Page 59 - ebook

Base editing of Arabidopsis thaliana DGAT1 gene for modification of seed oil
biosynthesis and 3D structure of DGAT1 protein
Won Nyeong Kimand Hyun Uk Kim*
Department of Bioindustry and Bioresource Engineering, Sejong University, Seoul, 05006 Republic of Korea

Abstract

In rising concern of climate change and dissipating petroleum, importance of vegetable oil ( triacylglycerol:TAG) is getting
highlight. Due to the structure similarity between petroleum and vegetable oil, many industrial products which traditionally
processed from petroleum can be replaced with phyto-based materials. To meet escalating demands of vegetable oil, appending
agricultural farm to produce more amount is simple but there are spatial huddles such as region specificity considering growth
condition of each oil producing plant. To overcome these issues, increase of vegetable oil producing amount per plant is the key.
At this point, diacylglycerol acyltransferase 1 (DGAT1) is crucial enzyme to solve the problem. DGAT1 is endoplasmic reticulu m
(ER) located transmembrane enzyme and take role in final transfer of acyl chain to diacylglycerol to make TAG. TAG are
accumulated between two layers of ER membrane forming monolayer oil body in seeds. In this study, we used base editing
technology to edit fifty-two target sites in seven functional domains of DGAT1 expecting change of enzyme properties in manner Fig 4. Changed base of Domain 3 treated
of increasing TAG produce. By far, total five type of base changed homo transgenic Arabidopsis were generated. We will line and its seed fatty acid profile
discuss the changes in oil composition and content in the seeds of these five DGAT1 variant mutants.
A) Chromatograph result of DNA
Sanger sequencing and substituted
Introduction amino acid occurred by domain 3 base
editing. B) Alignment of acyl-CoA
binding motif (Domain 3). Substituted
amino acid highlighted with yellow
shade. C) Fatty acid profile of D3-1 lines
using gas chromatography

Fig 1. Schematic construction of DGAT1 domains and process of Base Editing
A) Schematic Aradisopsis thatliana DGAT1 structure. Five red colored domains indicating predicted in early report. Remaining
Fig. 1 Schematic construction of DGAT1 domains and process of Base Editing
two green colored domains are highly studied its features or investigated by site -directed mutagenesis. Transmembrane
domains were predicted by TMHMM online tool. B) Principle of base editing. Base editor use nick or dead Cas9 which dead or
weakened on its cleavage activity. When n/dCas9 locate the deaminase domain on target sequence, deaminase domain
changes the base in window of base editor.
With this base editing system, we designed fifty -two targets to induce substitution of amino acids in those domains without Fig. 5 Changed base of Domain 5 treated line and its seed fatty acid profile
frame-shift. As result, five homogeneous Arabidopsis lines were obtained and analyzed by Gas Chromatography(GC) to A) Chromatograph result of DNA Sanger sequencing and substituted amino acid occurred by domain 3 base editing. B) Alignment o f
compare the fatty acid component or amount. The investigation of base edited lines were showed the difference between Col -0 acyl-CoA binding motif (Domain 5). Substituted amino acid highlighted with yellow shade. C) Qualitive, quantitative Fatty acid p rofile of
wildtype and dgat1 mutant. But unfortunately, not all targeted domain were substituted to different amino acid. Furthermore,
human DGAT1 enzyme, which highly similar in predicted structure with Ararbidopsis DGAT1, was reported its 3D structure and D5-1 and D5-2 lines using gas chromatography
biochemical features but we will discuss about these issues with our result.
Discussion

Fig 6. Predicted 3D structure of DGAT1 based on human
DGAT1 and schematic interaction of mutant DGAT1 and
acyl-CoA chain.
All 3D models were constructed by Phyer2 web tool that use
homology-based building algorithm. Based on human DGAT1
protein property, Arabidopsis DGAT1 makes dimer form as
normal status and have two main entrance to reaction chamber
which includes Histidine reaction residue. The two main
Fig 2. Schematic process of screening base edited Arabidopsis and example of base substitution through generation entrance is entry tunnel and lateral gate which gives
accessibility to cytosolic region and hydrophobic region of
To transfer sgRNA including vectors to Arabidopsis, we used floral dipping method or spraying method to inoculate membrane, respectively.
Agrobacterium. After transformation, harvested T1 seeds were screened by Hygromycin antibiotics. Through these process, A) 3D model of membrane-imbedded DGAT1 protein. Lateral
screened individual plants DGAT1 DNA sequences were send to company for analyze. Hetero individuals which showed mixed gate had indicated with red dotted circle. B) 3D model of
pick in sequencing results in T2 or T3 generation were selected to keep breeding. With generation continued, some bases were domain 1 base edited DGAT1. Entry tunnel had indicated with
edited to homogeneous status. Homogeneous lines were analyzed by gas chromatography to investigate the difference between green dotted circle. Cyan colored residue and line indicate
Col-0 wildtype and dgat1 mutants. substituted amino acid. Putative N-terminal also extended with
red dotted line. C) 3D model of domain 3 base edited DGAT1.
Red surface indicate substituted amino acid, which has different
binding angle to original one. D) 3D model of domain 5 base
Results edited DGAT1. Amino acid substitute affects negatively to acyl-
CoA binding indicated with red surface. Amino acid substitute
affects positively to acyl-CoA binding indicated with green
surface. Entrance of entry tunnel highligh ted green dotted circle.
Summary
➢ The activity of base edited DGAT1 seems weaken
On-going study ➢ By 3D modeling, edited regions are located.
➢ Gas Chromatography shows tag1-1 similar
➢ Comparison of Gene expression, protein accumulation
amount quantitative and qualitative result except 20:1 fatty
acid which imply base edited AtDGAT1 has
➢ Production of complementary AtDGAT1 transgenic line different mode of action from tag1-1 DGAT1
Fig 3. Changed base of Domain 1 treated line and its
seed fatty acid profile References
A) Chromatograph result of DNA Sanger sequencing
and substituted amino acid occurred by domain 1 base ➢ Xu, J., Francis, T., Mietkiewska, E., Giblin, E.M., Barton, D.L., Zhang, Y., Zhang, M. and Taylor, D.C. (2008), Cloning and
editing. B) Alignment of acyl-CoA binding motif (Domain characterization of an acyl‐CoA‐dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of
1) from various plant species including Homo sapiens. the functional motifs of the DGAT protein using site‐directed mutagenesis to modify enzyme activity and oil content. Plant
Substitu ted am ino acid high lighted wi th yellow shade. C) Biotechnology Journal, 6: 799-818. doi:10.1111/j.1467-7652.2008.00358.x
Fatty acid profile of D1-1 and D1-2 lines using gas ➢ Wang, L., Qian, H., Nian, Y. et al. Structure and mechanism of human diacylglycerol O-acyltransferase 1. Nature 581, 329–332
chromatography (2020). https://doi.org/10.1038/s41586-020-2280-2.
Sejong University, Seoul Korea