Background 

In order to improve the productivity of PHA, the culture condition optimisation was performed to investigate the ideal glucose concentration and propionic acid concentration. Currently, PHB and PHBV as biodegradable plastic candidates are being produced in a twostage glucose/propionate fed batch fermentation progress using Ralstonic eutropha or recombinant Escherichia coli (Masani et. al, 2009). In order to give the real-time data of PHA production during the cultivation, the new real-time productivity measurement was first established and tested with Nile red fluorescent dye through the Image J program, then assess the dry PHB weight.

Aims 

In this sub-project, the main objective is to find the effect of optimised culture conditions for E.coli harbouring phaCAB  operon on growth and PHA production. These results then indicate the best culture condition(s) for the rest of the group.

Materials and Methods

In the experiment, recombinant E. coli  harbouring phaCAB  were used to culture with different conditions up to 56 hours.

• Mmeasurement

Recombinant strains were cultured in M9 medium with 3% glucose and 25g/ml chloramphenicol. By measuring optical density of cells, the effect of different culture conditions on cell growth could be determined. The production of PHB is confirmed by Nile red culture staining and followed by quantitatively determination through the fluorescent intensity and PHB extraction for dry bioplastic mass.

Results and Discussion

The iGEM Edinburgh OG 2018 team decided to characterize the ideal glucose concentration for Hybrid promoter with PHA operon. E. coli cells were cultured with different concentrations of glucose at 37 ℃ and optical density was measured at cultivation time of 6 hours, 24 hours, 30 hours 48 hours, 56 hours and 72 hours. In order to assess the Real-time PHA detection by Semi-quantitative analysis (Nile Red Fluorescent intensity-through using Image J program). Cell cultures were harvested at different cultivation hours including 6 hours, 24 hours, 30 hours and 48 hours. The bars in blue and orange represented signal intensity of cells harboured pSB1C3-phaCAB and pSB1C3 respectively. The bars in yellow represented signal intensity resulting from PHA, which were calculated from the difference between intensity of pSB1C3-phaCAB and intensity of pSB1C3. The team then decided To determine the influence of glucose availability on PHA production level, in addition to the growth curves with different glucose concentrations.

Figure 1 Comparison of growth curve of recombinant E. coli harbouring pSB1C3-phaCAB for different concentration of glucose.

Figure 2 The fluorescent intensity of PHA produced with different glucose concentrations. (48 hours cultivation). Error bars represented standard deviations.

Table 1. Yield of PHA of pSB1C3-phaCAB with different glucose concentrations

Figure 3. Fluorescent intensity of cells harboured pSB1C3 or pSB1C3-phaCAB at different cultivation time.

In order to determine the tolerance of propionic acid, which allow the later investigation with the Bktb  gene, E. coli harbouring pSB1C3-phaCAB plasmids were cultured with different concentration of glucose and propionic acid for 56 hours.

Figure 4 Comparison of growth curves with different concentration of glucose and propionic acid. The time of adding propionic acid was pointed out by red arrow.

References 

• Masani, M.Y.A., Parveez, G.K.A. and Izawati, A.M.D. 2009. Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter.. Plasmid, , 62(3), pp.191-200..

Background 

Various gram-negative bacteria are known to synthesise PHAs, among which Ralstonic eutropha  is regarded as the model bacterium of PHA synthesis because three important genes of phaA, phaB  and phaC  are discovered in its genome (Moorkoth and Nampoothiri, 2016). These three genes which have been organised into an operon phaCAB  encode three essential enzymes for production of PHB. Some research showed that genes positioned closer to the promoter could have higher expression than the genes further away from promoter, and this expression level significant affect the activity of essential enzymes for PHB production, PHB molecular weight and accumulation level (Hiroe et al., 2012).

Aims 

In this sub-project, the main objective is to find the effect of gene order of phaCAB  on PHB production. Moreover, we aim to optimize the order of phaA , phaB  and phaC  genes for obtaining the most balanced PHB production.

Materials and Methods

• Construct establishment

All the gene fragments with different overhangs were amplified by PCR amplification. Then Gibson Master Mix was used to assemble all the fragments and build constructs in new gene orders.

Figure 2 The workflow of Gibson assembly - Fragments are amplified with overlap, followed by incubation at 50˚C for 15-60 min, with Gibson Assembly Master Mix, which includes three essential enzymes (5´ exonuclease, DNA polymerase and DNA ligase). Those essential enzymes enable new construct to be assembled (Chan).

• PHB production confirmation and measurement

Recombinant strains were cultured in M9 medium with 3% glucose and 25g/ml chloramphenicol. By measuring optical density of cells that harboured different constructs including phaCAB, phaCBA, phaACB, phaACB, phaACB and phaABC, the effect of gene order on cell growth could be determined. The production of PHB is confirmed by Nile red plate/culture staining and quantitatively determined by measuring dry weight of extracted PHB.

Results and Discussion

Four constructs including phaCAB, phaCBA, phaACB  and phaBCA  were successfully constructed while the establishment of phaACB  and phaBAC  failed due to the inappropariate primers design.  Nile red plate staining confirmed PHB production from cells that harboured four new constructs (phaCAB, phaCBA, phaACB  and phaBCA). By measuring the yield of produced PHB, the most balanced PHB production is from E. coli  that harboured original phaCAB   operon and phaACB  operon.

Table 1 The yield of produced PHB

Plasmid	Culture volume	Extracted PHB (g)	Yield of PHB (mg/ml)
pSB1C3	150ml	0	0
phaCAB	150ml	0.237	1.58
phaCBA	150ml	0.072	0.48
phaACB	150ml	0.201	1.34
phaBCA	150ml	0.021	0.14

Note: E. coli  that harboured different constructs were cultured in M9 medium with 3% glucose for 48 hours at 37 ℃ shaker.

 

Future Works

Further study about new constructs is remained be performed. For instance, the enzyme activities assays help to figure out how enzyme activities effected by the different gene order.

References 

• Geyer, R., Jambeck, J.R. and Law, K.L., 2017. Production, use, and fate of all plastics ever made. Science advances, 3(7), p.e1700782.
• Bonartsev, A.P., Zharkova, I.I., Yakovlev, S.G., Myshkina, V.L., Mahina, T.K., Voinova, V.V., Zernov, A.L., Zhuikov, V.A., Akoulina, E.A., Ivanova, E.V. and Kuznetsova, E.S., 2017. Biosynthesis of poly (3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: A precursor feeding strategy. Preparative Biochemistry and Biotechnology, 47(2), pp.173-184.
• Zakaria, M.R., Ariffin, H., Johar, N.A.M., Abd-Aziz, S., Nishida, H., Shirai, Y. and Hassan, M.A., 2010. Biosynthesis and characterization of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer from wild-type Comamonas sp. EB172. Polymer Degradation and Stability, 95(8), pp.1382-1386.
• Moorkoth, D. and Nampoothiri, K.M., 2016. Production and characterization of poly (3-hydroxy butyrate-co-3 hydroxyvalerate)(PHBV) by a novel halotolerant mangrove isolate. Bioresource technology, 201, pp.253-260.
• Siu-Hong Chan, Ph.D., New England Biolabs, Inc.: Restriction Endonucleases: Molecular Cloning and Beyond. https://international.neb.com/tools-and-resources/feature-articles/restriction-endonucleases-molecular-cloning-and-beyond 2018/8/10.
• Hiroe, A., Tsuge, K., Nomura, C.T., Itaya, M. and Tsuge, T., 2012. Rearrangement of gene order in the phaCAB operon leads to effective production of ultra-high-molecular-weight poly [(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Applied and environmental microbiology, pp.AEM-07715.

Figure 3 Schematic illustration of the pathways leading to the PHBV biosynthesis

Background

As a typical role in the PHAs family, poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) is more likely to be a potential candidate for thermoplastic because of its higher thermal stability and flexibility which could be optimised by adjusting 3-hydroxyvalerate (3HV) fractions (Yu et al., 2005). Escherichia coli is one of the best-studied bacteria and is used as an ideal host for PHB and PHBV production because of its well-studied genetics and metabolism. In addition, a high-cell-density cultivation strategy contributed to improve polymer yield and productivity (Shojaosadati et al., 2008). Whole genome analysis of R. eutropha H16 identified several genes as paralogous to the phaA, and the bktB was isolated from R. eutropha H16 as the most important 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). 

Aims

We aim to enhance the 3HV fraction in PHBV, by co-expressing paralog bktB with phaCAB operon. In order to improve the productivity of PHBV, the culture condition optimization was performed to investigate the ideal glucose concentration and propionic acid concentration. 

Materials and Methods

In this study, we constructed a PHBV synthesis pathway by introducing the phaA, phaB and phaC genes into E. coli BL21 (DE3). To improve the 3HV fraction in the copolymer, the phaA paralog bktB from R. eutropha H16 was introduced into E. coli as co-expression or replacement of phaA. To optimise the culture conditions for PHBV production, different concentrations of glucose and propionic acid were applied. The yield of accumulated intercellular PHA was first determined after the extraction and its thermal properties were fist determined by melting temperature measurement. 

Results and Discussion

E. coli strain BL21 (DE3) that harboured these two plasmids was spread on the Nile red agar plates with negative control (pSB1C3) respectively, and the two plates were exposed to blue light. Compared with negative control, strong Nile red fluorescence observed from strains that harboured either pSB1C3-phaCAB-bktB or pSB1C3-phaCB-bktB indicated that PHA (PHB and PHBV) production was assessed after 24 hours.

Figure 4 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 Chromatography remained to be done to analyse PHBV composition, lower melting temperature still gave strong suggestion that replacing 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 temperatures of PHA from various sources

	Tm 1(°C)	Tm 2 (°C)	Tm 3 (°C)
Pure PHB product from Sigma	170-179	168-176	168-174
PHBV with 12% 3HV from Sigma	159-161	160-160	161-164
PHA from pSB1C3-phaCAB	160-168	160-162	161-163
PHA from pSB1C3-phaCB-bktB	150-155	149-151	149-152
PHA from pSB1C3-phaCAB-bktB	155-159	156-161	157-159
PHB extraction	168-180	166-178	169-179

Future Works

Gas chromatography remains to be executed 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., Babaeipour, V. and Farnoud, A.M., 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.

Background

Low molecular weight protein phasin encoded by gene phaP, is able to bind to the surface of PHA granules and play essential roles in PHB sysnthesis and granule formation (York et al., 2001). The expression of phasin is regulated by the autoregulated repressor PhaR which is able to bind to the region of phaP promoter and its own promoter to regulate phasin and itself. In addition, PhaR was detected binding on the surface of PHA granules, which potential help cells to save energy by curtailing excessive expression of PHA biosynthesis pathways (Pötter et al., 2002). Protein can be secreted through the type I secretion pathway of E. coli by co-expressing HlyA signal peptide which is able to interact with HlyB/HlyD complex (Mergulhao et al., 2005). The sequence amplified from the BioBrick BBA_K390501 was confirmed to have a stop codon between phasin and hlyA by sequencing. Due to the existence of stop codon between phaP and hlyA, the biobrick of HlyA-depending PHB secretion from the previous iGEM team needs to be engineered.

Aims

We aim to investigate the effect of phasin and PhaR autoregulation system on PHB production and cell growth. In addition, we aim to investigate the influence of co-expressing phaP, phaR and hlyA on the PHB production. Moreover, the stop codon between phaP and hlyA was removed by PCR amplification.

Materials and Methods

Two biobricks BBa_K390501 and BBa_K 1149051 were used to establish new constructs including pSB3T5-ProR-phaR-ProP-phaP, pSB3T5-ProR-phaR-ProP-phaP-hlyA, pSB3T5-ProR-phaR and pSB3T5-ProP-phaP. New constructs were co-transformed with phaCAB operon to the E. coli BL21 (DE3) strain respectively. Optical density was measured to investigate the effect of pSB3T5-ProR-phaR-ProP-phaP and pSB3T5-ProR-phaR-ProP-phaP-hlyA on cell growth. The PHB production was confirmed by Nile red staining followed by measurement through plate reader. The total production of PHB consists of intracellular PHB and secreted PHB, which was measured and compared after the PHB extraction. 

Results and Discussion

The stop codon between phaP and hlyA was removed forming a part for new Biobrick that could be submitted to the iGEM registry. The results obtained from cell viability assay demonstrated that the expression of phaCAB operon was likely to pose extra pressure on cells, while the regulation of PhaR and phasin can release the burden in some extent. The PHB produced by E. coli that harboured pSB3T5-ProR-phaR-ProP-phaP-hlyA is the highest. And the comparison of PHB yield listed below, indicating that the presence of PhaR regulator effectively regulate the expression of phasin and moderate the biosynthesis of PHB.

 Table 3 The yield of PHB production

Plasmid	Culture volume (ml)	Intracellular PHB (g)	Secreted PHB (g)	Total PHB (g)	Yield of PHB (mg/ml)
phaCAB+pSB3T5	150ml	0.01	0	0.01	0.0667
phaCAB+pSB3T5-R-P	150ml	0.071	0.0015	0.0725	0.4833
phaCAB+pSB3T5-R-P-hlyA	150ml	0.043	0.0305	0.0735	0.4900
phaCAB+pSB3T5-R	150ml	0.071	0.002	0.073	0.4868
phaCAB+pSB3T5-P	150ml	0.071	0	0.071	0.4733

Note: E. coli that harboured different constructs were cultured in M9 medium with 3% glucose for 48 hours at 37°C shaker

 

References

York, G.M., Stubbe, J. and Sinskey, A.J., 2001. New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate. Journal of Bacteriology, 183(7), pp.2394-2397.

Pötter, M., Madkour, M.H., Mayer, F. and Steinbüchel, A., 2002. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology, 148(8), pp.2413-2426.

Mergulhao, F.J.M., Summers, D.K. and Monteiro, G.A., 2005. Recombinant protein secretion in Escherichia coli. Biotechnology advances, 23(3), pp.177- 202.

Background

There are many bio-based alternatives to the widely used petrochemical-based plastics, which are degradable and therefore are less damaging to the environment, and to health. One such ‘bio-plastic’ are polyhydroxyalkanoates (PHAs), which is a large family with a huge range of properties. However, only one type of microbe produced PHA is affordable – poly(3- hydroxybutyrate) (PHB) – but too brittle for widespread use. PHA co-polymers, such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are more flexible, and could fill niches that PHB does not. However, producing PHBV by microbes typically needs propionate to be supplied exogenously (Babu, et al., 2013). This is unsustainable as the propionate is normally sourced from unnatural sources. E. coli does encode many of the genes needed to produce propionate from glucose, in the Sleeping Beauty Mutase (SBM) operon, which encodes an incomplete pathway for propionate production (Kannan, 2008)

Figure 5 Proposed mechanism for propionate synthesis utilising the Sleeping beauty mutase operon (SBM) and Methylmalonyl-CoA epimerase (MCE) - Succinyl-CoA is converted into Methylmalonyl-CoA-R by the methylmalonyl- CoA mutase ScpA. Methylmalonyl-CoA-R is converted into Methylmalonyl-CoA-S by MCE or an uncharacterised, native pathway. Methylmalonyl-CoA-S is converted into propionyl-CoA by the methylmalonyl-CoA carboxylase ScpB. The CoA from Propionyl-CoA is transferred onto Succinate from the citric acid cycle by the Propionyl-CoA: Succinate CoA transferase ScpC, resulting in the production of propionate and Succinyl-CoA.

 

Aim

Our aim is to construct plasmids harbouring the SBM operon and MCE. These constructs were to be assayed for propionate production, followed by co-expression with the PHA operon and determination of the composition of the PHA. 

Materials and Methods

We designed and ordered DNA fragments from Integrated DNA Technologies, Inc. (IDT) to obtain Sleeping Beauty Mutase (SBM) which consist genes ScpA, ScpB, ScpC, and argK. Constructs of pSB3T5, pSB3T5:MCE, pSB3T5:SBM, and pSB3T5:MCE:SBM were established and grown in LB medium. Produced propionate was measured by detecting the change in absorbance at 410nm using spectrophotometer. 

Results and Discussion

Figure 6 Analytical digest of pSB3T5: SBM using XbaI and SpeI, followed by gel electrophoresis – Uncut sample showed 2 bands, one above 10kbp, and one between 8kbp – 6kbp. XbaI and SpeI single digests show a single band of 8790bp. XbaI and SpeI double digest shows two bands: a pSB3T5 backbone at 3250bp, and the SBM operon at 5540bp

Figure 7 Colony PCR of 7 colonies of pSB3T5: MCE: SBM – Samples 5, 6, and 7 all have bands between 6kbp – 8kbp, suggesting that they do possess PSB3T5: MCE: Sbm, but would have to be verified by sequencing. 

Future Work

In this work, constructs containing the SBM operon and MCE were developed. The constructs produced in this work need to be verified by sequencing, in order to determine whether there are any mutations that may impact their function.

 

References

Babu, R., O'Connor, K. & Seeram, R., 2013. Current progress on bio-based polymers and their future trends. Progress in Biomaterials,2(1), p.8.

Kannan, S., 2008. Studies on Methylmalonyl-CoA Mutase from Escherichia coli. Doctoral Dissertation. University of Westminster.

Background

As direct precursors, the amount of propionyl-CoA in the E. coli determines the accumulation of 3HV branches in the copolymer (Bhatia et al., 2015) and influences the PHBV yield. Thus, enhancement of propionyl-CoA pool is emphasized. The sucCD and sucAB are two genes participating the tricarboxylic acid (TCA) cycle, the former encodes succinyl-coA synthase catalyzing interconversion between succinate and succinyl-CoA, the latter encodes α-ketoglutarate dehydrogenase responsible for the formation of succinyl-CoA from α-ketoglutarate. According to Yu et al. (2006), either sucAB or sucCD is viable to produce enough succinyl-CoA, both are essential for cell viability. The interconversions of succinyl-CoA and propionyl-CoA are catalyzed by cluster of enzymes. Therein, playing important role in the conversion from succinyl-CoA and propionyl-CoA, both the proteins from sucAB and sucCD cooperate with other downstream enzymes, contribute to utilization of propionate, eventually to the PHBV production. 

Aims

In order to optimize yields and quality of PHBV which will help support the industrialization of PHBV production, we aim to obtain better PHBV production by enhancing the production of precursors via introducing exogenous genes SucAB and sucCD.

Materials and Methods

sucAB and sucCD were amplified from genome to construct new constructs including pSB3T5-sucAB, pSB3T5-sucCD, plasmid pSB3T5-X. These new constructs were transformed to E. coli BL21 (DE3) and cultured in M9 medium with 1% glucose, 0.01M propionic acid and 10M IPTG. Optical density was measured to give information for cell growth. Germinate multiple (GM), or the final cell concentration/inoculation cell concentration, was determined which represented proliferation capacity of cells. The utilization of propionic acid was determined by standard curve and equation between the absorbance and propionic acid concentration.

Results and Discussion

The presence of sucCD enable strains adapt environments with propionate meanwhile enhances the propionate utilization ability, thereby resulting in better PHBV production compared to existence of sucAB gene. The amount of propionic acid up taken by cell reflects the propionate utilization capacity of each strains in some extent, it can be told that all three strains present similar property in terms of propionic acid absorption, when the concentration is 0.03 M, every strain has peak absorption.

Figure 8 Propionic acid absorbed by three recombinant E. coli of pSB3T5-AB+ phaCAB, pSB3T5-CD+phaCAB and pSB3T5+phaCAB operon

Future Works

This investigation will be meaningful for practical issues like environment protection as well as industrial applications, for example, pot ale, a by-product from whisky distillery is rich in propionate. Currently, selling to local farmers and applying as fertilizer is the main by-products treatment method, PHBV production via recombinant strains fed with pot ale potentially can be a more environmentally-friendly and cost-effective alternative.

References

Bhatia, S.K., Yi, D.H., Kim, H.J., Jeon, J.M., Kim, Y.H., Sathiyanarayanan, G., Seo, H.M., Lee, J.H., Kim, J.H., Park, K. and Brigham, C.J., 2015. Overexpression of succinyl‐CoA synthase for poly (3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) production in engineered Escherichia coli BL 21 (DE 3). Journal of applied microbiology, 119(3), pp.724-735.

Yu, B.J., Sung, B.H., Lee, J.Y., Son, S.H., Kim, M.S. and Kim, S.C., 2006. sucAB and sucCD are mutually essential genes in Escherichia coli. FEMS microbiology letters, 254(2), pp.245-250.