Part:BBa_K4719009

pKARA_RT3 styrene monooxigenase

Introduction

Vilnius Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system in Komagataeibacter xylinus for in vivo bacterial cellulose polymer composition modification. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry.

Colorful cellulose was made by introducing styrene monooxygenase pKARA_RT3 BBa_K4719018 to K. xylinus. This enzyme can metabolize indole and its other derivatives into indigo dyes. Bacteria produce cellulose alongside pigments. Since they are not water soluble, the final product retains the color.

Usage and Biology

pKARA_RT3 is styrene monooxygenase. Styrene monooxygenases belong to oxidoreductases, specifically to an enzyme class 1.14.14-, which act on paired donors, with incorporation or reduction of molecular oxygen, which is donated by reduced flavin or flavoprotein. Styrene monooxygenases have been shown to produce indigo - a blue dye (1). Cells containing this enzyme are able to act as biocatalysts and convert indole to indigo. pKARA_RT3 is a basic part in BBa_K4719018, this construct is used for the production of colorful cellulose.

Characterisation

The sequence for pKARA_RT3 has been found by metagenomic analysis of a soil sample. To better understand the origin of this protein, we performed NCBI nucleotide BLAST analysis and with 99.11% identity, it was identified to be of Arthrobacter sp. StoSoilB22 DNA (Sequence ID: AP024652.1).

File:Https://static.igem.wiki/teams/4719/wiki/partai/bba-k4719009-pkara-rt3-nucleotide-blast-dot.png

Figure1: Dot plot of nucleotide sequence of pKARA_RT3 and identified sequence from Arthrobacter sp. StoSoilB22 DNA

For a better understanding of the function pKARA_RT3, we analyzed the amino acid sequence with UniProt BLAST analysis. The results showed with 93.2% identity to be Styrene monooxygenase StyA putative substrate binding domain-containing protein. This result was consistent with the experimental data of the activity of pKARA_RT3 to metabolize indigo to indole compounds.

File:Pkara-rt3-protein-blast-dot-plot.png

Figure1: Dot plot of nucleotide sequences of pKARA_RT3 and Styrene monooxygenase StyA putative substrate binding domain-containing protein

For further verification of pKARA_RT3 structure, we employed NCBI Domain architecture search. The two structures this tool identified were NAD-binding domain and a Fe-S cluster, as well Styrene monooxygenase A putative substrate binding domain. The results obtained by three different analysis were consistent and we were able to confirm the origin and function of the pKARA_RT3 protein.

References

1. O’Connor, K.E., Dobson, A.D. and Hartmans, S. (1997) ‘Indigo formation by microorganisms expressing styrene monooxygenase activity’, Applied and Environmental Microbiology, 63(11), pp. 4287–4291. doi:10.1128/aem.63.11.4287-4291.1997.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1171
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1171
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1171
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1171
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1171
  Illegal NgoMIV site found at 37
  Illegal NgoMIV site found at 79
  Illegal NgoMIV site found at 448
  Illegal AgeI site found at 634
  
• 1000
  COMPATIBLE WITH RFC[1000]