Identification and functional characterization of two acyl CoA:diacylglycerol acyltransferase 1 (DGAT1) genes from forage sorghum (Sorghum bicolor) embryo

Highlights

• Key genes controlling biosynthesis and accumulation of triacylglycerol (TAG) in forage sorghum grains are identified.

• Expression of SbDGAT1 genes in yeast (H1246) led to the restoration of TAG biosynthesis pathway.

• N-terminal deletion of SbDGAT1-1 exhibit lower TAG accumulation in yeast H1246.

• SbDGAT1-2 showed predominant expression pattern in developing bran and embryo of forage sorghum grains.

Abstract

Elevating the lipid content in high-biomass forage crops has emerged as a new research platform for increasing energy density and improving livestock production efficiency associated with improved human health beneficial meat and milk quality. To gain insights of triacylglycerol (TAG) biosynthesis in forage sorghum, two type-1 diacylglycerol acyltransferase (designated as SbDGAT1-1 and SbDGAT1-2) were characterized for its in vivo function. SbDGAT1-2 is more abundantly expressed in embryo and bran during the early stage of the grain development in comparison to SbDGAT1-1. Heterologous expression of SbDGAT1 genes in TAG deficient H1246 strain restored the TAG accumulation capability with high substrate predilection towards 16:0, 16:1 and 18:1 fatty acids (FA). In parallel, we have identified N-terminal intrinsically disordered region (IDR) in SbDGAT1 proteins. To test the efficacy of the N-terminal region, truncated variants of SbDGAT1-1 (designated as SbDGAT1-1_(39-515) and SbDGAT1-1_(89-515)) were generated and expressed in yeast H1246 strain. Deletion in the N-terminal region resulted in decreased accumulation of TAG and FA (16:0 and 18:0) when compared to the SbDGAT1-1 variant expressed in yeast H1246 strain. The present study provides significant insight in forage sorghum DGAT1 gene function, useful for enhancing the green-forage TAG content through metabolic engineering.

Introduction

Green forage plays an indispensable role in farming as it supplies the required nutrients for the livestock animals and in turn can contribute to a high quality of meat and milk available for human consumption. Plant oils and lipids are predominantly composed of glycerol esters of fatty acids (FA), also known as neutral lipid or triacylglycerol (TAG), which are highly concentrated sources of metabolic energy as they are reduced and anhydrous, containing double the energy of either carbohydrates or proteins (Thelen and Ohlrogge, 2002). Plant lipids are important sources of energy for the farm animals especially dairy cattle and also contribute directly to ~50% of the milk fat. Seed-grains like barley, wheat, oat, rye, and sorghum are excellent sources of energy that can fulfill the daily recommended dietary requirements of the dairy animals. However, their use as animal feed remains largely limited in most of the developing nations across the globe particularly in India. Poor energy-containing green forage is the major source of food for livestock animals, rendering the animals undernourished and lowering the production efficiency of milk and meat for human use. Development of strategies to boost up the energy content in green forage by means of enhancing the neutral lipid/oil content can lead to improved livestock production and in alleviating the prevailing dearth of high energy food resources for dairy cattle.

In plants, diacylglycerol acyltransferase (DGAT) catalyzes the final step of oil biosynthesis (the esterification of sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA) and is located in distinct but separate regions of the endoplasmic reticulum (ER) (Shockey et al., 2006). In the ER, TAGs are synthesized by the stepwise acylation of glycerol-3-phosphate, known as Kennedy pathway (Shen et al., 2010). In the beginning, fatty acyl moieties are esterified to the glycerol-3-phosphate backbone at the sn-1 and sn-2 positions. This esterification reaction is catalyzed by glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase, respectively to form phosphatidic acid (PA). Phosphatidate phosphatase further hydrolyzes PA to yield diacylglycerol (DAG) (Bhunia et al., 2016). DGAT is the last key rate limiting enzyme that synthesizes TAG by esterifying third acyl chain to DAG (Voelker and Kinney, 2001).

In eukaryotes, the DGATs are generally classified into three types, viz. DGAT1, DGAT2 and DGAT3, based on their differences in structure and function (Turchetto-Zolet et al., 2011). The DGAT1 has been reported to play an important role in biosynthesis and accumulation of TAG, whereas DGAT2 controls the content and composition of seed oils with unusual FAs (Chen et al., 2016). Both of these DGATs are ubiquitous and have unique structures, whereas DGAT3 is found to be soluble and cytosolic (Saha et al., 2006; Hernández et al., 2012; Rani et al., 2013; Aymé et al., 2018). Among the three types of DGATs, DGAT1 has been extensively used for enhancing the oil content in seeds (Vanhercke et al., 2013, 2014, 2017).

In maize and Arabidopsis, single amino-acid substitution (insertion of phenylalanine or mutation of serine to alanine) in DGAT1 protein, resulted in enhanced seed oil content by modulating the post-translational modifications (Zheng, 2008; Xu et al., 2008). DGAT1 proteins are predicted to contain eight to ten membrane spanning domains (Liu et al., 2012) preceded by the N-terminal region (Caldo et al., 2017). In recent studies, the N-terminal domain of DGAT1 protein was found to be actively involved in its functional regulation (Caldo et al., 2017; Panigrahi et al., 2018). Being mainly composed of intrinsically disordered region (IDR), the N-terminal domain functioned as a regulator based on the availability of acyl-CoA or DAG as a substrate for acyl coenzymeA (CoA)-dependent TAG biosynthesis (Caldo et al., 2017; Panigrahi et al., 2018). However, the N-terminal segment is not required for the activity of the enzyme (Caldo et al., 2017; Panigrahi et al., 2018). The role of N-terminal segment remains unclear in the context of SbDGAT1 functioning and needs to be analyzed for gaining useful insights into its regulatory mechanism.

Sorghum (Sorghum bicolor) is a high biomass crop and the fifth most important cereal crop worldwide (Mundia et al., 2019). Apart from grain and sweet sorghum, forage sorghum varieties are used worldwide to feed livestock animals and can grow in low water areas of tropical and subtropical regions of the world (Smith and Frederiksen, 2000). In addition, sorghum possesses higher photosynthetic energy conversion efficiency that leads to increase in leaf area and high biomass production (Slattery and Ort, 2015). The total oil content in sorghum grain and leaf is ~4% (grain dry weight) and ~0.2% (leaf dry weight), respectively (Vanhercke et al., 2019). Attempts have been made earlier to enhance the TAG content in sweet sorghum by ectopic expression of Umbelopsis ramanniana DGAT2 along with Zea mays WRI1 (Wrinkled 1) transcription factor and Sesamum indicum L-Oleosin (Vanhercke et al., 2019). Synergistic effect of all three genes in sorghum leaves enhances accumulation of oil which is stored as lipid bodies (LB) in the leaf mesophyll cells (Vanhercke et al., 2019). However, identification, functional role, expression pattern and evolution of endogenous SbDGAT1 genes of sorghum is still largely unexplored.

In this context, the in silico structural analysis, expression pattern and in vivo characterization of SbDGAT1 genes in Saccharomyces cerevisiae H1246 TAG mutant is evaluated. In parallel, the effect of full-length and two truncated N-terminal segment of SbDGAT1-1 variants were also examined. Our results strongly suggest that these two genes are potential candidates for altering TAG biosynthesis in seed and vegetative tissues.