Part:BBa_K4475000

CTB1 Gene from Cercospora beticola

This is the full exon only (cDNA) coding sequence for the CTB1 enzyme in the fungus cercospora beticola. The enzyme is the first in a multi step pathway for the production of cercosporin, a reactive oxygen species producing toxin in the presence of sunlight. FSU's iGEM team is looking to synthesize CTB1 in yeast as a proof of concept for cercosporin biosynthesis in a non-native and scalable organism, with the goal of producing cercosporin to combat algal blooms. The coding sequence has been codon optimized for Saccharomyces cerevisiae and modified to remove some illegal iGEM assembly cut sites via silent mutations. An HA tag is embedded on the N-terminus for expression verification.

Source: CB0940_00833 CTB1 [Cercospora beticola] https://www.ncbi.nlm.nih.gov/gene/35424645

Usage and Biology

This part is intended to be used as the first enzyme in the biosynthetic pathway of Cercosporin. It is intended to serve as an enzyme that can transform acetyl-CoA and 6 x malonyl-CoA into nor-toralactone. They were created from genome of C. beticola and sequentially inserted them into shuttle vectors for cloning in E.coli and episomal expression in S. cerevisiae.

Sequences and Features

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 3532
  Illegal BamHI site found at 3441
  Illegal BamHI site found at 5614
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 6121
  Illegal AgeI site found at 4195
  
• 1000
  COMPATIBLE WITH RFC[1000]

Modulation of Design

Choices of plasmid copy number and promoter strength were modulated to encourage maximum production in our chassis, resulting in several combinatorial designs. Molecular cloning was done using NEB HiFi DNA assembly, allowing DNA fragments for coding sequences and regulatory elements to be interchanged by annealing purposefully designed overlaps.

Enzyme and Product Detection

Assays were then implemented for enzyme and product detection

Characterization using HA Tagging

HA epitope tagging was used.

Characterization using HPLC

This analytical chemistry assay was used for product identification and quantification inspired by existing literature. (1,2,3)

References

(1) Newman AG, Townsend CA. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae. J Am Chem Soc. 2016 Mar 30;138(12):4219-28. doi: 10.1021/jacs.6b00633. Epub 2016 Mar 16. PMID: 26938470; PMCID: PMC5129747.

(2) Adam G. Newman, Anna L. Vagstad, Philip A. Storm, and Craig A. Townsend. Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming. Journal of the American Chemical Society 2014 136 (20), 7348-7362 DOI: 10.1021/ja5007299

(3) de Jonge R, Ebert MK, Huitt-Roehl CR, Pal P, Suttle JC, Spanner RE, Neubauer JD, Jurick WM 2nd, Stott KA, Secor GA, Thomma BPHJ, Van de Peer Y, Townsend CA, Bolton MD. Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum. Proc Natl Acad Sci U S A. 2018 Jun 12;115(24):E5459-E5466. doi: 10.1073/pnas.1712798115. Epub 2018 May 29. Erratum in: Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):E8324. PMID: 29844193; PMCID: PMC6004482.