Cao Viet Dang, Luo Guangbin, Korynta Shelby, Liu Hui, Liang Yuanxue, Shanklin John, Altpeter Fredy
Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida, IFAS, Gainesville, FL, USA.
DOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL, USA.
Biotechnol Biofuels Bioprod. 2023 Oct 14;16(1):153. doi: 10.1186/s13068-023-02393-1.
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.
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.
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.
通过代谢工程使营养组织中超量积累脂质是提高生物质作物能量密度和生物燃料产量的新策略。能源甘蔗因其高生物量产量以及在边缘条件下的适应能力，是该方法的优质原料。二酰甘油酰基转移酶（DGAT）催化三酰甘油（TAG）生物合成的最后一步且是唯一的关键步骤，可能是TAG生产的限速酶。
在本研究中，我们探究了内含子介导增强（IME）对能源甘蔗叶片和茎组织中DGAT1表达以及由此导致的TAG和总脂肪酸（TFA）积累的影响。为使脂质积累最大化，通过共表达生脂转录因子皱叶1（WRI1）和TAG保护因子油质蛋白（OLE1）来进行这些评估。在密码子优化的TmDGAT1中包含一个内含子，基于每个构建体的5个转基因株系，其转录本在叶片中的积累平均提高了7倍。具有WRI1（W）、含内含子的DGAT1（Di）和OLE1（O）表达的植株（WDiO）积累的TAG高达叶片干重（DW）的3.85%，与未改良的能源甘蔗（WT）相比增加了192倍，与无内含子基因组合（WDO）下的最高积累量相比增加了3.8倍。这对应着TFA积累量高达叶片干重的8.4%，分别比WDO或WT增加了2.8倍或6.1倍。WDiO的共表达导致茎中TAG积累量高达DW的1.14%或TFA积累量高达DW的2.08%，分别比WT高出57倍或12倍，比WDO高出两倍多。这些生脂“推拉护”因子的组成型表达与生物量减少相关。
内含子介导的DGAT表达增强（IME）使能源甘蔗的脂质积累发生了显著变化，并证实了在我们的实验条件下它是脂质积累的限速因素。IME应应用于其他生脂因子和代谢工程策略。本研究结果对于开发用于商业生产脂质和先进生物燃料的高生物量原料可能具有重要价值。
