus integration into the S. cerevisiae's genome[12]. The sequence was identified from personal communication with Dr. Gilles Truan.

URA3 selection marker (BBa_K3570013) is a gene commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and the URA3 gene in it) would be able to grow without histidine addition in the medium. The sequence was taken from FL38 plasmid [13].

Experiments

Summary and cloning strategy (<a href="https://parts.igem.org/Part:BBa_K3570002" target="_blank">BBa_K3570002</a>):

Our strategy was to divide our part into two plasmids: one composed of blocks B20, B221 and B22 in pUC19 and forms pUC19-B20B21B22; and another composed of B23 and B24 also in pUC19 leading to pUC19-B23B24. Then we would use pUC19-B23B24 as a template vector to insert B20B21B22.

Fig. 3: Cloning strategy for CrtYB and CrtI cloning

Results and discussion:

Built of pUC19-B20B21B22:

The blocks B20, B21 and B22 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (data not shown). pUC19 was digested by SbfI - XmaI and purified on gel. We proceeded to the InFusion reaction, transformation of Stellar cells, selection on ampicillin, and miniprep of clones. Unfortunately, this cloning repeatedly failed. So we decided to first clone B21 and B22 in the pUC19. The resulting colonies were checked by PCR (Figure 24).

Fig. 4: Verification by PCR of the block insertion

To verify our cloning we tested two different PCRs on our four clones, one showed us a 4300bp fragment and another a 300bp fragment. The line “F4000” is a fragment of 4000bp which serves as a reference. The two Crt - columns are negative controls.

We observed that 2 clones seemed to match our expectations. Both were sequenced and appeared to us as valid (or so we felt…). After following the same protocol to insert the last block B20, we obtained many colonies and tested the restriction profiles of six of them (figure 25).

Fig. 5: Verification by digestion with SbfI and XbaI of six ampicillin selected plasmids

The six columns contain different colonies that have been digested by restriction enzymes.

Among the 6 clones, only two presented the right size (1300 pb). We sent them to be sequenced by Eurofins, and the sequence again appeared as valid to us…

Built of pUC19-B23B24:

The blocks B23, and B24 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (data not shown). The pUC19 vector was digested by EcoRI - XmaI and purified on gel. We proceeded as usual to the InFusion reaction, transformation of Stellar cells, selection on ampicillin, and minipreps of a few, …well…, of the unique clone. Its plasmid was checked by restriction profiling (figure 26).

Fig. 6: Verification by digestion of our plasmid

We obtained only one clone on the petri dish after transformation. So we made several digestions on our miniprep to verify our cloning (line 1: SbfI and XmaI, line 2: XmaI and EcoRI, line 3: EcoRI and EcoRV).

Luckily enough (for a change !), this single clone got the right digestion profile so we sent it to be sequenced and it was fine.

Built of pUC19-CrtYB-CrtI:

The next step was to integrate B20B21B22 into pUC19-B23B24 as a vector with a classical cloning method. Sufficient plasmid quantities were first produced with the QIAGEN Plasmid Plus Mini Kit. Both plasmids were then digested with SbfI and XmaI and purified. The ligation was performed with T4 DNA ligase by NEB followed by a transformation into Stellar cells (ampicillin selection). The next day, we obtained hundreds of colonies so we chose six of them for a DNA extraction on the following day. To verify our colonies, we performed a PCR and a digestion. From the six clones tested by PCR, four were positive. We tested two of these clones by a series of digestion to validate our cloning.

Fig. 7: Verification by digestion of our plasmid

We performed three digestions with different enzymes, D1: SbfI and EcoRI, D2: SbfI and XmaI, D3: EcoRI and EcoRV. Our clones presented the right digestion profile, which allowed us to proceed to the transformation stage in the yeast that had previously integrated tHmg1-CrtE.

Yeast transformation:

We tried a first round of transformation in the previously obtained Hmg CrtE yeast strain. We were hoping for orange-colored yeast but our clones were desperately white. At this step, we had another careful look at our sequencing results. Knowing that something was wrong, we identified a mutation at the very beginning of block B22, that we thought was not significant because of its unusual location. Unfortunately, this mutation was real and triggered a shift in the reading frame, preventing the expression of CrtYB-CrtI. To solve this problem, we decided to undertake a directed mutagenesis of the plasmid by using the InFusion kit. The mutation is a success, the deletion is deleted, the reading frame is restored.

Because of the COVID-19 regulations and the university courses that are starting, we did not have time to continue our manipulation. After the correction of the mutation we just had to integrate the yeast to check if there is a production of β-carotene.

References

• [1]- Rabeharindranto, H., Castaño-Cerezo, S., Lautier, T., Garcia-Alles, L. F., Treitz, C., Tholey, A., & Truan, G. (2019). Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metabolic Engineering Communications, 8, e00086
• [2]- DIPLOCK, A. T., GREEN, J., EDWIN, E. E., & BUNYAN, J. (1961). Tocopherol, Ubiquinones and Ubichromenols in Yeasts and Mushrooms. Nature, 189(4766), 749–750. https://doi.org/10.1038/189749a0
• [3]- Ohya, Y., Qadota, H., Anraku, Y., Pringle, J. R., & Botstein, D. (1993). Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Molecular Biology of the Cell, 4(10), 1017–1025. https://doi.org/10.1091/mbc.4.10.1017
• [4]- Schaub, P., Yu, Q., Gemmecker, S., Poussin-Courmontagne, P., Mailliot, J., McEwen, A.G., et al., 2012. On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/ isomerase. PLoS One 7, e39550.
• [5]-Verdoes, J.C., Krubasik, P., Sandmann, G., Van Ooyen, A.J.J., 1999. Isolation and functional characterization of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous. Mol. Gen. Genet. MGG 262, 453–461.
• [6]-Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., Berg, J.A. van den, et al., 2007. High-level production of beta-carotene in saccharomyces cerevisiae by successive transformation with carotenogenic genes from xanthophyllomyces dendrorhous. Appl. Environ. Microbiol. 73, 4342–4350.
• [7]-Gao, S., Tong, Y., Zhu, L., Ge, M., Jiang, Y., Chen, D., et al., 2017. Production of βcarotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica. Biotechnol. Lett. 39, 921–927
• [8]-Xie, W., Ye, L., Lv, X., Xu, H., Yu, H., 2015. Sequential control of biosynthetic pathways for balanced utilization of metabolic intermediates in Saccharomyces cerevisiae. Metab. Eng. 28, 8–18.
• [9]- Monfort, A., Finger, S., Sanz, P., & Prieto, J. A. (1999). Evaluation of different promoters for the efficient production of heterologous proteins in baker's yeast. Biotechnology Letters, 21(3), 225–229. https://doi.org/10.1023/a:1005467912623
• [10]- SGD:S000003588
• [11]- Curran, K. A., Karim, A. S., Gupta, A., & Alper, H. S. (2013). Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metabolic Engineering, 19, 88–97. https://doi.org/10.1016/j.ymben.2013.07.001
• [12]- S. cerevisiae genome, chromosome IV, HO gene. GenBank: CP046084.1
• [13]- FL38 plasmid