Difference between revisions of "Part:BBa K4805002"

Latest revision as of 15:34, 12 October 2023

BmeTC-FL-BmeTC Y167A,D373C

description

BmeTC is a tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658). BmeTC_Y167A, D373C refers to a variant of BmeTC 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, the synergistic effect of BmeTC_Y167A, D373C and BmeTC in the cell-free system can produce the highest yield of ambrein with squalene as substrate. (Tsutomu S. et al, 2020) Thus we tried to construct a fusion protein with a flexible linker to tie BmeTC to BmeTC_Y167A, D373C, hoping that this strategy can improve the specifity of the cyclase. Our part can help and inspire other future teams to build and perfect the pathway of producing ambrein from squalene. It belongs to the terpenoids 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 and BmeTC_Y167A,D373C are both parts that our team uses to enhance the production of ambrein in S.Cerevisiae. Here we used a flexible linker to link the two enzymes together, pTDH3 and tTDH1 to enable its expression in yeast, hoping to reach a higher acticity of cyclization.

source

Bacillus megaterium

characterization

We tried to insert all three genes of BmeTC_Y167A, D373C and BmeTC into the 106 site to construct our strain Lv2a-1 (Figure 1A), and the successful integration were confirmed in strain as the positive PCR results shown in Figure 1C. Based on this synergistic effect that can promote the biosynthesis of ambrein, we presumed that using flexible linker to construct a fuison protein of BmeTC_Y167A, D373C and BmeTC could remarkably improve the yield of ambrein. To verify our assumption, we integrated BmeTC-Flexible Linker-BmeTC_Y167A, D373C into 106 site (Figure 1B, D).

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 different further modified strains. To be specific, the yield of squalene decrease up to 77.40% in strain Lv2a-1 and 90.69% in strain Lv2a-2. 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

references

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