Part:BBa_K2739010
Hybrid promoter-PhaCB-Bktb
This composite part is designed to allow the investigation the role of BktB with the PHA synthetic pathway (PHA operon) when the phaA is removed. It is known the expression of phaCAB operon allow the production of bioplastic polyhydroxyalkanoate (PHA), such as PHB and copolymer, PHBV. BktB is recognised as a phaA paralogous and promote the PHBV production.
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).
Replace phaA with bktB help to understand the effect of bktB on PHBV production directly. In this study, the strong ribosonme binding site (hybrid promoter and RBS) was deliberately chose for bktB expression because previous studies have showed that the expression level of genes in an operon drop the further away the genes is from the promoter. And bktB gene was introduced into the downstream of phaCAB operon while phaA gene is closer to the promoter, which might result in different transcription level of bktB and phaA. Although bktB showed higher specificity to catalyse condensation of acetyl-CoA and propionyl-CoA, the transcription level of two genes would affect the translation of enzymes and lower amount of enzyme mean that the overall product levels are affected. Produced PhaA might compete the substrates (acetyl-CoA) with BktB, which affected the second pathway for 3HV fraction.
Figure 1. Schematic illustration of the pathways leading to the PHBV biosynthesis.
Results and Discussion
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 curve
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.
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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