Part:BBa_K4273018

pTDH3-EEVS-tTDH1-pPGK1-MTOx-tPGK1

EEVS-MTOX are genes from zebrafish, Danio rerio, that support the biosynthesis of gadusol and gadusolate. EEVS and MTOx are expected to facilitate the pathway from SH7 to gadusol to block UV rays and mass produce antidioxants for sunscreen uses. (Osborn et al., 2015). In our experiment, EEVS converts sedoheptulose 7-phosphate (S7P)to 2-epi-5-epi-valiolnoe (EEV). MTOX converts 2-epi-5-epi-valiolnoe (EEV) to gadusol and gadusolate.

We create a collection including BBa_K4273017, BBa_K4273018, BBa_K4273019, BBa_K4273020, BBa_K4273021 , BBa_K4273016 and BBa_K4273000. This collection can provide inspiration and efficient methods to utilize the penta phosphate pathway or to produce other types of MAAs in S. cerevisiae for other teams.

Usage and Biology

Promoters pTDH3 and pPGK1 are selected due to their strong and stable expression in S. Cerevisiae as well as in YPD culture mediums (Apel et. al., 2016). pTDH3 is shown to has the highest stability and strength, followed by pPGK1. Therefore, we used pTDH3 for DDGS and pPGK1 for MTOX for the purpose of gadusol production.

For the production of gadusol

For the production of gadusol, we used promoters pTDH3 to express EEVS and pPGK1 to express MTOX. We also inserted these genes into L2 yeast at position 308 to obtain L4 strain. After testing the absorption spectrum of the supernatant broth after 72 hours of fermentation, a slight absorption peak was observed at around 290 nm. However, we subtracted the curve of the negative control from L4's absorption curve (Figure 15B) in order to better observe the absorption spectrum. As a result, the relative OD curve of L4 strain shows an obvious absorption peak at 290 nm, which suggests the production of gadusol.

Figure 1: Insertion of EEVS-OMT in order to produce MAA-like molecule gadusol. By transforming CRISPR-308 plasmid pCRCT-308, LA, EEVS, MTOx, and RA, EEVS should be inserted at 308 position after assembly in S. cerevisiae (A). We expanded the homogenous arms, and EEVS, MTOX genes through PCR and transformed them into L3. We performed colony PCR on the yeast colonies to determine the existence of LA-EEVS and MTOX (C) and verified this result through the sequencing testing (D), obtaining the L4 strai..

Figure 2: Gadusol production. Fermenting the L4 strain, using the L2 strain as a comparison. After 72 hours of culturing, the absorption curve became clear, and it can be seen that L4 has a peak at 290nm (A). We compared L4’s values to the control, obtaining a clear absorption peak for gadusol.

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 1202
  Illegal BglII site found at 1456
  Illegal BglII site found at 3450
  Illegal XhoI site found at 2102
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1384
  Illegal AgeI site found at 1990
  Illegal AgeI site found at 3750
  Illegal AgeI site found at 3972
  Illegal AgeI site found at 4401
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2348
  Illegal BsaI.rc site found at 3460
  

Reference

Park SH, Lee K, Jang JW, Hahn JS. Metabolic Engineering of Saccharomyces cerevisiae for Production of Shinorine, a Sunscreen Material, from Xylose. ACS Synth Biol. 2019;8(2):346-357.

Jin C, Kim S, Moon S, Jin H, Hahn JS. Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae. FEMS Yeast Res. 2021;21(7):foab053.

Chen M, Rubin GM, Jiang G, Raad Z, Ding Y. Biosynthesis and Heterologous Production of Mycosporine-Like Amino Acid Palythines. J Org Chem. 2021 Aug 20;86(16):11160-11168.

Osborn AR, Almabruk KH, Holzwarth G, Asamizu S, LaDu J, Kean KM, Karplus PA, Tanguay RL, Bakalinsky AT, Mahmud T. De novo synthesis of a sunscreen compound in vertebrates. Elife. 2015 May 12;4:e05919.

Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2017 Jan 9;45(1):496-508.

Zhang H, Jiang Y, Zhou C, Chen Y, Yu G, Zheng L, Guan H, Li R. Occurrence of Mycosporine-like Amino Acids (MAAs) from the Bloom-Forming Cyanobacteria Aphanizomenon Strains. Molecules. 2022 Mar 7;27(5):1734.

Cress BF, Toparlak ÖD, Guleria S, et al. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol. 2015;4(9):987-1000.