Part:BBa_K759004

phaA (beta-ketothiolase orgin from Ralstonia eutropha)

This gene codes the enzyme“Beta- ketothiolase (acetyl-CoA acetyltransferase)”which catalyzes the conversion of two acetyl-CoA molecules into acetoacetyl-CoA. It is an enzyme that catalyzes the first step of P3(HB) producing pathway like the picture below.

This gene was cloned from pGEM phbCAB which carries the synthetic operon genes (phaA,phaB,phaC) of Ralstonia eutropha. In the pGEM phbCAB phaA, we found a mutation in the 00th base according to the data base of NCBI.

Contribution by Vilnius-Lithuania 2023

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.
Bacterial cellulose-PHB composite was achieved by introducing PHB synthesis operon into K. xylinus. This way, bacteria are able to simultaneously produce both polymers and combine them into one composite cellulose-PHB polymer.

Usage and biology

This part was used in the polyhydroxybutyrate (PHB) operon, which gene expression was realized in K. xylinus. The construct BBa_K4719017, containing phaA, 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 procedures at boiling temperatures are required.
Bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength, resistance and accelerated rate of biodegradation [1].

Experimental characterization

Constitutive expression of PHB synthesis genes in K. xylinus

We have evaluated whether the phaA gene can by expressed in K.xylinus by designing PHB synthesis operon. 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 constitutive PHB synthesis construct should appear red under UV light.

Figure 1: 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.

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 121
  Illegal NgoMIV site found at 217
  Illegal NgoMIV site found at 225
  Illegal NgoMIV site found at 237
  Illegal NgoMIV site found at 334
  Illegal NgoMIV site found at 574
  Illegal NgoMIV site found at 715
  
• 1000
  COMPATIBLE WITH RFC[1000]