Overview
Our research project focuses on developing engineered Pseudomonas putida strains capable of producing high-value sesquiterpenes, specifically santalene and santalol isomers. We initiated our wet-lab experiments by analyzing the growth curves of transformed and untransformed P. putida strains grown in media supplemented with varying concentrations of terephthalic acid and ethylene glycol. These experiments revealed optimal growth conditions, paving the way for our next steps.
The main objective of our project is the design of biobricks for santalene production. Initially, the pCDF-Duet vector expressed SaSSy_FPPS genes involved in sesquiterpene synthesis. However, to enhance the efficiency of the pathway, we transitioned to pSEVA631_araBAD_SaSSy_FPPS (BBa_K5181014)plasmid, which facilitated improved production of santalene.
In the final phase of our project, we advanced the design of the CPR-P450 fusion protein, which is responsible for converting santalene into santalol. The expression of these eukaryotic transmembrane proteins is challenging in our prokaryotic chassis, and hence, we ideated and used computationally determined design strategies, such as truncation of the transmembrane domain and fusion protein (link to design and modelling page).
We assembled the truncated fusion candidate but couldn't finish due to time and experimental hurdles. However, this step represents another milestone in achieving P450 expression, thereby santalol production. We have shown through our P450-CPR docking studies that it can indeed function as a multi-substrate enzyme and provided docking results for the high abundance of alpha and beta santalol over the rest of the isomers.
Growth Curve
We set our goal to determine whether Pseudomonas putida TA7-EG could grow using terephthalic acid (TPA) and ethylene glycol (EG) as sole carbon sources which is a crucial step towards optimising the growth conditions. As per the suggestion given by Dr. Brandenberg, we prepared minimal M9 media containing TPA and EG in varying concentrations - 5 mM, 10 mM, 15 mM, and 20 mM. After many attempts at optimising the growth conditions of the bacteria on different concentrations of TPA and EG, we concluded that 20 mM TPA + 20 mM EG provided the optimal growth conditions for P. putida TA7-EG. While significant growth was observed within 36 hours from inoculation, this was not practical for real-world applications. We adopted a strategy to supplement the media with 5% glucose. We reduced its concentration using gradual serial dilutions, transitioning from 5% glucose supplementation and TPA + EG to reach glucose-free conditions and optimise the growth of the bacteria solely on TPA and EG. This enabled the bacteria to adapt to TPA and EG and utilise them as their sole carbon source within 10-12 hours.
While significant growth was observed within 36 hours from inoculation, this was not practical for real-world applications. We adopted a strategy to supplement the media with 5% glucose. We reduced its concentration using gradual serial dilutions, transitioning from 5% glucose supplementation and TPA + EG to reach glucose-free conditions and optimise the growth of the bacteria solely on TPA and EG. This enabled the bacteria to adapt to TPA and EG and utilise them as their sole carbon sources and grow within 10-12 hours.
Aim
To optimise the growth of Pseudomonas putida TA7-EG in terephthalic acid (TPA) and ethylene glycol (EG) as its sole carbon sources.
Results
- The analysis of the bacterial growth confirms the growth of wild type Pseudomonas putida TA7-EG in terephthalic acid and ethylene glycol media and displays the potential of P. putida to grow and utilise PET monomers TPA and EG as its sole carbon sources.
- P. putida TA7-EG exhibits growth 12 hours post inoculation.
Aim
To optimise the growth of transformed Pseudomonas putida TA7-EG with the ligated plasmid (pSEVA 631_SaSSy_FPPS) in terephthalic acid and ethylene glycol as its carbon source.
- Initially, we observed slower growth, possibly due to the stress induced by transformation - the bacteria took up to 36 hrs to reach the log phase compared to the wild type, which reached the log phase faster.
- We found that the bacterial growth improved significantly upon performing serial dilutions, and we successfully reduced the time required for the bacteria to grow to 24 hours by refining the dilution process.
- These adjustments showed positive improvements, but we can continue future experiments to optimize growth conditions like pH additional media supplements to increase product yield, and better growth.
Santalene Synthesis
Santalene synthesis using pCDFDuet-SaSSy_FPPS
Enzymes FPPS and SaSSy are required for the production of santalene.
Our primary objective was to successfully synthesise santalene in Pseudomonas putida TA7-EG by expressing pCDFDuet-SaSSy_FPPS, a breakthrough achievement for a budding research team like ours. We contacted Dr Bohlmann, who had previously expressed SaSSy using the pCDFDuet expression vector in E.coli to achieve santalene synthesis. We decided to test the expression of pCDFDuet-SaSSy_FPPS in our chassis, P.putida - bacterial system.
Transformation of pCDFDuet_SaSSy_FPPS into P.putida
Aim
To transform the Mach-1 E.coli electro-competent cells with pCDFDuet_SaSSy_FPPS plasmid.
Experiment
The pCDFDuet_SaSSy_FPPS plasmids were electroporated into the Mach-1 E.coli strain for amplification.
Results
- Colonies were observed in the petri dish containing Mach-1 E.coli cells transformed with pCDFDuet_SaSSy_FPPS, confirming the successful transformation of the plasmids into the bacteria.
- Plasmids were isolated from the bacteria and amplified to prepare stocks for future use.
Aim
To transform the isolated pCDFDuet_SaSSy_FPPS plasmids into P. putida TA7-EG electrocompetent cells.
Experiment
The plasmids were isolated using the Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit and further transformed into the engineered P. putida TA7-EG strain.
Results
Petri plates containing streptomycin show that the transformation of the pCDFDuet_SaSSy_FPPS plasmids in the P.putida TA7-EG cells was successful.
Expression of pCDFDuet_SaSSy_FPPS
Aim
To check the expression of His-tagged SaSSy protein in Pseudomonas putida TA7-EG .
Experiments
We grew our Pseudomonas putida TA7-EG transformed with pCDFDuet_SaSSy_FPPS in minimal M9 media supplemented with terephthalic acid (TPA) and ethylene glycol (EG) as the sole carbon source. 1mM of IPTG was used to induce the plasmid. The His-tagged SaSSy enzyme was purified using Ni-NTA chromatography technique with HisTrap resin. The purified protein was later analysed with SDS-PAGE gel and visualised using Coomassie staining.
Results
SDS-PAGE of the purified protein revealed a band at 65 KDa corresponding to the expected molecular weight of the Santalene Synthase (SaSSy) protein. The band observed confirms that SaSSy was expressed successfully in the engineered Pseudomonas putida TA7-EG strain.
Aim
Chemical Analysis:
To detect the presence of santalene using GC-MS analysis to provide functional evidence of protein expression.
Results
GCMS Analysis of Santalene Quantification
The GC chromatogram shows the intensities of detected peaks at their respective retention times, while the mass spectra depicted relative intensities versus m/z (Mass to Charge) values. Santalene showed a characteristic peak at 204 m/z in the chromatogram (as previously shown by Dr Bohlmann). Analysis of the MS spectrum confirmed the presence of santalene, which had an abundance of 1.14% in the sample. In contrast, the GC-MS analysis of the wild-type Pseudomonas putida TA7-EG strain showed no santalene peak at the corresponding retention time.
The absence of a peak corresponding to santalene in the wild type Pseudomonas putida also showed that pCDFDuet_SaSSy_FPPS expression was necessary to observe santalene production.
Below are the GC-MS results for the transformed pCDFDuet_SaSSy_FPPS in Pseudomonas putida:
Results for the untransformed wild type Pseudomonas putida:
The abundance of santalene observed was lower than expected, showing limited effectiveness of pCDFDuet_SaSSy_FPPS in our system. To overcome this issue simultaneously we designed a suitable plasmid with an appropriate promoter, RBS, and SaSSy genes codon optimised for P.putida. For more details refer to the part design BBa_K5181014.
Santalene synthesis using araBAD_SaSSy_FPPS (BBa_K5181014) in Pseudomonas putida TA7-EG
We designed a system of bicistronic inserts with appropriate promoters, RBS, inter-ribosomal entry site (IRES) and spacer sequences, which can be ligated and cloned with the pSEVA backbones for stable expression.
This experiment was designed to express the insert araBAD_SaSSy_FPPS (BBa_K5181014) in Pseudomonas putida TA7-EG growing on the PET monomers TPA and EG.
Aim
To assemble and amplify the araBAD_SaSSy_FPPS fragments.
Experiment
We assembled the three fragments to get a complete insert having araBAD promoter, RBS and SaSSy and FPPS genes using NEBuilder® HiFi DNA assembly. After the assembly, we performed bridge PCR using specific forward and reverse primers designed by our team.
Results
The gel results verified that the Gibson assembly was successful, confirming proper ligation of the araBAD_SaSSy_FPPS fragments. The size of our Gibson assembled insert was expected to be 4082 bp. Our results confirmed successful fragment assembly, as shown in the gel image. A 1 Kb ladder was used to check the size of bands.
Aim
Restriction digestion and ligation of PCR-amplified araBAD_SaSSy_FPPS insert with the pSEVA631 vector backbone.
Experiment
The plasmid backbone pSEVA631 was isolated from the LB culture using the Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit. The plasmid and the insert (araBAD_SaSSY_FPPS insert) were restriction-digested using the EcoRI-HF and Hind III endonucleases.
| Name of Fragment | Expected size (bp) |
|---|---|
| pSEVA 631 (backbone) | 3519 |
| araBAD_SaSSy_FPPS (insert) | 4082 |
Results
Upon running the gel, the 56 bp long MCS (Multiple Cloning Site) regions of the pSEVA631 backbone ran out of the gel, and the backbone reflected a size of 3159bp. Later, the gel was cut and cleaned using the Macherey-Nagel™ NucleoSpin™ Gel and PCR Clean-up Kit.
Aim
To ligate the insert araBAD_SaSSy_FPPS with the backbone pSEVA631
Experiment
- The PCR amplified samples of araBAD_SaSSy_FPPS were now cleaned up using the Macherey-Nagel™ NucleoSpin™ PCR Clean-up kit. Further, the insert was isolated after the PCR clean-up, and the pSEVA631 backbone was ligated using NEB T4 DNA Ligase.
- Single and double digestion (EcoRI and HindIII) of the ligated plasmid (pSEVA631_araBAD_SaSSy_FPPS) was performed to verify the ligation, followed by gel electrophoresis.
Results
- The 1% agarose gel electrophoresis confirmed the successful ligation of the insert into the backbone, ensuring proper assembly of the construct.
- In the case of single digestion, a thick band was found at 7Kb, corresponding to the size of our ligated plasmid (7005 bp), confirming the ligation process.
- Similarly, a single and double digestion of our ligated plasmid was performed to confirm ligation.
successful double digestion.
successful single digestion.
Transformation of ligated pSEVA631_araBAD_SaSSy_FPPS into P.putida TA7-EG
Aim
To transform the ligated araBAD_SaSSy_FPPS_pSEVA631 plasmid into Pseudomonas Putida TA7-EG.
Experiment
The ligated plasmids were subsequently transformed into the Mach1 E. coli electro-competent cells to amplify the plasmids. The amplified plasmids were isolated using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit. Finally, the plasmids were electroporated into Pseudomonas Putida TA7-EG electro-competent cells for transformation.
Results
Expression of pSEVA631_araBAD_SaSSy_FPPS into P.putida TA7-EG
Aim
To perform the expression of pSEVA631_araBAD_SaSSy_FPPS in P. putida TA7-EG.
Experiment
We transformed the ligated pSEVA631_araBAD_SaSSy_FPPS plasmid into P.putida TA7-EG in the minimum M9 media containing 20 mM TPA and 20 mM EG. The culture was induced with an optimised arabinose concentration of 0.7% and allowed to grow for 16 hours at 20°C. The bacteria was later pelleted and lysed using sonication.
Further, the Ni-NTA column was performed to purify the SaSSy and FPPS protein fragment, both of which were tagged with His tag. We performed SDS-PAGE, followed by Coomassie staining.
Results
We got a band at 65 KDa and 45 KDa corresponding to SaSSy and FPPS, respectively, confirming the expression of our desired proteins successfully.
Quantitative Analysis of Santelene through GC-MS
The GC chromatogram displayed intensities of the detected peaks at their respective retention times, while the mass spectra showed relative intensities versus m/z. The santalene standard produced peaks corresponding to 204 m/z in the chromatogram. A review of the mass spectrometry spectrum of the sample transformed with araBAD_SaSSy_FPPS_pSEVA631 plasmid revealed a distinct pattern, confirming santalene, which quantified a 10.25% abundance. It showed an abundance ten times greater than that of pCDF-Duet. In contrast, the GC-MS output for the wild type P. putida strain did not exhibit any peak at 204 m/z..The wild-type P. putida TA7-EG cells and the transformed P. putida TA7-EG showed different peaks at the 19-minute retention time, verifying that the 204 peak corresponds to the santalene m/z ratio, which is expressed by the pSEVA631_araBAD_SaSSy_FPPS plasmid incorporated into our chassis.
Consistent with our hypothesis, the modified P. putida strains expressing the SaSSy and FPPS proteins with the pSEVA631 backbone exhibited successful santalene synthesis, supporting our approach of achieving the first-ever santalene production in Pseudomonas putida TA7-EG
Results
Santalol Synthesis
CPR-P450
Expression of Cytochrome P450 Monoxygenase and Cytochrome Reductase
Building on our previous success with the expression of Santalene synthase (SaSSy) and Farnesyl pyrophosphate synthase (FPPS) in Pseudomonas putida TA7-EG, utilising PET-derived terephthalic acid and ethylene glycol as feedstock, we next aimed to incorporate the Cytochrome P450 Monooxygenase (CYP450) and Cytochrome Reductase system (CPR1). These enzymes, sourced from the Santalum album, catalyse the conversion of santalene to santalol. However, the expression of eukaryotic membrane-bound proteins in a prokaryotic host remains a big challenge due to the differences in membrane composition, enzyme anchoring, and varying post-translational modifications.
In this project phase, we focused on the expression of the CPR-P450 fusion protein in P. putida TA7-EG. Refer to Project design and part BBa_K5181015 for a detailed approach to designing a fusion protein.
Aim
To assemble CPR-P450 fragments using Gibson assembly.
Results
We performed Gibson assembly with three fragments synthesised by GenScript. A complete insert consisting of tet Promoter_RBS_P450-CPR fusion protein was assembled using NEBuilder® HiFi DNA assembly. Following the assembly, we performed bridge PCR using specific primers to confirm assembly and amplify the insert. We loaded the sample in 1% agarose gel and performed electrophoresis.
We observed bands around 4 Kb, which closely matched our expected assembled gene insert size of 4151 bp, confirming the success of the Gibson assembly. The detailed protocols for PCR amplification and Gibson assembly can be found on the protocols page.
Aim
To ligate the tet_CPR_P450 insert into the vector backbone pSEVA241.
Experiment
- The pSEVA241 vector backbone was isolated from E.coli Mach1 using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit.
- The PCR amplified CPR-P450 was PCR cleaned using Macherey-Nagel™ NucleoSpin™ PCR Clean-up XS kit.
- Both the insert and the vector backbone were restriction-digested using the endonucleases EcoRI-HF and HindIII. To remove the MCS region of the vector, the digested backbone was run on 1% agarose gel and later eluted from the gel.
| Name of Fragment | Expected size (bp) |
|---|---|
| pSEVA 241 (backbone) | 3252 |
| CPR-P450 ( insert) | 4125 |
Both the digested CPR-P450 fragment and the pSEVA241 backbone were ligated using NEB T4 DNA Ligase in a 1: 3 vector-to-insert ratio.
Results
- To confirm our ligation, we started off by transforming the ligated product into E.coli Mach-1. We grew them on Kanamycin plates, and the colonies formed were inoculated using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit to isolate the plasmid. Single digestion of the plasmid was done using EcoRI-HF. The digestion was run on 1% agarose gel.
- After analysing the gel results, we realised the presence of a smear instead of a thick band, indicating the degradation of the plasmid. We repeated the experiment multiple times after this, only to get the same result. In order to confirm the issue we switched to ligate the insert into pSEVA631 backbone.
Aim
Ligation of pSEVA631 backbone with tet_CPR_P450 insert.
Experiment
- The pSEVA631 vector backbone was isolated from E.coli Mach1 using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit.
- The PCR amplified tet_CPR_P450 insert was PCR cleaned up using Macherey-Nagel™ NucleoSpin™ PCR Clean-up XS Kit.
- Both the insert and the vector backbone were restriction digested using the endonucleases EcoRI-HF and HindIII. In order to remove the MCS region of the vector, the digested backbone was run on 1% agarose gel and further eluted from the gel.
| Name of Fragment | Expected size (bp) |
|---|---|
| pSEVA 631 (backbone) | 3519 |
| CPR-P450 ( insert) | 4125 |
The restriction digested tet_CPR_P450 insert, and the pSEVA631 backbone were ligated using NEB T4 DNA Ligase in a 1:3 vector-to-insert ratio. Gibson assembly was also used in an attempt to ligate the vector and backbone following a 2-fragment protocol.
Results
To confirm the ligation, we started off by running a gel for the ligated product.
From the gel results, a smear can be seen from 10 Kb to 8 Kb, where the expected band is 7 Kb hence this result indicates that the ligated plasmid might have been degraded. Regardless, we decided to transform the ligated product into E.coli Mach1 competent cells. We grew them on Gentamicin plates, and the colonies formed were inoculated, and the plasmid was isolated using Miniprep. Single digestion of the plasmid was done using EcoRI-HF. The digestion was run on 1% agarose gel. Upon analysis of the gel electrophoresis, we noticed that there was a smear instead of a thick band at the required size.
Based on a detailed structural analysis of our P450-CPR fusion protein,
- These proteins are stable under catalytic conditions. As per Expasy Protparam, the protein might be stable >10 hr in E.coli under in-vivo conditions. This opens new avenues for experimentation and establishing the expression of this fusion protein inserted into our chassis P. Putida.
- Evaluation of transmembrane tendencies of the fusion protein revealed that both domains have less tendency to get embedded in the membrane and hence are more likely to work as native prokaryotic P450. This analysis enhances our understanding of the workings of our bio-bricks and paves the way for promising results in the near future. (Refer to molecular docking and structural studies)
Conclusion
Results are beyond our expectations. We were successfully able to express the SaSSy-FPPS and synthesise santalene, a fragrant sesquiterpene responsible for the fragrance of sandalwood oil and the key precursor for santalol, which is the major component of sandalwood oil. We analytically detected santalene through GS-MS. Our main aim was synthesising santalene from PET, and we succeeded. Our second ambition was to oxidise (or convert) santalene to santalol, which was a lot more challenging as the expression of P450s in Pseudomonas putida, a prokaryote, is challenging.
While we were not able to express the designed P450 and CPR fusion protein in Pseudomonas putida due to time constraints, we successfully built a probable fusion protein and determined its structure via alpha fold and its catalytic domain via molecular docking with santalene and santalol isomers. Ultimately, we’ve shown that within a limited timeframe a small team of students can successfully engineer a microbe to produce a distinct scent.