Part:BBa_K2739009
Hybrid promoter-PhaCAB-Bktb
This is a composite part that was created to allow us to compare the functionality of bktB with phaA. The expression of phaCAB operon allow the production of bioplastic polyhydroxyalkanoate(PHA), such as PHB and copolymer, PHBV. In order to enhance the 3HV fraction in PHBV, paralog bktB was introduced into E. coli BL21 (DE3) with co-expression of phaCAB operon from Ralstonia eutropha.
Usage and Biology
This composite part is involved both the PHA operon and the Bktb, and under the control of a hybrid promoter. PHA operon is known to involve phaA, phaB and phaC and its expression allow the production bioplastic PHA. 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).
Co-expression of phaA and bktB could help to produce more PHBV and also improve the 3HV fraction in produced PHBV. phaCAB operon is enough for PHB production. Hou et al. (2013) suggested additional β-ketothiolases which catalysed reaction (1) shown in Fig. 1 was essential for PHBV production. bktB was isolated from R. eutropha H16 as the most important paralogous gene for PHBV production. It shows higher substrate specificity to the C5 monomer and is able to use 3-ketovaleryl-CoA more efficiently (Mifune et al 2010). The kinetic properties analysis presented that the specific activity of PhaA is 150 folds lower than BktB if 3-ketovaleryl-CoA was used as substrates, and the ability of PhaA and BktB to incorporate 3HV fraction in produced PHBV differed when growing in M9 minimal medium with 1 % glucose and different amount propionate (Slater et al., 1998).
Figure 1. Schematic illustration of the pathways leading to the PHBV biosynthesis.
Experiments and Results
In the following experiments, recombinant E.coli that harboured pSB1C3, pSB1C3-hybrid promoter-phaCAB (aka, pSB1C3-phaCAB, BBa_K1149051), pSB1C3-hybrid promoter-phaCAB-bktB (aka, pSB1C3-phaCAB-bktB, BBa_K2739009) and pSB1C3-hybrid promoter-phaCB-bktB (aka, pSB1C3-phaCB-bktB, BBa_K2739010) were used to assess the functionailty of BktB.
The effect of insert bktB gene on growth
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 in M9 medium with 3 % glucose to plot the growth curve (figure 2). E. coli strain BL21 (DE3) harbouring pSB1C3-phaCAB-bktB, pSB1C3-phaCB-bktB or pSB1C3 all 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.
The further investigation was performed to reveal whether the expression of bktB could help cells to tolerate higher concentration of propionic acid. Two concentrations of propionic acid were compared, the maximum optical density in 32 mM propionic acid was OD600 1.0 which was lower than that of cells cultured in 8mM propionic acid (OD600 2.2), indicating that the 32 mM propionic acid significantly affected the cell growth. Strains harbouring pSB1C3-phaCB-bktB showed best growth and reached highest optical density at stationary phase. Similar behaviour was performed as pSB1C3-phaCAB-bktB, the maximum optical density of OD600 1.9 was reached after 30 hours cultivation. The comparison of growth curves only indicated that bktB could help E. coli strain to grow better with 8mM propionic acid.
Figure 3. Comparison of cell growth of recombinant E. coli with different concentration of propionic acid concentration. Time of adding propionic acid was pointed out by red arrow. Optical density of cell culture was taken after 16 hours, 24 hours, 32 hours, 48 hours and 56 hours. Error bars represented the standard deviations.
Comparing the PHA production of E. coli expressing new construct
To confirm that the cell harbouring pSB1C3-phaCAB-bktB or pSB1C3-phaCB-bktB indeed produce PHA, 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 4. Nile red agar plate detection of PHA production.
PHA Extraction and Melting temperature measurement
After the fluorescent detection, which suggesting the recombinant E.coli with new constructs produce PHA, these strains (harboured pSB1C3-phaCAB, pSB1C3-phaCABbktB, pSB1C3-phaCB-bktB and negative control) grown with 3 % glucose and 8 mM propionic acid were harvested for PHA extraction. Compared PHA yield in two tables, the propionic acid significant affected the yield of PHA production.
Figure 5. Extracted PHA products. A. phaCAB. B. phaCB-bktb. C. phaCAB-bktb.
Table 1. Yield of PHA with 3 % glucose and 8 mM propionic acid
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 2. 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. Hou, J., Feng, B. and Han, J., 2013. Haloarchaeal type β-ketothiolases involved in poly (3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in Haloferax mediterranei. Applied and environmental microbiology, pp.AEM-01370. 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. Slater, S., Houmiel, K.L. and Tran, M.1998. Multiple β-ketothiolases mediate poly (β-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. Journal of bacteriology, 180(8), pp.1979-1987.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 979
Illegal BglII site found at 1804 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 285
Illegal NgoMIV site found at 356
Illegal NgoMIV site found at 956
Illegal NgoMIV site found at 1268
Illegal NgoMIV site found at 1547
Illegal NgoMIV site found at 2199
Illegal NgoMIV site found at 2221 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 4065
Illegal BsaI site found at 5108
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