Part:BBa_K2408020

ADH1_KP6_proToxin

KP6 protoxin for inhibiting wine spoilage by Brettanomyces.

Alcohol Dehydrogenase 1 (ADH1) is a strong constitutive promoter isolated from Saccharomyces cerevisiae. The toxin KP6 binds to the outer membrane of the Brett. cell with the use of receptors. By doing so, it damages the protective membrane of the yeast and thus causes cell death. This toxin does not affect the wine yeast (S.cerevisia) so there is no problem using it. It functions maximally between temperatures of 5-20 degrees Celsius and PH ranging from 2.5-5 and the research on the subject found that it affects many types of Brett. and therefore we decided to use it. We also added the α-mating factor secretion signal and a histidine tag.

Design:

Objective: Cloning the gene of the Brett. specific toxin into S.cerevisiae, giving it the ability to inhibit Brett., which is detrimental to the wine industry. KP6's gene was taken from the yeast U.maydis. KP6 can inhibit Brettanomyces but does not harm S. cerevisiae.
Difficulties: KP6 is in a state of protoxin. In the yeast strain it was isolated from, the protein is cleaved by a protease, into two subunits - alpha and beta. We don't know for sure if this process will occur in S.cerevisiae, therefore we chose to create three different parts. The first is the original protoxin. The second and third contain the alpha and beta subunits, separately (BBa_K2408021, BBa_K2408022).

In addition, we want the toxin to be both constitutive and inducible by ethanol or sugar so that the expression of the toxin can be controlled at the desired time of fermentation.

· Santos, A., Navascués, E., Bravo, E., & Marquina, D. (2011). Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis. International journal of food microbiology, 145(1), 147-154

· Tao, J. I. A. N. S. H. I., Ginsberg, I. D. I. T., Banerjee, N. A. N. D. I. T. T. A., Held, W., Koltin, Y. I. G. A. L., & Bruenn, J. A. (1990). Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins. Molecular and cellular biology, 10(4), 1373-1381.‏

. Lin-Cereghino, G. P., Stark, C. M., Kim, D., Chang, J., Shaheen, N., Poerwanto, H., ... & Huang, A. D. (2013). The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene, 519(2), 311-317.‏

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 93
  Illegal BsaI.rc site found at 1410