Part:BBa_K4719018

pKARA_RT3 styrene monooxigenase for indigo synthesis in K. xylinus

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1226
  Illegal SpeI site found at 37
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1226
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 37
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1226
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1226
  Illegal SpeI site found at 37
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1226
  Illegal SpeI site found at 37
  Illegal NgoMIV site found at 92
  Illegal NgoMIV site found at 134
  Illegal NgoMIV site found at 503
  Illegal AgeI site found at 689
  
• 1000
  COMPATIBLE WITH RFC[1000]

Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. Firstly, we chose to produce a cellulose-chitin copolymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. 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. Lastly, we have achieved a bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.
This specific part was used for production of colored cellulose. Color was introduced by styrene monooxygenase pKARA_RT3 ( BBa_K4719018)to K. xylinus. This enzyme can metabolize indigo and its other derivatives into indigo dyes. Bacteria produces cellulose alongside pigments. Since they are not water soluble, the final polymer retains the color.

Usage and Biology

The function of this construct is to introduce indigo synthesis into K. xylinus. It was achieved by styrene monooxygenase pKARA_RT3, capable of metabolizing indole and other substrates like 5-bromindoline, 7-nitroindole, 7-methylindole, 1,6,7,8-tetrahydrocyclopentan, 5-nitroindole indole from the growth medium to obtain colorful bacterial cellulose in one step.
Dyed bacterial cellulose has applications as an alternative to leather because of its material properties, low infrastructure needs and biodegradability. What is more, the conventional process of dyeing textiles is harmful to the environment. This problem can be solved with applying synthetic biology to produce already colorful material [1].
Since polymer production occurs in K. xylinus, a specific plasmid (pSEVA331-Bb) backbone for successful replication is required. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable plasmid backbone for Komagateibacter species. We performed PCR of the plasmid eliminating mRFP to preserve Anderson promoter J23104 (BBa_J23104), RBS (BBa_B0034) and terminator ( BBa_B0015). pKARA_RT3 was assembled into the backbone by Gibson assembly.

Experimental characterization

Production of in situ dyed bacterial cellulose

In situ dyed bacterial cellulose is synthesized by K. xylinus grown in the Glucose Yeast Extract broth (GYB) while shaking at 180 rpm at 28°C, for 7 days. As a carbon source, we used 2% glucose. Substrates indole (0.5mM), 5-nitroindole (0.25mM), bromindoline (0.5mM), 7-nitroindole (0.25mM), 7-methylindole (0.25mM), 1,6,7,8-tetrahydrocyclopentan indole (0.25mM), were added to the growth medium for dye production by pKARA_RT3.

Figure 1. Dyed bacterial cellulose in the medium. A - bacterial cellulose grown on 5-bromindoline (0.5mM). B - bacterial cellulose grown on indole (0.5mM). C - bacterial cellulose grown on 1,6,7,8-tetrahydrocyclopentan indole (0.25mM). D - bacterial cellulose grown on 5-nitroindole. E - bacterial cellulose grown on 7-nitroindole. F - unmodified bacterial cellulose.

K. xylinus, which was transformed with a construct containing styrene monooxygenase pKARA_RT3, produced a range of indigoid compounds. As can be seen in Figure 1, some of the pigments were water-soluble, while others were contained in the structure of the bacterial cellulose. After purification, cellulose retained its color (Figure 2).

Figure 2. Dried dyed bacterial cellulose. A - bacterial cellulose grown on 5-bromindoline (0.5mM). B - bacterial cellulose grown on indole (0.5mM). C - bacterial cellulose grown on 7-nitroindole D - bacterial cellulose grown on 7-methylindole (0.25mM). E - bacterial cellulose grown on 1,6,7,8-tetrahydrocyclopentan indole (0.25mM). F - bacterial cellulose 5-nitroindole (0.25mM). G - unmodified bacterial cellulose.

Pigment HPLC-MS analysis of self dyed bacterial cellulose

We performed HPLC-MS analysis of the brightest colored samples, including bacterial cellulose produced by K. xylinus expressing pKARA_RT3 styrene monooxygenase when growth medium was supplemented with 7-methylindole, 5-nitroindole, 7-nitroindole.

Figure 3. Color comparison of DMSO and indigoid pigments extracted from self-dyed bacterial cellulose, grown in media supplemented with A - 5-nitro-indole, B - 7-methylindole or C - 7-nitroindole.

Testing pigments produced when 7-methylindole was used for cellulose’s self-dyeing revealed characteristic indigo UV absorption of 602 nm and molecular mass equivalent of 291 m/z (Figure 4 A), meaning that the registered substance might be 7,7-dimethylindigo (Figure 4 A). However, there seems to be a high amount of byproduct having similar UV absorption but possessing 16 Da smaller mass (Figure 4 A). Based on the UV absorption of 538 nm and molecular mass equivalent of 351 m/z (Figure 4 B), the substance extracted from cellulose when 5-nitroindole was present in GYB might be 5,5-dinitro-indigo (Figure 4 B). Although when 7-nitroindole is used as pKARA_RT3 substrate a characteristic molecular mass of 7,7-dinitroindigo (Figure 4 C) (351 m/z) and UV absorption is observed, a significant amount of byproducts is also seen (Figure 4 C). The high amount of byproducts may result from further indigo’s nitro groups modification by K. xylinus.

Figure 4. Results of HPLC-MS analysis. A - 7-methylindole, B - 5-nitroindole, C - 7-nitroindole metabolites produced by K. xylinus expressing pKARA_RT3.

Growth burden

In order to work with E. coli for designing constructs and testing synthetic biology parts, the growth burden of said constructs has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified E. coli DH5α. The composite of indigo synthesis did not inhibit the growth of E. coli as seen in Figure 5.

Figure 5. growth burden of pKARA_RT3 composite.

References

1.Walker, K.T. et al. (2023) Self-dyeing textiles grown from cellulose-producing bacteria with engineered tyrosinase expression [Preprint]. doi:10.1101/2023.02.28.530172.