Team:Manchester/Fragrance
FRAGRANCE
Ah - the sweet, sweet smell of success: Manchester iGEM 2019 is proud to announce that, to the best of our knowledge, we have achieved for the first time in the iGEM history the production of crucial intermediates in the vanillin biosynthesis pathway from tyrosine. Vanilla or Citrus fruits - take your pick: we have them both!
Our Achievements …
In 5 bullet points
1.
Integrated Human Practices: Odorant Production- was included into our developing project following feedback from the Science Symposium. Scientists raised concerns that the smell of growing E. coli would turn people away from Cutiful. (Human Practices, Act I: The Symposium)
2.
Integrated Modelling: Detection Thresholds of odorant molecules were compared, following simple isochoric sphere diffusion calculations. Limonene was selected because of its regular occurrence in hair care products, as well as in-house availability, and vanillin for its remarkably low Odour Detection Threshold (ODthresh) value. (Modelling, Act II: Fragrance)
3.
New Parts Expressed and Characterised - expression and characterisation of codon optimised and His-tagged TAL, C3H and COMT enzymes (genes are sam8, sam5 and comt respectively), the first three enzymes in the vanillin biosynthesis pathway.
4.
Proof of New Part Functionality- Production of 213.954 µM, 1.207 µM and 3.914 µM for p-coumaric acid, caffeic acid and ferulic acid respectively, the first three intermediates in the vanillin pathway, detected and quantified using UPLC-MS/MS.
5.
Limonene from Non-Standardised Pathway- production of 0.6 mg/L of limonene from GPPS and LimS enzymes detected by GC-MS.
Fragrance – seven enzymes: two scents
Or: where our goal is to make the most possible so it can all evaporate off, and we discover that tears, sweat and blood are not good organic solvents. Sara wants to cry, but ends up laughing, and together with Ewan they go where no one has been before.
Prologue
”A complex blend of private and public selves” – Mary Gaitskill
The olfactory side of our project was influenced by discussions with scientists at the MIB synthetic biology symposium. (Human Practices, Act I: The Symposium) Many researchers brought up the unpleasant smell our dye-secreting E. coli would produce whilst growing on the hair. Our solution was to engineer the E. coli to produce volatile molecules vanillin or limonene, leaving a nice scent around an individual's hair. Vanillin was chosen because our modelling showed that it would be optimally perceived at long distances, due to its low smell detection threshold, and limonene because it is commonly used in hair care products. (Modelling, Act II: Fragrance)
Microbial genes involved in the phenylpropanoid pathway have been used for vanillin biosynthesis, mimicking a natural pathway of vanillin biosynthesis in plants. This includes five genes to synthesize vanillin from L-tyrosine (Figure 1). sam8 encodes tyrosine ammonia lyase (TAL) which catalyses the non-oxidative deamination of L-tyrosine to p-coumaric acid. 4-Coumarate 3-hydroxylase (C3H), encoded by sam5, converts p-coumaric acid to caffeic acid by hydroxylation of the benzene ring. Caffeate O-methyltransferase (COMT), encoded by comt, converts caffeic acid to ferulic acid which is then converted to feruloyl-CoA by feruloyl-CoA synthase (FCS) encoded by fcs. Finally, enoyl-CoA hydratase (ECH), encoded by ech, converts feruloyl-CoA to vanillin.
Figure 1. Vanillin biosynthesis pathway from L-tyrosine consisting of five enzymes (adapted from Ni et al.).
Terpenoids, a diverse class of naturally occurring compounds, are synthesized from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPP are universal C5 isoprenoid precursors produced by the mevalonate‐dependent (MVA) pathway or the methylerythritol 4‐phosphate (MEP) pathway. The former is present in all higher eukaryotes and the latter is present in most bacteria and plant plastids. Both these pathways lead to the production of geranyl diphosphate (GPP) catalyzed by geranyl diphosphate synthase (GPPS), the sole substrate for all monoterpenoid synthases/cyclases. Production of the monoterpenoid limonene from GPP is then performed by limonene synthase (LimS) (Figure 2). Therefore, limonene biosynthesis can be achieved depending on the endogenous MEP pathway of E. coli and addition of exogenous GPPS and LimS genes.
Figure 2. Mevalonate‐dependent (MVA) pathway and methylerythritol 4‐phosphate (MEP) pathway produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate DMAPP which are converted to geranyl diphosphate (GPP) by geranyl diphosphate synthase (GPPS) . Limonene synthase then converts GPP to limonene (adapted from Leferink et al.).
Act I: Design
”Perfume is the art that makes memory speak” – Francis Kurkdjian
The genes and their origins used for vanillin and limonene biosynthesis are listed in table 1.
| Gene | Length (bp) | Enzyme | Origin | Our parts |
|---|---|---|---|---|
| Vanillin | ||||
| sam8 | 1533 | Tyrosine ammonia lyase (TAL) | Saccharothrix espanaensis | BBa_K2906011 |
| sam5 | 1539 | 4-Courmarate-3-hydroxylase (C3H) | Saccharothrix espanaensis | BBa_K2906013 |
| comt | 1092 | Caffeate O-methyltransferase (COMT) | Arabidopsis thaliana | BBa_K2906015 |
| fcs | 1770 | Feruloyl-CoA synthetase (Fcs) | Pseudomonas putida KT2440 | BBa_K2906021 |
| ech | 831 | Enoyl-CoA hydratase/aldolase | Pseudomonas putida KT2440 | BBa_K2906023 |
| Limonene | ||||
| GPPS | 894 | Geranyl diphosphate synthase | Abies grandis | Leferink et al. |
| LimS | 1632 | Limonene synthase | Mentha spicata | Leferink et al. |
Scene 1: Vanillin
Of our smell molecules, vanillin, was more challenging to produce as its biosynthesis requires five genes, of which sam8 was not previously characterized by iGEM teams. To increase the chances of successful expression, we decided to codon optimise all five genes using GeneArt GeneOptimiser to lower the GC content and reduce repeats, increasing transcriptional accuracy and translation rate. Moreover, due to the large size of these genes, a T7 promoter was placed upstream of each of the five genes to ensure expression.
Cloning the entire pathway into one plasmid can cause metabolic burden on the transformed cells in terms of protein expression and plasmid replication. We instead chose to clone the genes into separate vectors to first prove the expression and functionality of the genes. sam8 and sam5 were each cloned individually into pETM-11. comt and fcs+ech (ordered as one g-block composing both enzymes) were cloned into pETDuet-1, a backbone with two multiple cloning sites (MCSs).
These plasmids (pETM-11 and pETDuet-1) were chosen as they contain a T7 promoter and an RBS homologous to the strong g10-L RBS, which would stimulate a regulated high expression of our genes by addition of IPTG. The plasmid also provides a N-terminal His-tag upon cloning of sam8, sam5 and comt allowing purification and functional analysis of the respective proteins.
E. coli DH5α were used for cloning procedures whereas BL21 (DE3) cells were used for expression, as DH5α lack the phage-derived T7 RNA polymerase required for transcription of T7 promoter.
Figure 3. Schematic diagram of the parts containing the enzymes involved in vanillin biosynthesis. The T7 promoter, RBS, His-tag, TEV site and terminator are introduced to sam8 and sam5 upon cloning into pETM-11 plasmid. The T7 promoter, RBS and His-tag upstream comt and fcs are from the pETDuet-1 plasmid.
Scene 2: Limonene
We chose to use the GPPS-LimS genes without the precursor genes as we did not want to put a high metabolic burden on our E. coli As we had the in house GPPS-LimS pathway cloned into a pBbB2a plasmid gifted to us, we transformed the construct into DH5α and induced the cells with 50 μM IPTG and 25 nM of anhydro-tetracycline (aTet). Limonene expression could be confirmed by Gas Chromatography - Mass Spectrometry (GC-MS). pBbB2a-GFP was used as a negative control as it expresses GFP and not limonene. pJBEI-6410 was used as the positive control as it contains the optimised limonene production pathway, including an inducible hybrid MVA pathway and the idi gene increasing the pool of GPP (Figure 2).
Figure 4. Schematic diagram of the genes involved in limonene biosynthesis.
Act II: Experimental and Results
”Music is the fragrance of the universe” – Guiseppe Mazzini
Scene 1: Vanillin
Cloning
pETDuet-comt
After PCR amplification, comt and pETDuet-1 were sequentially digested with BamHI-HF and PstI-HF restriction enzymes (NEB). To prevent plasmid re-ligating, DpnI digestion was carried out to digest any leftover template plasmid, then the PCR product was purified. Quick ligaseTM (NEB) was then used for ligation. Our constructs were then transformed into DH5α before being plated onto 50 µg/mL carbenicillin plates and left to incubate overnight at 37°C.
From this transformation, one colony was obtained, and so colony PCR was performed (Figure 5). The positive control was the pETM-11 plasmid and the negative control was water. Restriction digestion (Figure S2) and sequencing were done for verification of cloning of comt into pETDuet-1.
Figure 5. Colony PCR of pETDuet-1-comt single colony. The expected amplified product of pETDuet-1-comt is 1548 bp.
pETM-11-sam8 and pETM-11-sam5
Even after extensive troubleshooting, sam8 and sam5 restriction cloning was still not achieved, so we switched to Gibson assembly instead for cloning. After PCR of sam8, sam5 and pETM-11 (Table S1), the HiFi assembly mixtures were set up following the manufacturer's protocol with a 3:1 insert to vector ratio (50 ng vector). The samples were then kept at 50°C for 30 minutes before being transformed into DH5ɑ cells and plated onto 50 µg/mL kanamycin plates.
Colony PCR was performed to test for cloning of sam8 and sam5 into pETM-11 plasmids. This was then verified by restriction digestion (Figure S3) and sequencing.
Figure 6. Gel after colony PCR of pETM-11-sam8 and pETM-11-sam5. The expected size of pETM-11-sam8 is 1861 bp and of pETM-11-sam5 is 1867 bp + 980 bp due to multiple annealing sites of primers in pETM-11-sam5. The positive control is pETM-11 yielding a 996 bp band, and the negative control is water.
RESULTS:
Expression of sam8, sam5 and comt
Pre-cultures of BL21(DE3) carrying pETM-11-sam8, pETM-11-sam5 and pETDuet-1-comt were prepared for SDS-PAGE and Western blot. BL21(DE3) cells containing either pETM-11 (empty vector) or pETM-11-ABU58587 (expressing a His-tagged protein about 36 kDa in size) were also prepared as negative and positive controls respectively.
Following overnight incubation, 10 mL cultures (1/100) were used to make working cultures which were kept at 37°C with shaking. After OD600 of 0.6 was reached, the cultures were induced with 100 μM IPTG. The working cultures were incubated at different temperatures: 16°C, 20°C, 26°C and 30°C to check the protein expression.
The samples were centrifuged at 6000 x g for 10 min at 4°C and subsequently resuspended in either PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4; pH 7.9) or Buffer A (25 mM Tris-HCl, pH 7.9; 500 mM NaCl; 20 mM imidazole pH). Next, samples were sonicated to lyse the cells and centrifuged again at 18500 xg for 30 min at 4°C so that the soluble and insoluble fractions would be separated. These were then analysed by SDS-PAGE. Western blots for the samples were carried out to confirm the presence of all the His-tagged proteins (Figure 7). The negative control is pETM-11 empty vector and the positive control is pETM-11-ABU58587 expressing a 36 kDa His-tagged protein.
Figure 7. SDS-PAGE gels (right) and western blots (left) of TAL, C3H and COMT under specified conditions. The expected sizes of the proteins are 57.4 kDa, 60.49 kDa and 41.39 kDa for TAL, C3H and COMT respectively. The negative control (-ve) is the pETM-11 (empty vector) and the positive control (+ve) is the pETM-11-ABU58587 expressing a 36 kDa His-tagged protein.
Expression of the proteins is higher in the insoluble fraction than in the soluble fraction (Figure 7). COMT is soluble if resuspended in Buffer A whereas TAL (encoded by sam8) is soluble at 16 °C, 20 °C and 26 °C - but not 30 °C - if resuspended in PBS. C3H, encoded by sam5, is insoluble in both Buffer A and PBS. This demonstrates that the buffers used influence the solubility of these proteins.
Metabolite Analysis
For metabolite analysis, cultures containing plasmids carrying sam8, sam5 and comt were mixed to generate multiple 10 mL co-cultures as shown in table 2. Samples were induced with 100 µM IPTG and incubated at 26 °C for 3 days. Then samples were diluted 1000x with water and analysed by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) to detect (Figure 8) and quantify (table 2) of p-coumaric acid, caffeic acid and ferulic acid, the first three intermediates in the vanillin biosynthesis pathway, formation of which are catalysed by TAL, C3H and COMT respectively.
Figure 8. UPLC–MS/MS chromatograms of p-coumaric acid, caffeic acid and ferulic acid (a) from co-cultures: sam8:sam5:comt 1:1:1 (b), sam8:sam5:comt 1:2:2 (c), sam8 (d), sam5:comt 1:1 + 3 mM p-Coumaric acid (e) and pETM-11 empty vector (f). X-axis show retention time.
Table 2. Amounts of p-Coumaric acid, Caffeic acid and Ferulic acid from the respective co-cultures determined by UPLC-MS/MS.
| Co-cultures (ratio of respective components) | p-Coumaric acid (µM) | Caffeic acid (µM) | Ferulic acid (µM) |
|---|---|---|---|
| sam8:sam5:comt (1:1:1) | 58.906 | - | - |
| sam8:sam5:comt (1:2:2) | 41.018 | - | - |
| sam8 | 213.954 | - | - |
| sam5:comt (1:1) with 3 mM p-coumaric acid | 2653.056 | 1.207 | 3.914 |
| pETM-11 | - | - | - |
All cultures containing sam8 showed production of p-Coumaric acid from tyrosine in LB broth showing that TAL, the product of the sam8 gene, is functional. Caffeic acid and Ferulic acid are only detected in the sam5:comt co-culture. Therefore, it is likely that p-Coumaric acid is not secreted by the cells which is recovered upon addition of 3 mM p-coumaric acid. It is worth mentioning that the low amount of caffeic acid detected could be due to the instability and membrane-bound nature of C3H. The western blots show that our proteins are insoluble, yet UPLC-MS/MS results show the products are produced, suggesting that the enzymes are functioning in vivo conditions.
Scene 2: Limonene
pBbB2a-GPPS-LimS, pJBEI-6410 and pBbB2a-GFP were transformed into DH5α E. coli, plated onto 100 µg/mL carbenicillin plates of which colonies were picked and inoculated into a glucose-rich TB media mix. The cultures were then grown at 37°C and 180 rpm shaking incubator. Next the cultures were induced with 50 μM IPTG or 25 nM anhydro-tetracycline and overlain with 20 %(v/v) n-nonane. The cultures were further incubated shaking at 180 rpm and 30°C for 72 hours.
After incubation, OD600 readings were taken. Each sample’s n-nonane layer was removed and centrifuged at 18400 xg for 3 minutes. Following this it was dried over anhydrous MgSO4 and mixed 1:1 with ethyl acetate (with internal standard 0.1 % Sec-Butylbenzene). Products were then analysed by GC-MS (Figure 8) and then quantified (Figure 9) using 5 mM limonene as the standard. The LimS-GPPS plasmid produced a total of 0.6 mg/L whereas the positive control pJBEI-6410 plasmid shows about 1000x higher limonene production than the LimS-GPPS.
Figure 9. Chromatogram results (right) and MS spectrum results (left) of GFP negative control (a), pJBEI-6410 positive control (b) and LimS-GPPS (c) aligned with 5 mM limonene. Molar mass of limonene is 136.12 g/mol.
Figure 10. Limonene quantification (mg/l) of DH5α cells carrying pBbB2a-GFP (negative control), pJBEI-6410 and pBbB2a-GFP (positive control) and pBbB2a-GPPS-LimS. Numbers represent the average of three replicates. SD= 0, ±0.928 and ±0.34 for GFP, pJBEI-6410 and LimS-GPPS respectively.
Act III: Conclusion and Future Works
”the unseen, unforgettable, ultimate accessory” – Coco Chanel
Scene 1: Vanillin
We successfully achieved protein expression and characterisation of TAL, C3H and COMT enzymes, the first three enzymes involved in the vanillin biosynthesis. By expressing these enzymes in co-cultures of E. coli with our constructs, 213.954 µM, 1.207µM and 3.914 µM of p-coumaric acid, caffeic acid and ferulic acid were respectively detected and quantified by UPLC-MS/MS. To the best of our knowledge, we are the first iGEM team to express and characterize sam8 which encodes TAL.
All enzymes were found to be insoluble in western blots possibly due to overexpression. Furthermore, proteins might vary in solubility depending on the type of buffer used for resuspension as TAL was soluble in PBS while COMT was soluble in Buffer A. With more time we would want to troubleshoot the buffers to further test protein solubility. For example, TAL has been shown to be soluble on a SDS-PAGE gel if resuspended in a 50 mM Na3PO4, 300 mM NaCl, 150 mM imidazole; pH 8.0 buffer.
In the future, cloning fcs and ech genes into pETDuet-1-comt would allow the full synthesis of vanillin from L-tyrosine which could be tested by growing co-cultures of pETM-11-sam8, pETM-11-sam5 and pETDuet-1-comt-fcs-ech, to then be analysed by UPLC-MS/MS. Moreover, we would want to clone all genes involved in the vanillin pathway into one plasmid in order to synthesize vanillin from a tyrosine precursor. In case of vanillin being under the detection threshold identified by our modelling, (Modelling, Act II: Fragrance) vanillin production can be improved by overproducing the precursor tyrosine. This can be achieved by knocking out the tyrR gene, the product of which represses the genes responsible for biosynthesis of aromatic amino acids. Another way of increasing tyrosine is by increasing the carbon flow into the aromatic amino acid biosynthesis pathway (the shikimate pathway), possibly by overexpressing phosphoenolpyruvate synthetase (PEPS) encoded by ppsA and transketolase (TKT) encoded by tktA, both involved in the shikimate pathway. These approaches can further be exploited to improve vanillin biosynthesis.
Although we did not produce vanillin, we codon optimised and His-tagged sam8, sam5 and comt , each regulated by a T7 promoter, and characterised them. To the best of our knowledge, we are the first team to characterize sam8 from Saccharothrix espanaensis and the new part comt from the plant Arabidopsis thaliana. As the last two enzymes FCS and ECH were expressed and characterized by British Columbia 2013, these major improvements will make it possible for future iGEM teams to be able to produce vanillin from a tyrosine precursor by combining both sets of Biobricks.
Scene 2: Limonene
By expressing GPPS and LimS in E. coli, we produced 0.6 mg/L of limonene which we detected by GC-MS. This is lower than the amount produced by Leferink et al., titre of 530.6 mg L org -1, who coexpressed the pBbB21-GPPS-LimS plasmid with the pMVA plasmid, leading to improved IPP and DMAPP supply in the cell (Figure 2). Although 0.6 mg/L is close to the OD of limonene, which is 0.56 mg/L, if we wanted to further improve limonene expression we could increase the pool of precursors by upregulating the genes involved in the MVA or MEP pathway.
The citrus scent of limonene was covered by the natural smell of E. coli which arises from the tnaA gene that produces tryptophanase, converting L-tryptophan into indole. To remove this smell which overpowers and hides the scent of limonene, we could delete the tnaA gene as done by a previous iGEM team (Paris Saclay 2014, Odor-free E.coli).
Scene 3: Overall
In the future, we would also want to clone vanillin- or limonene-producing plasmids to our naturally attaching dye-secreting bacteria, to test the volatility of these compounds and the distance at which they are perceived, which would validate our modelling predictions. (Modelling, Act II: Fragrance) Having the genes encoding the dyeing, scenting and repairing molecules in separate plasmids will allow the consumer to mix and match according to preference, making our product highly customizable. In a day and age where mass production is suffering a social backlash, this is an edge Cutiful has over most chemical dyes.