Part:BBa_K3052001

(4S)-limonene synthase

The limonene synthase (LS) sequence used throughout our project which converts GPP to limonene is an E. coli codon-optimized version of a truncated sequence from M. spicata previously described(Hyatt et al., 2007).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Background

In our study, we aim to achieve limonene and linalool synthesis in E.coli DH5α. According to 2018 GreatBay_China team’s experience, no target product was detected using gas chromatography when carrying out shake-flask fermentation with this strain induced by 25uM IPTG for 24 hours due to the lack of endogenous MVA pathways wherein GPP is produced. Thus we decided to co-express an MVA pathway. 2018 GreatBay_China generously gave us one plasmid pJBEI6409, which contains a MVA pathway in addition to an GPPs-LS operon. We reconstructed this plasmid and get a plasmid only contains a MVA pathway.

Figure 1: MVA pathway

Figure 2: GPP production and synthesis of linalool and limonene

We have used MVA synthesis pathway which is common in plants. Since limonene and linalool have the same synthetic precursor GPP, we have divided the synthesis pathway into two parts:

1. (BBa_K3052001) IPTG inducible precursor circuit which contains eight enzymes of MVA pathway enable conversion from AcCoA to GPP: atoB, HMGS, HMGR, MK, PMK, PMD, idi, trGPPs.

2. (BBa_K3052004, BBa_K3052010) two Production Circuits: limonene synthase or linalool synthase regulated by a ptrc promoter in E. coli.

Results

For validation of these parts, we did both RT-PCR and SDS-PAGE to detect the expression, Gas Chromatography to detect limonene and linalool production. Click to see our [http://2019.igem.org/Team:XJTU-CHINA protocol].

RT-PCR

Figure 3: Relative mRNA level of 3 experimental group ( pGPP’s mRNA level is defined 1)

SDS-PAGE

Figure 4: 3 experimental group and 1 control group pGPP (+: with IPTG induction / -: without IPTG induction)