Part:BBa_K4719017

phaCAB operon for polyhydroxybutyrate synthesis in K. xylinus
Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 37
  Illegal PstI site found at 824
  Illegal PstI site found at 1397
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal SpeI site found at 37
  Illegal PstI site found at 824
  Illegal PstI site found at 1397
  Illegal NotI site found at 200
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 642
  Illegal BamHI site found at 3039
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 37
  Illegal PstI site found at 824
  Illegal PstI site found at 1397
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 37
  Illegal PstI site found at 824
  Illegal PstI site found at 1397
  Illegal NgoMIV site found at 253
  Illegal NgoMIV site found at 368
  Illegal NgoMIV site found at 602
  Illegal NgoMIV site found at 914
  Illegal NgoMIV site found at 1193
  Illegal NgoMIV site found at 1606
  Illegal NgoMIV site found at 1673
  Illegal AgeI site found at 341
  
• 1000
  COMPATIBLE WITH RFC[1000]

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. Firstly, we chose to produce a cellulose-chitin polymer 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 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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), which is synthesized by K. xylinus.

We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into K. xylinus BBa_K4719017. The bacteria simultaneously produce both polymers combined into the same material during the purification process.

Usage and Biology

This construct is a polyhydroxybutyrate synthesis operon (phaC, phaA, phaB) producing PHB along with bacterial cellulose in K. xylinus. PHB is stored in bacteria intercellularly while cellulose is secreted outside of the cell. To combine both of these polymers washing procedure at boiling temperatures is required.

Bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strenght and resistance, accelerated rate of biodegradation [1].

Since polymer production occurs in K. xylinus requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool 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. phaC, phaA, phaB was assembled into the backbone by Gibson assembly.

Experimental characterization

Polymer production

Bacterial cellulose and polyhydroxybutyrate composite 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.

Figure 1: A - bacterial cellulose control group grown on 2% glucose. B - bacterial cellulose-PHB composite. The pellicles of the composite are much more opaque and connected, reminisced of the appearance of bioplastic.

FTIR spectra of bacterial cellulose-polyhydroxybutyrate composite

For verification that the approach of transforming K. xylinus with phaCAB operon produces bacterial cellulose-PHB composite we performed FTIR analysis to identify chemical moieties present in the material. Since PHB is composed of different monomers than cellulose (Figure 1), the spectra are quite different (Figure 2). https://static.igem.wiki/teams/4719/wiki/partai/phb-ir-celiuliozes-struktura.png

Figure 1: chemical structure of PHB and bacterial cellulose

Figure 2: Bands at 1589 and 1637 cm^-1 indicate absorbtion of OH group. While 1728 cm ^-1 corresponds with C=O group and 1537 cm ^-1 with CH₂ [3]. When comparing spectra of bacterial cellulose-PHB composite to control of bacterial cellulose, it can be seen that the composite was achieved successfully.

Constitutive expression of PHB synthesis genes in K. xylinus

To verify the synthesis of PHB, we supplemented growth media with 2.5µl/ml Nile red A. Nile red A is used to determine the presence of PHB by fluorescence. Colonies containing working constitutive PHB synthesis construct should appear red under UV light.

Figure 3: Left - bacterial cellulose control group grown on 2% glucose (negative control). Right - constitutive gene expression construct producing bacterial cellulose-PHB composite. K. xylinus can be identified as producing PHB.

Chracterization of polymer surface with SEM

To verify bacterial cellulose-PHB composite structural differences from natural bacterial cellulose, we performed scanning electron microscopy (SEM).

Figure 4: A - bacterial cellulose control group grown on 2% glucose. B - bacterial cellulose-PHB composite. The fibrils of the bacterial cellulose-PHB composite are more connected and do not have a pronounced mesh pattern as natural cellulose. This can be explained by the incorporation of PHB granules into cellulose structure.

Regulated PHB production in K. xylinus

We wanted to regulate the amount of PHB in the bacterial cellulose-polyhydroxybutyrate copolymer therefore, the production of PHB was put under an inducible araC-pBAD promoter. Gene expression of the PHB synthesis construct could be induced after a sufficient amount of bacterial cellulose has grown. For the characterization of this improved part, we selected to test different sugars as carbon sources for K. xylinus as glucose is known to inhibit pBAD promoter [2]. Additionally, varying concentrations of L-arabinose were tested to see if this had an effect on PHB production.

Figure 4: A - bacterial cellulose-PHB composite grown on 2% sucrose, gene expression induced with 4% L-arabinose. B - bacterial cellulose-PHB composite grown on 1% sucrose and 1% glucose, gene expression induced with 4% L-arabinose. C - bacterial cellulose-PHB composite grown on 1% fructose and 1% glucose, gene expression induced with 4% L-arabinose. D - control group of unmodified K. xylinus . E - control group of modified K. xylinus grown on media without Nile red A. F - bacterial cellulose-PHB composite grown on 2% fructose, gene expression induced with 4% L-arabinose. G -bacterial cellulose-PHB composite grown on 1% fructose and 1% sucrose, gene expression induced with 4% L-arabinose. H - bacterial cellulose-PHB composite grown on 2% glucose, gene expression induced with 4% L-arabinose. I - bacterial cellulose-PHB composite grown on 2% fructose, gene expression induced with 6% L-arabinose. J - control group of constitutive expression of PHB synthesis genes.

The best conditions for inducible PHB synthesis operon were carbon sources of 1% glucose and 1% sucrose, 1% fructose and 1% sucrose. Since a 6% concentration of L-arabinose did not produce significantly different results we decided to test lower concentrations.

Figure 6: A - bacterial cellulose control group grown on 2% glucose, 4% L-arabinose and Nile red A. B - bacterial cellulose-PHB composite grown on 1% sucrose and 1% glucose, gene expression was not induced. C - bacterial cellulose-PHB composite grown on 2% glucose, gene expression induced with 4% L-arabinose. D - bacterial cellulose-PHB composite grown on 1% sucrose and 1% glucose, gene expression induced with 0.5% L-arabinose. E - bacterial cellulose-PHB composite grown on 1% sucrose and 1% glucose, gene expression induced with 1% L-arabinose. F - bacterial cellulose-PHB composite grown on 1% sucrose and 1% glucose, gene expression induced with 2% L-arabinose. G - bacterial cellulose-PHB composite grown on 1% sucrose and 1% glucose, gene expression induced with 4% L-arabinose.

The plate where gene expression was not induced showed that the araC-pBAD promoter is slightly leaky. The optimal conditions to obtain the highest content of PHB after induction were 1% sucrose and 1% glucose as a carbon source, where gene expression was induced with 1% L-arabinose.

References

1. Ding, R. et al. (2021) ‘The facile and controllable synthesis of a bacterial cellulose/polyhydroxybutyrate composite by co-culturing Gluconacetobacter xylinus and Ralstonia eutropha’, Carbohydrate Polymers, 252, p. 117137. doi:10.1016/j.carbpol.2020.117137.
2. Teh, M.Y. et al. (2019) ‘An expanded synthetic biology toolkit for gene expression control in acetobacteraceae’, ACS Synthetic Biology, 8(4), pp. 708–723. doi:10.1021/acssynbio.8b00168.
3. López, J.A. et al. (2012) ‘Biosynthesis of PHB from a new isolated bacillus megaterium strain: Outlook on future developments with endospore forming bacteria’, Biotechnology and Bioprocess Engineering, 17(2), pp. 250–258. doi:10.1007/s12257-011-0448-1.