Difference between revisions of "Part:BBa K2739009"

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Hybrid promoter-PhaCAB-Bktb

This is a composite part that was created to allow us to test the functionality of BktB. PHA operon is known to produce bioplastic PHB. The coexpression of the BktB allow the production of 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 produce bioplastic PHB. 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

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 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.

The further investigation was performed to reveal whether the expression of BktB could help cells to tolerate higher concentration of propionic acid. Figure 3.15 showed cell growth curves of different constructs with 3 % glucose and 8 mM or 32 mM 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). When comparing the strains grown in 8mM propionic acid, strains harbouring pSB1C3-phaCB-bktB showed best growth and reached highest optical density at stationary phase. The empty plasmid pSB1C3-phaCAB-bktB as control demonstrated similar behaviour and maximum optical density of OD600 1.9 was reached after 30 hours cultivation. Cells that expressed pSB1C3-phaCAB reached stationary phase earlier with maximum optical density of OD600 1.87 which was lower than that of cell harbouring constructs with bktB inserted.

Comparing the same recombinant E. coli grown with glucose and 32mM propionic acid with their corresponding strains grown in glucose only, cells that harboured pSB1C3-phaCAB represented most significant differences of the maximum optical density. Without adding 32 mM propionic acid, cells harboured pSB1C3-phaCAB could reach final optical density of OD600 1.6 while cells grown with 32 mM propionic acid showed maximum optical density of OD600 0.5. Similar behaviour was performed as negative control, the maximum optical density drooped from OD600 0.9 to OD600 0.5 if 32 mM propionic acid was added as co-substrate. There were no big differences when compared the other two constructs. When comparing constructs grown with glucose and 8 mM propionic acid with corresponding constructs which grown with glucose only. All constructs reached higher optical density, among which the highest optical density of OD600 2.2 and the lowest of OD600 1.3 were observed from the cells that harboured pSB1C3-phaCB-bktB and pSB1C3 respectively.

After the 56 hours cultivation, the PH of culture was determined by PH strip tester. The PH of culture that contained glucose only was estimated to be approximately 4.8 while cells cultured with propionic acid showed higher PH approximately 5.0. There was no big difference among different constructs.

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.

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