Contents 1 Lemon Scent 1.1 Countdown 1.2 Summary 1.3 Introduction 1.3.1 Limonene 1.3.2 Pinene 1.3.3 Citral 1.4 Principle of our Constructions 1.4.1 Construction of pPS1 1.4.2 Construction pPS2 1.4.3 Construction of pPS3 1.4.4 Construction of pPS4 1.4.5 Construction of pPS5 1.5 Réferences

Lemon Scent

Countdown

This page is under Melanie's responsibility

• Deadline: 12/oct
  • Final review by Sylvie.

Summary

In the part of the project, we want to express the main components of the lemon scent (limonene, β-pinene and citral) in the odor-free E. coli we constructed. For this, we plan to engineer a plasmid constructed by the team of Taek Soon Lee, bearing a complete mevalonate pathway, a geranyldiphosphate synthase and a (S)-limonene synthase, to express (i) a β-pinene synthase (BBa_K517003), (ii) a geraniol synthase (GES) and a cinnamyl alcohol dehydrogenase (CAD) to produce β-pinene and citral respectively. We also would like to characterize the limonene synthase biobrick constructed by the Edinburgh team (BBa_K722100) and verify whether it is functional by replacing the intial (S)-limonene synthase by the biobrick.

We managed to construct two plasmids, the plamsid containing the β-pinene synthase gene and the plasmid containing the geraniol synthasegene. Due to lack of time, We could not obtain the other plasmids.

Introduction

The lemon scent is due to many volatile compounds, but the three main components are the limonene, pinene and citral. All these molecules belong the terpene family of metabolites.[1]

Limonene

There are two isomeric forms of the limonene molecules: The odors of these two isomers are different: the (R)-Limonene has a orangy smell whereas the (S)-Limonene has a strongly lemon scent. [2]

Pinene

There are also two isomeric forms of pinene: alpha and beta pinene. In the lemon oil, β-pinene represents between 10-17% of the total compounds [3]. In contrast, there is no trace of α-pinene.

Citral

This is the principal component of the lemon grass and lemon smell and it is present in many other plants such as orange, verbena... Here again there are two stereoisomeric forms, geranial and neral. The trans isomer, geranial or citral A, has a strong lemon scent. On the contrary, the cis isomer named neral or citral B has sweaker lemon scent. These compounds are often used in the perfume industry to reproduce the smell of lemon.

All these compounds derive from a common precursor, the geranylpyrophosphate (GPP). This precursor is converted in one or two steps in the molecules we are interested in obtaining.

The desoxyxylulose 5-phosphate (DXP) parthway is the common pathway used by prokaryote cells to produce GPP. E. coli synthesizes GPP using the DXP pathway but in low quantity [4]. However, another pathway, the mevalonate pathway (MEV) exists in eukaryote and some prokaryotes. Moreover, plants have the two pathways to produce GPP in higher quantity. To copy the plants' strategy, we chose to add the MEV pathway to our engineered E. coli in order to produce higher quantity of GPP and thereby have a stronger lemon smell. The figure below illustrates the MEV pathway:

To tackle the problem of the low GPP bioavailability in E. coli, the Taek Soon Lee’s research group designed and constructed a plasmid with a complete biosyntheticmevalonate pathway for the production of GPP [3]. They then expressed the Abies grandis (S)-limonene synthase (LS) gene in the same plasmid, named pJBEI6409. With this construction, they were able to produce 400 mg/L of (S)-limonene.

We decided to make use of their construction for the production of limonene, β-pinene and citral in E. coli. We thank the team of Taek Soon Lee for the gift of the pJBEI6409 plasmid.

Principle of our Constructions

For our project, we need to replace the (S)-limonene synthase (LS) gene by the (R)-limonene synthase gene (BBa_K762100), the β-pinene synthase gene (BBa_K517003) and the geraniol synthase (GES) and cinnamyl alcohol dehydrogenase (CAD) genes.

However, pJBEI6409 was constructed using a 3A assembly method, meaning that there are no restriction sites flanking the LS gene. We therefore decided to replace the (S) Limonene Synthase gene by an apramycin resistance cassette flanked by PacI and SalI restriction sites by double homologous recombination. The resulting plasmid (pPS1) will then be used to clone the various genes (limonene synthase – β-pinene synthase – geraniol synthase and cinnamyl alcohol dehydrogenase).

Construction of pPS1

Replacement of the LS gene by an apramycin cassette

The first step of this sub-project is to replace the (S)-limonene synthase (LS) gene by an apramycin-resistant cassette. This cassette is flanked by PacI and SalI restriction sites to facilitate the cloning of the different genes.

The (S)-LS starting gene is replaced by the apramycin cassette by PCR targeting.

1. PCR amplification of the apramycin cassette with oligonucleotides iPS70 and iPS71 that contain the restriction sites PacI and SalI. The ends of these oligonucleotides are homologous with the sequence that constitutes the LS gene.
2. Transformation of the E. coli DY330 strain [5] (hyperrecombinant strain) by the pJBEI6409 plasmid.
3. Transformation of the E. coli DY330/pJBEI6409 with the PCR product after induction of the recombination genes.
4. Verification of the clones by BglII digestion.

We call this plasmid pPS1.

Construction pPS2

Replacement of the apramycin cassette by the (R)- LS gene, Bba_K722100

This step consists in replacing the apramycin resistance gene cassette introduced in pJBEI6409 by the (R)-LS gene.

1. PCR amplification of Bba_K722100 with oligonucleotides iPS66 and iPS67 that contain the restriction sites PacI and SalI.
2. TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (LS Topo plasmid).
3. Verification of the clones LS-TOPO by PCR with the oligonucleotides iPS66 and iPS67 and sequencing.
4. Digestion of the LS-TOPO plasmid by PacI and by MscI to eliminate the topo vector.
5. Digestion of pPS1 by PacI.
6. Ligation of the LS gene into pPS1/PacI.
7. Transformation of E. coli DH5α with the ligation product.
8. Verification of the clones by PCR with the oligonucleotides iPS72 and iPS96

Here we failed to obtain an insert in the correct orientation.

Construction of pPS3

Replacement of the apramycin cassette by the β pinene gene, BBa_K517003

This step consists in replacing the apramycin resistance gene cassette introduced in pJBEI6409 by the (R)-LS gene.

1. PCR amplification of Bba_K517003 with oligonucleotides iPS68bis and iPS69 that contain the restriction sites PacI and SalI.
2. TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (PS Topo plasmid).
3. Verification of the clones LS-TOPO by PCR with the oligonucleotides iPS68bis and iPS69 and sequencing.
4. Digestion of the LS-TOPO plasmid by PacI and by PstI to eliminate the topo vector.
5. Digestion of pPS1 by PacI.
6. Ligation of the PS gene into pPS1/PacI.
7. Transformation of E. coli DH5α with the ligation product.
8. Verification of the clones by PCR with the oligonucleotides iPS72 and iPS93.

Construction of pPS4

Replacement of the apramycin cassette by the geraniol synthase gene,

This step consists in replacing the apramycin resistance gene cassette introduced in pJBEI6409 by geraniol synthase gene (obtained from Marc Fischer, from the Grapevine Secondary Metabolism team of the ‘grapevine health and wine quality' research unit (Colmar)).

1. PCR amplification of Bba_K722100 with oligonucleotides iPS81bis and iPS82 that contain the restriction sites PacI and SalI.
2. TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (GS Topo plasmid).
3. Verification of the clones LS-TOPO by PCR with the oligonucleotides i iPS81bis and iPS82 and sequencing.
4. Digestion of the LS-TOPO plasmid by PacI and PstI to eliminate the topo vector.
5. Digestion of pPS1 by PacI.
6. Ligation of the GES gene into pPS1/PacI.
7. Transformation of E. coli DH5α with the ligation product.
8. Verification of the clones by PCR with the oligonucleotides iPS72 and iPS96

Construction of pPS5

Cloning of the CAD gene in pPS4

This step consists in cloning the Cinnamyl alcohol deshydrogenase (CAD) gene in the SalI site of pPS4

1. PCR amplification of the truncated CAD gene (synthetic gene with oligonucleotides iPS79 and iPS80 that contain the restriction sites PacI and SalI).
2. TA-cloning of the PCR product in the PCR 4 Blunt-Topo vector (CAD Topo plasmid).
3. Verification of the clones LS-TOPO by PCR with the oligonucleotides i iPS79 and iPS80 and sequencing.
4. Digestion of the LS-TOPO plasmid by SalI and PvuII to eliminate the topo vector.
5. Digestion of pPS4 by SalI.
6. Ligation of the CAD gene into pPS4/SalI.
7. Transformation of E. coli DH5α with the ligation product
8. Verification of the clones by PCR with the oligonucleotide

Réferences

• [1] Marcelo Carnier Dornelas - A genomic approach to characterization of the Citrus terpene synthase gene family - Genet. Mol. Biol. vol.30 no.3 suppl.0 São Paulo 2007
• [2] http://www.societechimiquedefrance.fr/produit-du-jour/limonene-et-monoterpenes.html
• [3] http://booksofdante.wordpress.com/tag/beta-pinene/
• [4] Jorge Alonso-Gutierrez and al - Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production – metabolique engineering 2013
• [5] Daiguan Yu and al - An efficient recombination system for chromosome engineering in Escherichia coli – 2000)