Part:BBa_K849000

3-Hydroxy-3-Methyl-Glutaryl-CoA-Reductase from Yeast

This is a truncated version of the 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme-A-Reductase (HMG-CoA-R) from Saccharomyces cerevisiae. It catalyses the reaction of 3-Hydroxy-3-Methyl-Glutaryle-Coenzyme-A to Mevalonate which occurs in the Mevalonate Pathway to Isopentylpyrophosphate which is the Precurser of all Isoprenoides. The enzyme is naturally a membrane bound enzyme. The membrane anchor is situated N-terminal. This region is also essential for the degradational control of the enzyme. By deleting the N-terminal part to Aminoacid (inclusive) the enzyme is expected to be water soluble and deregulated.

Trunctuation had been done according to the following publication: http://aem.asm.org/content/63/9/3341.short

K A Donald, R Y Hampton and I B Fritz: Effects of overproduction of the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase on squalene synthesis in Saccharomyces cerevisiae . Appl. Environ. Microbiol. 1997, 63(9):3341.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 685
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1456
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization-Team Nagahama 2017-

Fig.1 Overview of the ergosterol biosynthetic pathway in S. cerevisiae and the carotenogenic pathway in X. dendrorhous. The carotenogenic pathway in X. dendrorhous consists of GGPP synthase encoded by crtE, the bifunctional enzyme phytoene synthase and lycopene cyclase encoded by crtYB, and phytoene desaturase encoded by crtI. S. cerevisiae contains a GGPP synthase, encoded by BTS1, which is able to convert FPP into GGPP. HMG1 encodes HMG-CoA reductase, which is the main regulatory point in the ergosterol biosynthetic pathway in many organisms. IPP:isopentenyl diphosphate DMAP:dimethylallyl diphosphate GPP:geranyl diphosphate.

HMG1 encodes HMG-CoA reductase and catalyzes conversion of HMG-CoA to mevalonate, which is a rate-limiting step in sterol biosynthesis. Because mevalonate is synthesized, it is easy to flow to subsequent pathways. Therefore,β-carotene production increase. Color of colony was changed yellow from orange when tHMG1 was inserted into S. cerevisiae synthesizing carotenoid.(Fig.2,3,4) This result showed that tHMG1 was inserted.

tHMG1 has a characteristic of changing color of S. cerevisiae. This result to be due to the fact that as a result of massive synthesis of mevalonate, β-carotene was produced in large amount.

In order to investigate whether tHMG1 increased β-carotene productions, color of S. cerevisiae and β-carotene were compared. It is considered that the amount of β-carotene synthesized increased because the color of yeast introduced tHMG1 was closed the color of 1μg of β-carotene dissolved in 1 ml of hexane.

Fig2.S. cerevisiae synthesizing carotenoid by inserting crtYB, crtI and crtE into genomic DNA of S. cerevisiae.

Fig3.Result of colour of colony by inserting crtYB, crtI,crtE and tHMG1 into genomic DNA of S. cerevisiae.

Fig4.Result of the counterpart control S. cerevisiae which was transformed crtYB, crtI, crtE and empty vector isinto genomic DNA of S. cerevisiae.

Fig5.a:fig1,b:fig2,c:fig3,d:β-carotene

Fig6.S. cerevisiae was transformed with pYES2 plasmid inserted sod 2

S. cerevisiae transformed was grown onto sd-ura medium. This figure shows that the pYES2 plasmid inserted sod 2 was introduced into S. cerevisiae.

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 Marxianus.

Overall metabolic pathway of Astaxanthin

Figure 1. The Matabolic pathway of Astaxanthin production.

Phenotyping

Figure 2. The phenotyping of our strain for the third generation.

Quantitative check

Figure 3. The HPLC test for the third generation.

Reference

[http://aem.asm.org/content/73/13/4342.full]Verwaal R, Wang J, Meijnen JP, Visser H, Sandmann G, van den Berg JA, van Ooyen AJ (2007) High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol 73(13):4342–4350

Lin, Y. J., Chang, J. J., Lin, H. Y., Thia, C., Kao, Y. Y., Huang, C. C., & Li, W. H. (2017). Metabolic engineering a yeast to produce astaxanthin. Bioresource technology, 245(Pt A), 899–905. https://doi.org/10.1016/j.biortech.2017.07.116