Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues

Abstract

Background Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. Results In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction. Conclusions Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels. Graphical abstract

Triacylglycerol, total fatty acid, and biomass accumulation of metabolically engineered energycane grown under field conditions confirms its potential as feedstock for drop‐in fuel production

“…hybrid) is an ideal feedstock to fuel the emerging bioeconomy with its exceptional biomass production, and carbon sequestration (Alexander, 1985;Carvalho-Netto et al, 2014;Diniz et al, 2019;Richard, 2010). However, due to its recalcitrance to tissue culture and genetic transformation, there are only three reports to date of metabolically engineered energycane (Cao et al, 2023;Luo et al, 2022;Padilla et al, 2020). Metabolic engineering for elevated oil content in vegetative tissues of high biomass crops represents a substantial opportunity for boosting lipid production and biodiesel yields (Ohlrogge & Chapman, 2011).…”

“…DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), catalyzes the final and only committed step in TAG synthesis which leads to a formation of an ester linkage between a fatty acyl CoA and the free hydroxyl group of diacylglycerol (Lassner, 1997). We recently confirmed that DGAT1 is a rate-limiting factor for TAG accumulation in vegetative tissues of energycane (Cao et al, 2023). OLEOSIN1 (OLE1) codes for a structural protein that stabilizes lipid droplets (LD) by preventing them from coalescing and reducing oil hydrolysis by lipases through steric hindrance (Parthibane et al, 2012;Siloto et al, 2006).…”

“…Three multigene expression vectors were created using Golden Gate cloning (Lebedenko et al, 1991;Szybalski et al, 1991) as described by Cao et al (2023). The first vector contains expression cassettes of WRI1 from Sorghum bicolor controlled by the Brachypodium distachyon elongation factor 1-α promoter (pBdEF1α) and Panicum virgatum ubiquitin (tPvUbiII) terminator, and the selectable marker gene neomycin phosphotransferase (nptII) driven by the maize ubiquitin promoter (pZmUbi) and Sorghum bicolor heat shock protein terminator (tSbHSP; Figure 1a).…”

“…Two transgenic lines L2 and L13, each expressing WRI1, DGAT1, and OLE1, were chosen for field phenotyping. Lines L2 and L13 were selected for this study due to similar WRI1 expression and difference in DGAT1 expression and TAG and TFA accumulation under greenhouse conditions (Cao et al, 2023). When the plants in the greenhouse achieved a height of approx.…”

“…However, metabolic engineering strategies combining the simultaneous optimization of fatty acid synthesis, TAG assembly and protection from degradation termed "push, pull, protect strategy" resulted in more drastic increases of TAG levels in vegetative tissues (Vanhercke, El Tahchy, et al, 2014). To date, vegetative tissues of both model plants (Fan et al, 2013;Vanhercke et al, 2013;Vanhercke, El Tahchy, et al, 2014;Vanhercke, Petrie, et al, 2014;Yang et al, 2015) and high biomass plants such as sugarcane, energycane, maize, sorghum, potato, duckweed, and perennial ryegrass (Alameldin et al, 2017;Beechey-Gradwell et al, 2020;Cao et al, 2023;Hofvander et al, 2016;Liang et al, 2023;Liu et al, 2017;Luo et al, 2022;Parajuli et al, 2020;Vanhercke et al, 2019;Xu et al, 2012;Zale et al, 2016) have been metabolically engineered for hyperaccumulation of TAG. This involved overexpression of WRI1, DGAT1, and OLE1, and/or suppression of SUGAR-DEPENDENT1 (SDP1) and TRIGA LAC TOS YLD IAC YLG LYCEROL1 (TGD1) or PEROXISOMAL TRANSPORTER1 (PXA1).…”