A study of DGAT enzymes from Tropaeolum majus with focus on oil biosynthesis

Abstract

An alternative energy and chemicals must be found to meet the future demand. A possible source of renewable energy is plant oil since it has a close chemical structure to fossil oil with less refining requirement. However, vegetable oil has not made a significant contribution to replace fossil oil as an energy source because of high demand of fossil oil. It is possible to substitute fossil-based chemicals and materials. One of the prospective chemical is erucic acid. The derivates of erucic acid, erucamide and behenic acid, are widely used in many oleochemical industries, such as plastic, surfactant and lubricant industries.
Besides having an attractive flower appearance, Tropaeolum majus is identified as the only known species that naturally possesses ultra-high erucic acid content (>75%) in its seed oil. Highly concentration of erucic acid in oil will reduce the refining process cost. T. majus is not capable to be cultivated as an industrial oil crop due to low oil content (5-10%) and agricultural reasons. Nevertheless, it can be a model for studying oil biosynthesis, especially high erucic acid content. One of the responsible enzymes in plant oil biosynthesis is DGAT (Diacyl Glycerol Acyl Tranferase).
The aim this study is to clone DGAT genes from Indian cress (later called TmDGAT1 and TmDGAT2 gene) and express DGAT in a yeast mutant strain H1246MATα with all 4 genes knocked out that are responsible in TAG biosynthesis, Δdgai1, Δlro1, Δare1, and Δare2 which encoding gene DGAT, PDAT, ACAT1, and ACAT2 respectively. The activity of the cloned TmDGAT1 and TmDGAT2 enzyme in oil synthesis was assayed in vitro. The main challenge of this thesis project was that the full-length sequence of TmDGAT2 gene was unknown before this study and could not be found in any transcriptomic database. However, by the help of cDNA RACE synthesis, TmDGAT2 gene sequence was identified.
The results of the study show that TmDGAT1 and TmDGAT2 has functional protein motif sites as DGAT enzyme. Both, TmDGAT1 and TmDGAT2 also have activity as DGAT enzyme and can transfer fatty acids to the sn-3 position of DAG, to produce oil. However, acyl-CoA specificity was very different between TmDGAT1 and TmDGAT2. TmDGAT1 has a broad preference of transferring acyl-CoA into [14C] 6:0-DAG. TmDGAT2 has a high specificity in transferring C18:3-CoA into [14C] 6:0-DAG, not C22:1-CoA as hypothesized. The specificity may be influenced by acyl-CoA availability, the fatty acid content in DAG and the enzymes itself. Each enzyme may have different regulation leading to oil synthesis, even though they have similar protein sequences. Therefore, it is required to conduct further research to acquire a full characteristic of TmDGAT enzymes.
Key words: Tropaeolum majus, erucic acid, TAG, DAG, DGAT, enzyme specificity, acyl-CoA

Popular Abstract

Besides having an attractive flower appearance, Tropaeolum majus, also called Indian cress, is identified as the only known species that naturally possesses ultra-high erucic acid content (>75%) in its seed oil. It is around 40% higher than the erucic acid content in commercial High Erucic Acid Rapeseed oil (HEAR). More interestingly, the seed oil mainly consists of trierucin, which cannot be found in HEAR. Highly concentration of trierucin in oil will reduce the refining process cost.
The derivates of erucic acid, erucamide and behenic acid, are widely used in many oleochemical industries, such as plastic, surfactant and lubricant industries. The recent years, plant oil has become more popular to be used in the oleochemicals industry compared to the biofuel industry. The reasons for using plant oil in the oleochemical industry is driven by limitation supply of fossil oil, environmental reason and the high value of specialty chemicals.
Indian cress is not capable to be cultivated as an industrial oil crop due to low oil content (5-10%) and agricultural reasons. Nevertheless, Indian cress can be a model for studying oil biosynthesis, especially high erucic acid content. Indian cress genes that are responsible for oil synthesis are possible to clone into industrial oil crops, such as B. rapa and C. abyssinia, in purpose of fulfilling the erucic acid demand in oleochemical industry. One of the responsible enzymes in plant oil biosynthesis is DGAT (Diacyl Glycerol Acyl Tranferase).
In this master thesis study, DGAT genes from Indian cress are cloned (later called TmDGAT1 and TmDGAT2 gene) and expressed in a yeast mutant strain devoid all enzyme that can contribute to oil (TAG, Tri Acyl Glycerol) production. The activity of the cloned TmDGAT1 and TmDGAT2 enzyme in oil synthesis was assayed in vitro. The main challenge of this thesis project was that the full-length sequence of TmDGAT2 gene was unknown before this study and could not be found in any transcriptomic database. However, by the help of cDNA RACE synthesis, TmDGAT2 gene sequence was identified.
The results of the study show that TmDGAT1 and TmDGAT2 has functional protein motif sites as DGAT enzyme. Both, TmDGAT1 and TmDGAT2 also have activity as DGAT enzyme and can transfer fatty acids to the sn-3 position of DAG, to produce oil. However, acyl-CoA specificity was very different between TmDGAT1 and TmDGAT2. The specificity may be influenced by acyl-CoA availability, the fatty acid content in DAG and the enzymes itself. Each enzyme may have different regulation leading to oil synthesis, even though they have similar protein sequences. Therefore, the results in this master thesis project will be the basis of future research of TmDGAT enzymes.
Key words: Indian cress, Tropaeolum majus, erucic acid, TAG, DGAT, enzyme specificity

• author: Rivani, Meta
• supervisor: Nélida Leiva Eriksson; Ida Lager
• organization: Pure and Applied Biochemistry
• course: KBKM01 20181
• year: 2018
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Tropaeolum majus, erucic acid, TAG, DAG, DGAT, enzyme specificity, acyl-CoA, applied biochemistry, tillämpad biokemi
• language: English
• id: 8950534
• date added to LUP: 2018-06-18 14:28:53
• date last changed: 2018-06-18 14:28:53