Part:BBa_K4719017

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

Assembly Compatibility:

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INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 37
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INCOMPATIBLE WITH RFC[12]
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INCOMPATIBLE WITH RFC[21]
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INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 37
Illegal PstI site found at 824
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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
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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 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 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 of bacterial cellulose and polyhydroxybutyrate (PHB) composite, 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

To verify 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 2), the spectra are quite different (Figure 3).

**Figure 2:** chemical structure of PHB and bacterial cellulose

**Figure 3:** 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 4:** **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 5: 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 6: 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 was a carbon source of 1% glucose and 1% sucrose. Since a 6% concentration of L-arabinose did not produce significantly different results, we decided to test lower concentrations.

PHB accumulating cell staining with Nile red A

Bacterial cellulose-PHB composite producing cells were grown in GYB while shaking at 180 rpm at 28°C, for 7 days. Then the cells were stained with Nile red A, and fluorescence signal strength was measured to determine araC-pBAD promoter induction under different concentrations of L-arabinose.

**Figure 7:** This data shows that the araC-pBAD promoter in the PHB synthesis operon transformed to *K. xylinus* does not work when bacteria is grown on 2% glucose. However, when 1% glucose and 1% sucrose was used as a carbon source it was possible to achieve a different amount of PHB in the bacterial cellulose-PHB composite. **The best concentration of L-arabinose for the highest incorporation of PHB was 2%**, as the fluorescence signal corresponding to the content of stained PHB was the strongest. What is more, where gene expression was not induced, it can be seen that the araC-pBAD promoter is leaky on 2% glucose but not on 1% glucose and 1% sucrose.

Growth burden

In order to work with E. coli for designing constructs and testing synthetic biology systems, the growth burden of said synthetic biology tools 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 constitutive or inducible PHB synthesis did not inhibit the growth of E. coli as seen in Figure 8 and 9.

**Figure 9:** growth burden of inducible PHB synthesis composite.

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.