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
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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
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INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 642
Illegal BamHI site found at 3039
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INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 37
Illegal PstI site found at 824
Illegal PstI site found at 1397
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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 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 of bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.
This part was specifically designed for incorporating PHB into bacterial cellulose-PHB polymer. This was achieved introducing this PHB synthesis operon into K. xylinus. Therefore, after transformation with plasmid, containing this operon, bacteria are able to simultaneously produce both polymers and combine them into one composite cellulose-PHB polymer.

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 strength and resistance, accelerated rate of biodegradation [1].
Since polymer production occurs in K. xylinus, it requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We chose 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). phaC, phaA, phaB genes were 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. Bacterial cellulose-PHB composite. 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 transformation of 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 different (Figure 3).

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

**Figure 3. FTIR spectra of bacterial cellulose-PHB composite compared to natural bacterial cellulose.** 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 with fluorescence. Colonies containing working constitutive PHB synthesis construct should appear red under UV light.

**Figure 4. Nile red A staining of bacterial cellulose-PHB composite.** **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.

Characterization 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. Bacterial cellulose-PHB composite surface characterization by SEM. 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 araBAD 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 araBAD promoter [2]. Additionally, varying concentrations of L-arabinose were tested to see if this had an effect on PHB production.

**Figure 6. Characterization of the best carbon source for araBAD promoter in *K. xylinus*. 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 use of 1 % glucose and 1 % sucrose as a carbon source. 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 araBAD promoter induction under different concentrations of L-arabinose.

**Figure 7. Results of *K. xylinus* staining with Nile red A.** This data shows that the araBAD 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. Also, when gene expression was not induced, it can be seen that the araBAD 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 parts 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.