Part:BBa_K3904102

surface layer protein A promoter (slpA) + 4-coumarate-CoA ligase (4CL)

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.

In our genetically engineered Escherichia coli Nissle 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.

Usage and Biology

The Lactobacillus acidophilus ATCC 4356 slpA promoter demonstrates the highest levels of transcription in all Lactobacillus strains examined in the study conducted by A. McCracken and colleagues. Combined with data from other researchers indicating that the slpA promoter is highly efficient in the natural host and L. casei ATCC 393, this suggests that the slpA promoter sequence harbours motifs that interact effectively with RNA polymerase of several lactobacilli. This has striking implications for the development of heterologous gene-expression cassettes in lactobacilli by offering a powerful tool for achieving high levels of foreign protein production [2]. Construct containing slpA promoter and Glycine max 4CL gene optimized for naringenin synthesis in E. coli and L. casei should efficiently synthesize 3,4',5-trihydroxystilbene from trans-4-coumarate.

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
  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.
2. McCracken, A., Turner, M. S., Giffard, P., Hafner, L. M., & Timms, P. (2000). Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species. Archives of microbiology, 173(5), 383-389.