Difference between revisions of "Part:BBa K3052010"

Revision as of 01:15, 22 October 2019

Precursor Circuit -- a series of enzymes used for GPP production

This circuit contains eight enzymes in plant's MVA pathway to produce geranyl pyrophosphate(GPP),a precursor to various metabolites unique in plants like linalool and limonene. All of the genes in this circuit are inducible by IPTG since all of them are under control of Ptrc or Plac.

Reference [1]Alonso-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.

Sequence and Features

Validation

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

Precursor Circuit converts carbon sources like glucose to geranyl pyrophosphate(GPP), ready to be catalyzed into limonene and linalool. We constructed the circuit by modification of pJBEI6409. The 11523 bp-length vector is constructed based on pSB1C3 backbone and each operon is under the regulation of Ptrc, and the digestion verification result of two endonucleases is as follows.

Figure 2:Endonuclease digestion verification of precursor circuit

Since the product of this circuit, GPP is unstable to be detected via HPLC etc., we performed SDS-PAGE analysis to compare the expression level of key enzyme--GGPs with and without IPTG induction to test if this circuit works properly, and the result is shown below. Click to see our protocol.

Figure 3:SDS-PAGE result of precursor circuit (+:with IPTG induction/-: without IPTG induction)

The results of SDS-PAG clearly demonstrate the high expression of the GPPs in the precursor circuit, indicating its functionality and eventual GPP synthesis.