Part:BBa_K4805000

BmeTC

Description

BmeTC is a tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium(Genbank Accession: CP001982.1, 2130781–2132658). It encodes the cyclase that intends to produce Bicyclic structure based on the substrate of squalene. It can convert squalene to 8α-hydroxypolypoda-13,17,21-triene(Bicyclic structure), subsequently,14β-hydroxyono-cera-8(26)-ene onoceranoxid(Bicyclic-Bicyclic structure).
It is reported that BmeTC can collaborate with other enzymes that tend to catalyze one side of squalene into monocyclic structure, ultimately forming monocyclic-bicyclic structure, which is known as ambrein. So far, it has been heterologously expressed in yeast to biosynthesize ambrein, including P. pastoris and S. cerevisiae.
Our part can help and inspire other future teams to build the pathway of producing ambrein from squalene. It belongs to the part collection we have established for the production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012.

Usage and Biology

BmeTC is tetraprenyl-B-curcumene cyclase from Bacillus megaterium (GenBank Accession: CP001982.1, 2130781-2132658), is a widely used multifunctional terpenoid cyclase. This cyclase, BmeTC, has been characterized to be able to convert squalene into the bicyclic 8α-hydroxypolypoda-13,17,21-triene. Utilising such usage, it is usually co-expressed with SHC_D377C in P. pastoris and S. cerevisiae for the production of ambrein. But such strategy can produce the lower yield of ambrein than the another strategy of only expressiong its variant, BmeTC_D373C (Harald P. et al, 2018; Harald P. et al, 2019). However, Tsutomu Sato's research team used cell-free system to find out that synergistic effect of BmeTC_Y167A, D373C and BmeTC can reach about 9.77-fold higher activity than BmeTC_D373C (Tsutomu S. et al, 2020). 
This year, we co-expressed BmeTC and BmeTC_Y167A, D373C under the control of pPGK1 and pTDH3 respectively.

Source

Bacillus megaterium

Characterization

We tried to insert BmeTC_Y167A, D373C and BmeTC into 106 site to construct our strain Lv2a-1(Figure 1A), and the successful integration can be seen in strain as it is shown in Figure 1C.

Figure 1: (A) Schematic strategy of BmeTC_Y167A, D373C and BmeTC integration into the site 106. (B) Schematic strategy of BmeTC_Y167A, D373C-Flexible Linker-BmeTC integration into the site 106. (C) These two genes were confirmed to be inserted into site 106 in the strain 6 of Lv2a-1.(D) These two genes were confirmed to be inserted into site 106 in the strain 2-8 of Lv2a-2.

Unfortunately, we did not observe any peaks suspected of ambrein. But the reaction substrate, squalene, can be detected at 9.70-9.83 min. Compared with strain Lv1, the yield of squalene significantly decreased in the further modified strains. To be specific, the yield of squalene decrease up to 77.40% in strain Lv2a-1. Although there's no ambrein can be detected, the decrease of squalene might indicate the synthesis of some potential intermediates. We will optimize our testing method and the specificity of cyclase to reach the goal of ambrein production.

File:Https://static.igem.wiki/teams/4805/wiki/engineering-success/es10.png

Figure 2: Figure 2. (A) Analysis of ambrein and squalene, accumulated in strain Lv1, Lv2a-1 and Lv2a-2 by GC-MS. (B) The yield comparison of squalene in different strains based on GC-MS.

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 676
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 79
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Moser, S., Leitner, E., Plocek, T. J., Koenraad Vanhessche, & Pichler, H. (2019). Engineering of Saccharomyces cerevisiae for the production of (+)‐ambrein. Yeast, 37(1), 163–172. https://doi.org/10.1002/yea.3444 Ueda, D., Hoshino, T., & Sato, T. (2013). Cyclization of Squalene from Both Termini: Identification of an Onoceroid Synthase and Enzymatic Synthesis of Ambrein. Journal of the American Chemical Society, 135(49), 18335–18338. https://doi.org/10.1021/ja4107226 Yamabe, Y., Kawagoe, Y., Okuno, K., Inoue, M., Chikaoka, K., Ueda, D., Tajima, Y., Yamada, T. K., Kakihara, Y., Hara, T., & Sato, T. (2020). Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein. Scientific Reports, 10(1), 19643. https://doi.org/10.1038/s41598-020-76624-y Moser, S., Strohmeier, G. A., Leitner, E., Plocek, T. J., Vanhessche, K., & Pichler, H. (2018). Whole-cell (+)-ambrein production in the yeast Pichia pastoris. Metabolic Engineering Communications, 7, e00077. https://doi.org/10.1016/j.mec.2018.e00077