Part:BBa_K2739010
Hybrid promoter-PhaCB-Bktb
This composite part is designed to allow the investigation of PHBV production using PHA synthetic pathway (PHA operon) with phaA replaced by BtkB.
Usage and Biology
This composite part is involved both Bktb, phaCB, and under the control of a hybrid promoter. phaCB is part of the PHA operon, which is known to produce bioplastic PHA, contributing to the final two steps of the PHA production. The BktB was isolated from R. eutropha H16 and being recognised as a phaA paralogous, which allows the formation of 3-ketovaleryl-CoA. Bktb is recognised as a important phaA paralogous gene for PHBV production since it showed higher substrate specificity to the C5 monomer and used 3-ketovaleryl-CoA more efficiently (Mifune et al 2010).
Figure 1. Schematic illustration of the pathways leading to the PHBV biosynthesis.
Results and Discussion
In order to investigate the effect of produced protein BktB on cell growth, cells that harboured pSB1C3-phaCAB, pSB1C3-phaCAB-bktB or pSB1C3-phaCB-bktB were cultivated with 3 % glucose and various concentrations of propionic acid. Cell growth over time for strains expressing three different constructs and negative control with 3 % glucose was showed in Figure 3.14, in which E. coli strain BL21 (DE3) harbouring pSB1C3-phaCAB-bktB, pSB1C3-phaCB-bktB and pSB1C3 reached maximum optical density after 30 hours of cultivation and their maximum optical densities were estimated to be approximately OD600 1.0. Cells that harboured pSB1C3-phaCAB reached stationary phase after 50 hours with highest final optical density of OD600 1.5.
Figure 2. Time course of cell growth for different construction plasmids. Glucose was added into culture medium as carbon resource with final concentration of 3 %. OD600 was taken after 16 hours, 24 hours, 32 hours, 48 hours and 56 hours. Standard deviation was showed as error bar.
E. coli strain BL21 (DE3) that harboured these two plasmids was spread on the Nile red agar plates with negative control (pSB1C3) respectively, and two plates were exposed to blue light. Compared with negative control, the strong Nile red fluorescence from strains that harboured either pSB1C3-phaCAB-bktB or pSB1C3-phaCB-bktB, indicating that PHA (PHB and PHBV) production was assessed after 24 hours.
Figure 3. Nile red agar plate detection of PHA production.
Paralogous gene bktB represented similar function with phaA gene in the pathway, which showed higher specificity to C5 monomers contributed to the PHBV productivity and 3HV fraction. Although gas chromatograph remained to be done to analyse PHBV composition, lower melting temperature still gave strong suggestion that replace phaA gene with bktB could significantly increase the PHBV content in PHA production and co-expression of two genes would show small increase of PHBV production. Combined with the culture condition optimisation, cells harbouring pSB1C3-phaCB-bktB showed great potential to improve production of PHBV with higher 3HV fraction.
Table 1. Melting Temperature Measurement
Future work
Gas chromatograph analysis remain to be done to give more specific information about the composition of extracted PHA products including the percentage of PHBV content and the fraction of 3HV in PHBV, which are essential for confirming the effect of bktB on PHBV production.
References
Yu, S.T., Lin, C.C. and Too, J.R., 2005. PHBV production by Ralstonia eutropha in a continuous stirred tank reactor. Process Biochemistry, 40(8), pp.2729-2734. Shojaosadati, S.A., Varedi Kolaei, S.M. and Babaeipour, V. 2008. Recent advances in high cell density cultivation for production of recombinant protein. Iranian Journal of Biotechnology, 6(2), pp.63-84. Mifune, J., Nakamura, S. and Fukui, T., 2010. Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polymer Degradation and Stability, 95(8), pp.1305-1312.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 69
Illegal NheI site found at 92 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1041
Illegal BglII site found at 1866 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 347
Illegal NgoMIV site found at 418
Illegal NgoMIV site found at 1018
Illegal NgoMIV site found at 1330
Illegal NgoMIV site found at 1609
Illegal NgoMIV site found at 2768 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2836
Illegal BsaI site found at 3898
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