Part:BBa_K4805001

BmeTC_Y167A, D373C

Description

BmeTC_Y167A, D373C refers to a variant of the Tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658) that contains two specific mutations: Y167A and D373C. Although BmeTC can only catalyze the bicyclic structure at the end of squalene, its variant BmeTC_D373C and BmeTC_Y167A, D373C possess the ability of catalyzing both monocyclic and bicyclic structure, which can produce ambrein. So far, it has been used in cell-free system with squalene as substrate, and can produce the highest yield of ambrein in all single enzyme reactions. (Tsutomu S. et al, 2020)
Our part can help and inspire other future teams to build and perfect 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_Y167A, D373C refers to a variant of the Tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium. It possess the ability of catalyzing both monocyclic and bicyclic structure, which can produce ambrein. So far, it has been used in cell-free system with squalene as substrate, and can produce the highest yield of ambrein in all single enzyme reactions. In order to achieve the goal of producing more ambrein, BmeTC_Y167A, D373C has a higher affinity for bicyclic substances(8α-hydroxypolypoda-13,17,21-triene) compared to squalene. In a cell-free system, this enzyme produces 2.9 times more ambrein than squalene when a bicyclic substance is used as a substrate. And the synergistic effect of BmeTC_Y167A, D373C and BmeTC can reach 53.26% higher activity than BmeTC_Y167A, D373C. This inspired us to attempt to co-expressed BmeTC and BmeTC_Y167A, D373C in S.cerevisiae.

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.

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.

sequencing 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]

reference

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