Modern life is reliant on the use of plastics, and since their production began on a large scale over 8.3 million tons have been produced, of which over 6.3 million tons has been thrown away, with the majority accumulating in landfill, or the environment (Geyer et al., 2017). Drawn by increasing demand of plastics and sustainable development request, the general mindset was shifted towards developing completely natural biodegradable plastics. Polyhydroxyalkanoate (PHA) is a huge family of bio-derived and biodegradable polymers belonging to the polyesters class that are also termed “Microbial Plastics” (Bonartsev et al., 2017). The biopolymers are commonly produced among a wide array of gram-positive and gram-negative bacteria but the highest occurrence has been attributed to Cupriavidus necator (C. necator, previously known as Rastonia eutropha), which is the main chassis for current commercial production of PHA-based bioplastics.
Because of the characteristics of being water insoluble, nontoxic and degradable, poly(3- hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) became two major members of this family that have attracted our attentions most (Zakaria et al., 2010). PHB is the most prevalent biopolymer of this family and has been investigated fervently as a bio-based alternative to petrochemical-derived plastics. Within specific conditions of nutrients and carbon sources, co-polymers – that is, polyesters comprising of more than one type of monomer – can also be synthesized. PHB is frequently the predominant monomer but various co-polymers exist that are built up from larger species (i.e. polyhydroxyvalerate, polyhydroxyhexanoate, etc.). PHA co-polymers are currently an area of interest due to the diversity of properties (e.g. elasticity, crystallinity, melting point, etc.) that emerge as a result of their relative monomer compositions and their corresponding side chain moieties. It has been shown that a higher molar ratio of the hydroxyvalerate component resulted in increased ductility, strength and a larger thermal processing window. Effectively this means that across a range of monomer compositions, PHBV can be modified to become usable in 3D printers (which operate within specific melting temperatures of the bioplastic “ink), used in consumer packaging or even applied in the biomedical context as components of slow-release drug formulations and orthopedic devices.
In this project, we aim to construct PHB and PHBV synthesis pathways by introducing phaCAB operon, SBM operon, and other functional genes into E. coli to improve the efficiency in production of PHB/PHBV.
Figure 1 Comparison between the molecular structure of PHB and PHBV
When investigating the effect of phaCAB on PHB production, the effect of different culture conditions on recombinant E. coli harbouring the phaCAB operon should be known. Herein, culture conditions with different glucose concentration were assessed to determine the optimised condition(s) on growth and PHA production. Moreover, due to the importance of propionic acid in producing PHBV, its effect on E.coli growth was also assessed and the result was used as a reference for the Bktb gene research project. Comparison of the different growth curves indicated that cells with 1 %, 2 % and 3 % glucose reached stationary phase with the same level after 50 hours cultivation while culture conditions with glucose concentration higher than 3% or input of propionic acid will compress the cell growth.
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 two-stage glucose/propionate fed batch fermentation progress using Cupriavidus necator or recombinant E. 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 followed by assessment of the dry PHB weight.
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.
In the experiment, recombinant E. coli harbouring phaCAB were used to culture with different conditions up to 56 hours.
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.
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.
When investigating the effect of gene order of phaCAB on PHB production, five new constructs were established by Gibson assembly and succeed constructs were transformed to E. coli BL21 (DE3) strains. Those five recombinant E. coli strains would be cultured and compared with strains that harboured original phaCAB operon. The highest yield of PHB among the new constructs was achieved by E. coli that expressed phaACB plasmid, which was similar gene order with the original phaCAB operon, while none of new operons got higher yield than original operon.
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).
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.
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).
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.
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
phaCAB
0.237
1.58
phaCBA
0.072
0.48
phaACB
0.201
1.34
phaBCA
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.
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.
In this study, we constructed a PHBV synthesis pathway (shown in Figure 3) by introducing the phaA, phaB and phaC genes into E. coli BL21 (DE3). 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. And the effect of bktB on PHBV synthesis was further investigated by replacing phaA with bktB. With the cell culture condition optimisation, 3 % glucose and 8 mM propionic acid was proper feeding strategy for PHBV production. Cells harbouring pSB1C3-phaCB-bktB showed great potential to improve production of PHBV with higher 3HV fraction.
Figure 3 Schematic illustration of the pathways leading to the PHBV biosynthesis
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).
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.
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.
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
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.
When investigating the influence of PhaR autoregulation system coupled with phasin on PHB production and cell growth, phaR and phaP which encode PhaR regulation system and phasin respectively were introduced to E. coli strain BL21 (DE3) with phaCAB operon, forming new constructs pSB3T5-ProR-phaR-ProP-phaP. Furthermore, another construct pSB3T5-ProR-phaR-ProP-phaP-hlyA was established where the hlyA was expressed with PhaR regulation system and phasin to investigate the effect of hlyA depending secretion system coupled with PhaR regulation system and phasin on PHB production. The highest yield of PHB was achieved by strain that expressed pSB3T5-ProR-phaR-ProP-phaP-hlyA. In addition, the presence of PhaR regulator could effectively regulate the expression of phaP and moderate the biosynthesis of PHB.
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.
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.
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.
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
Culture volume (ml)
Intracellular PHB (g)
Secreted PHB (g)
Total PHB (g)
phaCAB+pSB3T5
0.01
0.0667
phaCAB+pSB3T5-R-P
0.071
0.0015
0.0725
0.4833
phaCAB+pSB3T5-R-P-hlyA
0.043
0.0305
0.0735
0.4900
phaCAB+pSB3T5-R
0.002
0.073
0.4868
phaCAB+pSB3T5-P
0.4733
Note: E. coli that harboured different constructs were cultured in M9 medium with 3% glucose for 48 hours at 37°C shaker
Methylmalonyl-CoA Epimerase (MCE) was introduced in the proposed pathway (as shown in Figure 5) resulting to high production of propionate. By expressing the Sleeping Beauty Mutase (SBM) operon and MCE with genes for PHA production in E. coli would result in the production of PHBV with a high ratio of valerate to butyrate. In this study, vectors harbouring the SBM operon with and without MCE was achieved.
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.
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.
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.
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.
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.
In this project, various genes including gene sucAB and sucCD from Escherichia coli were introduced to E. coli and co-expressed with phaCAB operon from Rastonis eutropha. The engineered strains are used to investigate the effects of these genes on PHBV production and strain growth, therein, different effects caused by sucCD and sucAB respectively are compared, as well as the PHBV yield inside.
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.
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.
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.
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
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.
