Team:GreatBay China/Basic Part

GBC-Basic_Part


mCATNIP2018@outlook.com
@Igem2018C

Best Basic Part



This part is the coding sequence of geraniol synthase (GES) from the species Ocimum basilicum (sweet basil)[1] and it’s codon-optimised for the use in E. coli. GES catalyses the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used and is commercially important in favour and fragrance industries[2], and it’s an intermediate to the biosynthesis of nepetalactol which is an active ingredient in catnip.


===Characterization===
Figure. 1 The biosynthetic route for obtaining geraniol from acetyl-coA, which requires either a MVA or MEP pathway, a GPPS, and a GES.

In our study, we aim to achieve geraniol synthesis in E. coli, so at the beginning, we expressed a geraniol synthesis operon (http://parts.igem.org/Parts:BBa_K2753016) containing an Abies Grandis geranyl pyrophosphate synthase (http://parts.igem.org/Parts:BBa_K2753002) and an Ocimum basilicum geraniol synthase (http://parts.igem.org/Parts:BBa_K2753003) regulated by a pTac promoter in E. coli. Assuming that higher expression leads to higher yield, the operon was assembled on a high copy vector pUC20 whose copy number is approximately 500 per cell. However, when carrying out shake-flask fermentation with this strain, after induced by 25uM IPTG for 24 hours2, no geraniol production was detected using gas chromatography. (see detailed methods in our notebook: https://2018.igem.org/Team:GreatBay_China/Notebook)


We infer that the failure to produce geraniol was due to the lack of substrates IPP and DMAPP. Thus we decided to co-express an MVA pathway in addition to the geraniol synthesis operon. The pathway can either be added by cotransformation of a plasmid containing only the MVA pathway and the other plasmid with only GPPS and GES, or by constructing an all-in-one plasmid containing the MVA pathway, GPPS, and GES. We obtained a plasmid pMVA-only which was a gift from Taek Soon Lee, and have added GPPS and GES onto pMVA-only to acquire a pMVA-GPPS-GES.



Figure. 2 Geraniol production analysis by gas chromatography. (A) GPPS and GES are arranged in operon regulated by pTac, placed on a high copy vector pUC20. (B) The MVA pathway is split into two clusters and placed on a low copy vector with the upper cluster containing three genes and the downstream containing four. (C) A combined plasmid with GPPS&GES operon and MVA pathway with a low copy p15A origin. (D) Gas chromatography for geraniol produced by E. coli expressing pMVA only (middle trace) or pMVA-GPPS-GES (bottom trace) whose peaks coincide with a geraniol standard (top trace). (E) Geraniol yield is noticeable with only the heterologous MVA pathway. Upon introduction of GPPS and GES, the yield doubled relative to the negative control pMVA only.


Under the same fermentation condition, pMVA-GPPS-GES gave a geraniol titer of 11.4mg/L while the negative control pMVA-only as well produced geraniol titer of about 5.65mg/L. Then we are assured that a stable supply of precursors IPP and DMAPP are crucial for geraniol production. But still, there is room for higher geraniol yield. Therefore, to gain maximum flux to geraniol, we tuned the expression level of GPPS and GES using three promoters:


And three vectors of unique copy number:


  • pUC20: ~500
  • pR6K: ~15
  • pSC101: ~1

Figure. 3 Geraniol production assay of TALEsp relative to pTac IPTG-inducible promoter. (A) Twelve combinations of promoters and plasmid backbones used to express a geraniol synthesis operon for characterization. pSC101 is the low copy vector, pR6K is the medium copy vector and pUC20 is the high copy vector. The combinations are transformed and tested in E. coli already harboring pMVA plasmids. (B) Geraniol yield achieved under the regulation of pTac, TALE1 sp1, TALE2 sp1, and TALE2 sp6 across different copy numbers is shown. The vertical error bars represents standard deviations of geraniol yield measured, and the horizontal error bars signify the reported range of copy number of plasmid backbones used. The portion of graph belong to TALE2sp6 is not shown as there is no detectable geraniol production.


Having transferred the constructs above along with pMVA-only into strain E. coli DH5α, the same shake-flask fermentation experiment was conducted. The result shows detectable geraniol production by each strain.


With pTac promoters, geraniol yield increased with the copy number of the vector, showing positively correlated relation. However, when TALE stabilized promoter (TALEsp) was used, higher the copy number of the vector, lower the production of geraniol, being the opposite of pTac. And the stronger TALEsp promoter, TALE1sp1 gave generally reduced yield compared to its weaker counterpart TALE2sp1. Interestingly, the graphs of the two TALEsp promoters appeared to be seemingly parallel to each other.

Figure. 4 Gas chromatography analysis for geraniol produced regulated by pTac and TALE2sp1 compared with the peak of geraniol standard. (A) Geraniol production accomplished by pTac regulation across low (pSC101), medium (pR6K) and high (pUC20) copy number. The area of the peak which appears at around 4.41s concurring with that of the geraniol standard increases along the copy number. (B) Geraniol yield assay using TALE2sp1 with the same set of copy number. The largest peak of geraniol emerges on the low copy vector pSC101. The peak shrinks in size on higher copy.

We surmised that the yield of geraniol was affected by two factors: the expression level of enzymes and the cellular burden. As for enzyme expression, there exists an optimal gene expression level that produces just enough enzyme to metabolize all the substrate. The yield would be the greatest at this level. But if lower than this level, production would increase with enzyme expression since there is a surplus of substrates. And if the enzyme expression is higher, the more enzymes now becomes a cellular burden as it no longer contributes to more product.

In the case of pTac, it fits into the scenario when the gene expression is lower than the optimum: higher copy produced more enzymes to catalyze geraniol synthesis reaction. As for TALE stabilized promoters, the expression level of enzymes would remain unchanged regardless of copy number due to the stabilization nature of pTALE (LINK more about pTALE). So in low copy vector where the cellular burden isn’t significant, the product yield is only related to the strength of the promoter, explaining why pTALE sp2 has better performance than pTALE sp1 as it’s closer to the optimum. But when the copy number increases, the expression level is unaffected while the cellular burden rises sharply because much more TALE would be made to stabilize expression, leaving less energy available for geraniol synthesis. And it in turns answers why a negative trend of production is shown when regulated by pTALE.


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TALE1:


TALE2:


Table. 1 The source of all promoters in the library
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All promoters are placed on the standard biobrick assembly compatible backbone pSB1C3, with a RBS (BBa_B0034) reporter gene sfGFP (BBa_K1679038) downstream of the promoters in between the restriction sites of SpeI and PstI. So teams can change reporter genes as they wish.


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For TALE stabilised promoters repressed by the same TALE, the only difference between them is the sequence of the core promoter which the TALE binds to. By replacing this promoter, new TALE stabilised promoters could then be easily created. For making the process more convenient, we designed this composite part in which a genetic circuit encoding for amilCP, a blue pigment, replaces the core promoter sequence, and downstream are a RBS, a coding sequence of sfGFP, and a terminator. Two restriction sites of BsaI are put on the two ends of the amilCP circuit. By adding the same digestion sites on the promoter to be assembled, the amilCP circuit could be substituted by Golden Gate. The blue colour of amilCP and green colour of sfGFP provide a rapid distinguishment of the successful construct. If the amilCP circuit is not replaced, the colony grown would be blue as amilCP is being expressed. But if a promoter sequence substitutes amilCP circuit, sfGFP would be expressed instead of amilCP, making the colony fluorescent and easy to recognise.


Except for TALE1sp1 and TALE2sp1 which were synthesized de novo by Genscript, we assembled the rest of TALEsps by first obtaining core promoters using SOE PCR then replacing the amilCP circuit with them.


Characterization


In order to verify the stabilization effect of the pTALE promoter family, we assembled all promoter members onto three backbones:

pUC20: copy number ≈ 500
pR6K: copy number ≈ 15
pSC101: copy number ≈ 1


An identical sfGFP is placed downstream of all promoters as a reporter gene. Three constitutive promoters: J23119, J23101, J23105, with the same sfGFP downstream were characterized along with the pTALE promoters as a reference of promoter strength. The green fluoresce was measured by flow cytometry. (see methods in our notebook).


     


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To decide whether pTALE can perform as expected in metabolic pathways, maintaining the pre-set ratio of production enzymes for ensuring optimal flux to the product, we tested pTALE1 sp1 and pTALE2 sp1 by comparison to pTac, a frequently-used IPTG inducible promoter, using an geraniol synthesis operon (<a href="http://parts.igem.org/Part:BBa_K2753015">Part:BBa_K2753015</a>) containing a GPPS (<a href="http://parts.igem.org/Part:BBa_K2753002">Part:BBa_K2753002</a>) and a GES (<a href="http://parts.igem.org/Part:BBa_K2753003">Part:BBa_K2753003</a>). Similar to the characterisation of green fluoresce characterization, three backbones: pUC20, pR6K, pSC101 are used.


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