Part:BBa_K3904109

CHI

In our genetically engineered Escherichia coli Nissle 1917 and Lactobacillus casei BL23 probiotic strains naringenin is synthesized from L-tyrosine with the action of four enzymes, i.e., tyrosine ammonia-lyase (TAL) 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) [1]. To assure continuous and efficient naringenin production, we had to guarantee the proper expression of naringenin synthesis enzymes in our probiotic strains. To reach the maximum efficiency of the natural synthesis pathway, we decided to manipulate the expression rates of these proteins by finding the promoters of optimal strength for the expression of each of these enzymes. Since both organisms do not share the same codon frequency, but using the shuttle vector for protein synthesis calls for optimal calculations for protein synthesis, Since we attempted to optimize proteins needed for naringenin synthesis for two organisms E. coli and L. casei.

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

Usage and Biology

Medicago sativa CHI gene optimized for naringenin synthesis in E. coli and L. casei should efficiently catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. This enzyme is responsible for the isomerization of 4,2',4',6'-tetrahydroxychalcone (also termed chalcone) into naringenin.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 667
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 667
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 667
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 667
  Illegal NgoMIV site found at 627
  Illegal AgeI site found at 420
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 214
  

References

1. Dunstan, M. S., Robinson, C. J., Jervis, A. J., Yan, C., Carbonell, P., Hollywood, K. A., ... & Scrutton, N. S. (2020). Engineering Escherichia coli towards de novo production of gatekeeper (2 S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol. Synthetic Biology, 5(1), ysaa012.