Difference between revisions of "Part:BBa K3570000"

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GGPP production enhancement in S. cerevisiae

Introduction

The purpose of this biobrick is to enhance the mevalonate pathway in S. cerevisiae to increase the pool of geranylgeranyl pyrophosphate (GGPP). The surplus of GGPP can be used to make S. cerevisiae produces provitamin A (𝛽-carotene), geraniol or limonene using BBa_K3570001, BBa_K3570002 or BBa_K3570003 biobricks respectively (figure 1), or any other product of interest.

Fig. 1: Metabolic pathway of 𝛽-carotene, limonene and geraniol from Acetyl-CoA in engineered yeast.

Design

According to Rabeharindranto et al. 2019, the enhancement of the mevalonate pathway can be achieved by overexpressing the HMG1 and CrtE genes. The construction as it is presented here differs from the publication in the choice of the promoter. We thus created the plasmids containing a truncated version of the HMG1 (tHMG1) gene from S. cerevisiae and the CrtE gene from X. Dendrorhous as on figure 2.

Fig. 2: tHMG1-CrtE part. The integrative locus used is DPP. The selective locus used is HIS. The genes coding for the truncated version of HMG1 comes from S. cerevisiae. The genes coding for CrtE comes from X. dendrorhous. The bidirectional TDH3-TEF1 promoter and the terminators CYC1 and PGK1 are used.

The HMG1 (3-hydroxy-3-methylglutaryl coenzyme A) enzyme is considered as a rate-limiting step in the mevalonate pathway. To counteract this, authors [2] amplified it's catalytic domain and named it tHMG1. The overexpression of tHMG1 and CrtE (GGPP synthase) in S. cerevisiae led to a significant improvement of carotenoid production because the direct precursor GGPP was increased[3].

The choice of a couple of promoters was essential for the optimal functioning of the construct since tHMG1 and CrtE needed to be expressed at a constant and similar level. TDH3 and TEF1 promoters proved themselves to have a non-significant difference in the expression level of the downstream gene, and to be quite versatile under different carbon sources for yeast[4]. TDH3 promoter is a gene-specific promoter from the yeast TDH3 gene[5], in parallel, TEF1 promoter is a gene-specific promoter from the yeast TEF1 gene[6]. The bidirectional TDH3-TEF1 promoter was designed for this construction. The sequence was identified from personal communication with Dr. Gilles Truan.

CYC1 and PGK1 terminators are chosen because of their large usage in yeast biotechnological manipulations[7]. The sequence was identified from the personal communication with Dr. Anthony Henras.

DPP1 upstream and downstream homology arms (BBa_K3570006 and BBa_K3570007) 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[8]. The sequence was identified from personal communication with Dr. Gilles Truan.

Finally, the HIS3 selection marker (BBa_K3570008) is a gene commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and the HIS3 gene in it) would be able to grow without histidine addition in the medium. This sequence was taken from RS313 plasmid [9].

Experiments

The cloning strategy consists to combine 6 blocks all together (B14 to B19). This has been divided in two constructions, one grouping B14, B15 & B16, the other B17, B18 & B19 (Figure 3).

Fig. 3: Cloning strategy of the part BBa_K3570000.

Construction of pUC19-B14B15B16
The gblocks B14, B15 and B16 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 4).

Fig. 4: PCR verification of the digested pUC19 and the three gblocks B14, B15 and B16 The expected strands are at 2.6kb, 0.4kb, 1.8kb and 1.0kb respectively.

pUC19 was digested by SbfI - BamHI and prepared to receive the PCR products B14, B15 and B16 by InFusion. After transformation of Stellar cells, selection on ampicillin, and minipreps of 8 clones, we checked the restriction profiles of the constructions. The results were then verified by digestion with the enzyme SacI (Figure 5).
We had six clones that had the expected profile.Since the sequence was valid, we had successfully obtained the first plasmid of our tHmg1-CrtE construction.

Fig. 5: Infusion verification: the expected sizes were 4.8kb and 1.2kb.

Built of the pUC19-B17B18B19
The gblocks B17, B18 and B19 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 6).

Fig. 6: PCR verification of the digested pUC19 and gblocks B17, B18, B19.

We digested the pUC19 vector by BamHI and EcoRI was done and purified the digested vector on gel. We proceeded to the InFusion reaction, transformation of Stellar cells, selection on ampicillin, and minipreps from 6 clones. The plasmids were assessed by restriction profiling with the enzymes BamHI and EcoRI.
Only one clone had the expected profile (figure 7). We sent it to be sequenced by Eurofins and it was fortunately valid. We also had successfully obtained the second plasmid of our tHmg1-CrtE insert.

Fig. 7: InFusion verification: the expected sizes were 4.8kb and 2.6kb.

Built of tHmg1-CrtE insert
The next step was to combine both plasmids by subcloning the fragment B14B15B16 into plasmid pUC19-B17B18B19.
To do this, we first extracted the DNA with the QIAGEN Plasmid Plus Midi Kit. Then, we digested both plasmids with SbfI and BamHI and purified with the Monarch Genomic DNA Purification Kit by NEB. The fragments were ligated together with T4 DNA ligase by NEB followed by a transformation into Stellar cells (ampicillin selection). Over the eight assessed colonies, two presented the expected restriction profile when digested with SbfI and EcoRI (Figure 8).

Fig. 8: Ligation verification: the expected size is 6.6kb and 2.6kb.

Yeast transformation
Since the construction was successful, we proceeded to the yeast transformation. The plasmid was digested with enzymes SbfI and EcoRI and purified to transform the yeast Saccharomyces cerevisiae. The yeast was then grown on YNB leu+, ura+, met+ his- and 2% of glucose for 3 days. At the third try, we were able to observe around 20 colonies in our yeast transformation, about the same on the positive control and none on the negative control plate.
To verify our colonies we performed a genomic PCR using the TaKaRa PCR Amplification Kit, so we randomly chose eight clones from our transformation and one from the positive control plate (Figure 9). All clones have the expected size (1.2kb), and the control, where we inserted pRS313 does not show any band , which means that BBa_K3570000 part is well integrated in the yeast genome. The integration in the yeast genome is a success that means that the parts BBa_K3570008 (HIS3 selective marker), BBa_K3570006 and BBa_K3570007 (for DPP1 homologous sequence) work. Our modified strain is BY4741 DPP1::tHMG1-crtE.

Fig. 9: Verification of the integration of BBa_K3570000 part into the yeast genome by PCR. BBa_K3570000 size is 1.2kb.

Analysis
The integration of the BBa_K3570000 part in the yeast genome should enhance the flow of the mevalonate pathway. This modification should result in an increase of the GGPP pool. GGPP have been extracted from BY4741 DPP1::tHMG1-crtE and analysed by LC-MS.
Our strain BY4741 DPP1::tHMG1-crtE produced GGPP five times than the wild type yeast. The BBa_K3570000 part therefore works.

Fig. 10: Results of the GGPP analysis from culture on YNB with 2% Glucose by LC-MS.

References

• [1]- Hery Rabeharindranto, Sara Castaño-Cerezo, Thomas Lautier, Luis F. Garcia-Alles, Christian Treitz, Andreas Tholey, Gilles Truan, 2019. Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metab Eng Commun. 2019 Jun; 8: e00086.
• [2]- Polakowski, T., Stahl, U., & Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology, 49(1), 66–71. https://doi.org/10.1007/s002530051138
• [3]- Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350. https://doi.org/10.1128/aem.02759-06
• [4]- Peng, B., Williams, T. C., Henry, M., Nielsen, L. K., & Vickers, C. E. (2015). Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microbial Cell Factories, 14(1). https://doi.org/10.1186/s12934-015-0278-5
• [5]- SGD:S000003424
• [6]- SGD:S000006284^
• [7]- 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
• [8]- S. cerevisiae genome, chromosome XVI, Ty4 LTR region. GenBank: CP046096.1
• [9]- RS313 plasmid

Contribution: PTSH-Taiwan 2023

Group: PTSH-Taiwan
Authors: Jen-Hsien,Liu
Summary: We identified the role of tHMG-1 in the production of Astaxanthin, and presented our results.
Documentation:

The astaxanthin biosynthesis pathway design includes a variety of essential enzymes that drive the conversion of acetyl-CoA into astaxanthin. This design aligns with the yeast's monocistronic system and relies on the Adh1 constitutive promoter and Adh1 terminator to facilitate gene expression within yeast cells. Within this pathway, genes like chyB, crtE, crtI, CrtYB, and bkt play a crucial role in encoding catalytic enzymes such as beta-carotene 3-hydroxylase, geranylgeranyl diphosphate synthase, phytoene dehydrogenase, Bifunctional lycopene cyclase/phytoene synthase, and β-carotene ketolase, respectively, all of which contribute to astaxanthin production.

To assist in the selection process, the genetic construct includes a hygromycin-resistance gene, hph, allowing for the identification and maintenance of yeast cells that have successfully integrated the desired genetic material.Notably, the naturally occurring gene tHMG-1 is also present in the Kluyveromyces Marxianus' genome.tHMG-1 plays a significant role in this process, as it is involved in the synthesis of acetyl-CoA, a crucial starting material for astaxanthin production.

The complete genetic construct is integrated into the pklac2 expression vector, which possesses the ability to replicate in E. coli and stably integrate into the yeast genome, particularly in kluyveromyces lactis.

Overall metabolic pathway of Astaxanthin

Phenotyping

Quantitative check