van Erp Harrie, Shockey Jay, Zhang Meng, Adhikari Neil D, Browse John
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 (H.v.E., N.D.A., J.B.);Southern Regional Research Center, United States Department of Agriculture-Agricultural Research Service, New Orleans, Louisiana 70124 (J.S.); andDepartment of Plant Biology, Michigan State University, East Lansing, Michigan 48824-1312 (M.Z.).
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 (H.v.E., N.D.A., J.B.);Southern Regional Research Center, United States Department of Agriculture-Agricultural Research Service, New Orleans, Louisiana 70124 (J.S.); andDepartment of Plant Biology, Michigan State University, East Lansing, Michigan 48824-1312 (M.Z.)
Plant Physiol. 2015 May;168(1):36-46. doi: 10.1104/pp.114.254110. Epub 2015 Mar 4.
One goal of green chemistry is the production of industrially useful fatty acids (FAs) in crop plants. We focus on hydroxy fatty acids (HFAs) and conjugated polyenoic FAs (α-eleostearic acids [ESAs]) using Arabidopsis (Arabidopsis thaliana) as a model. These FAs are found naturally in seed oils of castor (Ricinus communis) and tung tree (Vernicia fordii), respectively, and used for the production of lubricants, nylon, and paints. Transgenic oils typically contain less target FA than that produced in the source species. We hypothesized that competition between endogenous and transgenic isozymes for substrates limits accumulation of unique FAs in Arabidopsis seeds. This hypothesis was tested by introducing a mutation in Arabidopsis diacylglycerol acyltransferase1 (AtDGAT1) in a line expressing castor FA hydroxylase and acyl-Coenzyme A:RcDGAT2 in its seeds. This led to a 17% increase in the proportion of HFA in seed oil. Expression of castor phospholipid:diacylglycerol acyltransferase 1A in this line increased the proportion of HFA by an additional 12%. To determine if our observations are more widely applicable, we investigated if isozyme competition influenced production of ESA. Expression of tung tree FA conjugase/desaturase in Arabidopsis produced approximately 7.5% ESA in seed lipids. Coexpression of VfDGAT2 increased ESA levels to approximately 11%. Overexpression of VfDGAT2 combined with suppression of AtDGAT1 increased ESA accumulation to 14% to 15%. Our results indicate that isozyme competition is a limiting factor in the engineering of unusual FAs in heterologous plant systems and that reduction of competition through mutation and RNA suppression may be a useful component of seed metabolic engineering strategies.
绿色化学的一个目标是在作物中生产具有工业用途的脂肪酸（FAs）。我们以拟南芥（Arabidopsis thaliana）为模型，重点研究羟基脂肪酸（HFAs）和共轭多烯脂肪酸（α-桐酸 [ESAs]）。这些脂肪酸分别天然存在于蓖麻（Ricinus communis）和油桐（Vernicia fordii）的种子油中，并用于生产润滑剂、尼龙和油漆。转基因油中通常含有的目标脂肪酸比源物种中产生的要少。我们推测内源性和转基因同工酶对底物的竞争限制了拟南芥种子中独特脂肪酸的积累。通过在一个在其种子中表达蓖麻脂肪酸羟化酶和酰基辅酶A:RcDGAT2的品系中引入拟南芥二酰甘油酰基转移酶1（AtDGAT1）的突变来验证这一假设。这使得种子油中HFA的比例增加了17%。在该品系中表达蓖麻磷脂：二酰甘油酰基转移酶1A使HFA的比例又增加了12%。为了确定我们的观察结果是否更广泛适用，我们研究了同工酶竞争是否影响ESA的产生。在拟南芥中表达油桐脂肪酸共轭酶/去饱和酶在种子脂质中产生了约7.5%的ESA。VfDGAT2的共表达将ESA水平提高到了约11%。VfDGAT2的过表达与AtDGAT1的抑制相结合，使ESA积累增加到14%至15%。我们的结果表明，同工酶竞争是异源植物系统中非常规脂肪酸工程的一个限制因素，通过突变和RNA抑制减少竞争可能是种子代谢工程策略的一个有用组成部分。
