Part:BBa_K4743034

Astaxanthin production set

The engineered Kluyveromyces marxianus exhibits a red coloration as a result of successful genetic modification, indicating the accumulation of astaxanthin within the cells. This color change serves as a visual confirmation of the genetic engineering success. Yeast cells reproduce through a process known as budding, wherein some newly formed buds may still be attached to the parent cell. While these buds may lack the desired genes, they can survive the antibiotic selection process due to the antibiotic resistance conferred by the engineered parent cell. It was observed that the cell sizes within the culture vary, and not all of them display the red coloration, suggesting that not all cells in the culture contain the desired genes.

To address this variation and further refine the culture, a three-generation streak selection method was employed, taking advantage of the easily observable red phenotype in the engineered 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.

References

1.Jin, J., Wang, Y., Yao, M., Gu, X., Li, B., Liu, H., Ding, M., Xiao, W., & Yuan, Y. (2018). Astaxanthin overproduction in yeast by strain engineering and new gene target uncovering. Biotechnology for biofuels, 11, 230. https://doi.org/10.1186/s13068-018-1227-4

2.Chang, J. J., Ho, C. Y., Ho, F. J., Tsai, T. Y., Ke, H. M., Wang, C. H., Chen, H. L., Shih, M. C., Huang, C. C., & Li, W. H. (2012). PGASO: A synthetic biology tool for engineering a cellulolytic yeast. Biotechnology for biofuels, 5(1), 53. https://doi.org/10.1186/1754-6834-5-53

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 8176
  Illegal PstI site found at 8266
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 8176
  Illegal NheI site found at 627
  Illegal NheI site found at 915
  Illegal NheI site found at 1119
  Illegal NheI site found at 2274
  Illegal NheI site found at 9773
  Illegal NheI site found at 10061
  Illegal NheI site found at 10265
  Illegal PstI site found at 8266
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 8176
  Illegal BglII site found at 725
  Illegal BglII site found at 5611
  Illegal BglII site found at 9871
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 8176
  Illegal PstI site found at 8266
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 8176
  Illegal PstI site found at 8266
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 4377
  Illegal BsaI.rc site found at 12941