Part:BBa_K3904108

CHS

In our genetically engineered Escherichia coli Nissle 1917 and Lactobacillus casei BL23 probiotic strain 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

Arabidopsis thaliana CHS gene optimized for naringenin synthesis in E. coli and L. casei should efficiently catalyze the conversion of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 1186
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 1186
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 1186
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 1186
  Illegal NgoMIV site found at 511
  
• 1000
  COMPATIBLE WITH RFC[1000]

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.