Part:BBa_K3052004

(S)-linalool synthase: DHJ

The linalool synthase is used to convert geranyl pyrophosphate (GPP) to linalool, which has aroma of lavendar.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 490
  Illegal PstI site found at 571
  Illegal PstI site found at 994
  Illegal PstI site found at 1060
  Illegal PstI site found at 1150
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 490
  Illegal PstI site found at 571
  Illegal PstI site found at 994
  Illegal PstI site found at 1060
  Illegal PstI site found at 1150
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 490
  Illegal PstI site found at 571
  Illegal PstI site found at 994
  Illegal PstI site found at 1060
  Illegal PstI site found at 1150
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 490
  Illegal PstI site found at 571
  Illegal PstI site found at 994
  Illegal PstI site found at 1060
  Illegal PstI site found at 1150
  Illegal NgoMIV site found at 21
  
• 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(Keasling et al., 2013), 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: (A)MVA pathway. (B)Synthesis of limonene and linalool

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.

Reference [1]lonso-Gutierrez, J., Chan, R., Batth, T. S., Adams, P. D., Keasling, J. D., Petzold, C. J., & Lee, T. S. (2013). Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production. Metabolic engineering, 19, 33-41.

Characterization

Ptrc and linalool synthase genes was obtained by PCR flanked two Type IIS sites BsaI and was used for Golden Gate Assembly to construct the expression vectors. The length of linalool synthase DHJ is 1632bp. Here is the gel results confirming success amplifications.

Figure 2: Gel results of PCR amplification of CS

The expression of limonene synthase and linalool synthases are under the regulation of Ptrc. RT-qPCR and SDS-PAGE were conducted to test their expressions, as well as Gas Chromatography analysis was used to detect limonene or linalool yields. Click to see our protocol. We measured the transcription level of CS and LSs of four different strains, E.coli harboring only pGPP vector, E.coli with dual plasmid pGPP+pDHJ (Stain B in Fig 3), pGPP+pLGF (Stain C in Fig 3), pGPP+pCS (sStain A in Fig 3),and relative mRNA levelst compared with the blank strain only harboring pGPP are shown in Fig 4.

Figure 3:RT-qPCR result of linalool synthase DHJ, LGF and limonene synthase CS

As shown in Fig 4, compared to blank strain, the relative mRNA level of three strains were much higher, which DHJ were most actively transcripted, and CS was relatively less, but all indicated the efficient transcription of these three genes in engineered E.coli. We also conducted SDS-PAGE of whole cell proteins to ensure their expression. As shown in Fig 6, compared to blank strain only with pGPP vector, CS protein was expressed but yields were not high, and because of the leaky expression, expression level with IPTG induction were just a little bit higher than the level without IPTG induction.

Figure 4:SDS-PAGE result of DHJ: Control group: sample from E.coli transformed with pGPP; Experimental group: sample from E.coli transformed with pGPP and pDHJ (+:with IPTG induction/-: without IPTG induction)

Both the results of above RT-qPCR and SDS-PAGE demonstrate the expression of the linalool synthase DHJ.

Figure 5:Comparison of linalool production of E. coli strain harboring LS gene with linalool standard, confirmed its production.

GC analysis will confirm whether the expressed protein is functional. Firstly we analyzed a positive limonene-containing control that yielded a strong peak as indicated by the black line with the retention time abound 1.44 minutes. The results show that there is no overlap of retention time between n-hexane and linalool, and indicates the GC method is effective for testing. In comparison, we expressed the limonene generator with trc promoter in DH5α E. coli and lysed the cell culture, extracted with n- hexane, and the extraction was subjected to gas chromatography mass spectrometry. All in all, the compressive analysis of RT-qPCR, SDS-PAGE and GC-MS have demonstrated that the fragrance molecule linalool can be successfully synthetized by engineered E.coli with our selected enzymes.