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. 2021 Feb 4:8:621127.
doi: 10.3389/fbioe.2020.621127. eCollection 2020.

High-Level Patchoulol Biosynthesis in Artemisia annua L

Xueqing Fu  1 , Fangyuan Zhang  1   2 , Yanan Ma  1 , Danial Hassani  1 , Bowen Peng  1 , Qifang Pan  1 , Yuhua Zhang  3 , Zhongxiang Deng  3 , Wenbo Liu  3 , Jixiu Zhang  3 , Lei Han  3 , Dongfang Chen  3 , Jingya Zhao  1 , Ling Li  1 , Xiaofen Sun  1 , Kexuan Tang  1
Affiliations

High-Level Patchoulol Biosynthesis in Artemisia annua L

Xueqing Fu et al. Front Bioeng Biotechnol. .

Abstract

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

Keywords: Artemisia annua L.; patchoulol; sesquiterpenoids; synthetic biology; terpenes.

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Conflict of interest statement

YZ, ZD, WL, JZhang, LH, and DC were employed by company Firmenich Aromatics (China) Co. Ltd. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
A depiction of the terpene biosynthesis, along with a conceptualization for patchoulol biosynthesis to the cytoplasm (blue) and to the chloroplast (red) compartments. HMGR, 3-hydroxy-3- methylglutaryl coenzyme A reductase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose5-phosphate reductase; FPS, farnesyl diphosphate synthase; FPP, farnesyl pyrophosphate; PTS, patchoulol synthase.
Figure 2
Figure 2
Engineering patchoulol biosynthesis in the cytoplasm of A. annua leaves. (A) Relative expression of FPS and PTS in FPS+PTS transgenic A. annua lines. (B) The patchoulol content in FPS+PTS transgenic A. annua lines. ACTIN was used as internal control. T0 transgenic lines were used for analysis. The error bars represent the means ± SD from three biological replicates. All data represent the means ± SD of three replicates. **P < 0.05, *P < 0.01, student's t-test.
Figure 3
Figure 3
Blocking the artemisinin biosynthesis increased patchoulol content in ADSi+ FPS+PTS transgenic A. annua plants. (A) Relative expression of ADS in ADSi+ FPS+PTS transgenic A. annua lines. (B) The artemisinin content in ADSi+ FPS+PTS transgenic A. annua lines. (C) Relative expression of FPS and PTS in ADSi+ FPS+PTS transgenic A. annua lines. (D) The patchoulol content in ADSi+ FPS+PTS transgenic A. annua lines. T0 transgenic lines were used for analysis. ACTIN was used as internal control. The error bars represent the means ± SD from three biological replicates. All data represent the means ± SD of three replicates. **P < 0.05, *P < 0.01, student's t-test.
Figure 4
Figure 4
The subcellular localization of heterologous proteins. (A) Subcellular localization of FPS-GFP and PTS-GFP in tobacco leaf epidermal cells. (B) Subcellular localization of tpFPS-GFP and tpPTS-GFP in tobacco leaf epidermal cells. GFP: green fluorescent protein, Bars = 20 μm.
Figure 5
Figure 5
Engineering the patchoulol biosynthesis in the chloroplast compartment increased the patchoulol content in tpFPS+tpPTS transgenic A. annua plants. (A) Relative expression of FPS and PTS in tpFPS+tpPTS transgenic A. annua lines. (B) The patchoulol content in tpFPS+tpPTS transgenic A. annua lines. ACTIN was used as internal control. The error bars represent the means ± SD from three biological replicates.
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