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肉桂醇脱氢酶CAD1和咖啡酰莽草酸酯酶CSE作用于杨树木质素合成

已有 6834 次阅读 2017-9-11 08:54 |个人分类:每日摘要|系统分类:科研笔记

Different metabolic routes for coniferaldehyde and sinapaldehyde with CINNAMYL ALCOHOL DEHYDROGENASE1 deficiency

First author:Rebecca Van Acker; Affiliations: Ghent University (根特大学): Gent, Belgium

Corresponding author: Wout A Boerjan

In search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification (糖化) efficiency and is therefore a prime target. Here, lignin content and composition were modified in poplar (Populus tremula × P. alba) by specifically downregulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1; 肉桂醇脱氢酶) by a hairpin-RNA-mediated (发夹RNA) silencing approach. These transgenic lines showed no biomass penalty (处罚) despite a 10% reduction in Klason lignin content (硫酸木质素，又称Klason木质素) and severe shifts in lignin composition. NMR spectroscopy (核磁共振光谱) and thioacidolysis (硫代酸解) revealed a strong increase in sinapaldehyde (芥子醛) incorporation into lignin, whereas coniferaldehyde (松柏醛) was not markedly increased. Accordingly, ultrahigh-performance liquid chromatography-mass spectrometry-based phenolic profiling (超高效液相色谱-质谱法) revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals (芥子醛基). However, no additional cinnamaldehyde (肉桂醛) coupling products could be detected. Instead, the CAD1-deficient lines accumulated a range of hydroxycinnamate-derived (羟基肉桂酸) metabolites of which the most prominent accumulation was observed for a compound that was identified by purification (提纯) and NMR as syringyl lactic acid hexoside (紫丁香基乳酸己糖苷). Our data suggest that upon downregulation of CAD1, coniferaldehyde is converted into ferulic acid (阿魏酸) and derivatives (衍生物), whereas sinapaldehyde is either oxidatively (氧化) coupled into S′(8-8)S′ and lignin, or converted to sinapic acid (芥子酸) and derivatives. The most prominent sink of the increased flux to hydroxycinnamates (羟基肉桂酸) is syringyl lactic acid hexoside(紫丁香基乳酸己糖苷). Furthermore, low-extent saccharification assays (糖化试验), under different pretreatment conditions, showed strongly increased glucose (葡萄糖) and xylose (木糖) release, suggesting that downregulating CAD1 is a promising strategy for improving lignocellulosic (木质纤维素) biomass for the 'sugar platform' industry.

对于搜索可再生能源，显示遗传工程是一种可以提高植物细胞壁组成用以生物燃料和生物产品再生的较为有希望的策略。木质素是糖化效率主要影响因素，因此也是主要的研究目标。本文，作者通过利用发夹RNA介导的沉默技术特异下调肉桂醇脱氢酶CAD1基因来改变杨树的木质素含量与组成。这些转基因株系在不影响生物量的前提下硫酸木质素含量下降了10%，并且在木质素组成上也有所改变。核磁共振光谱和硫代酸解试验揭示了木质素整合的芥子醛含量严重下降，但松柏醛的含量并为显著提高。另外，超高效液相色谱-质谱法显示一个新鉴定的由两个芥子醛基8-8耦合的化合物含量提升了近2,4000多倍。然而，并未检测到其它的肉桂醛耦合产物。相反，CAD1缺陷株系积累了一系列的羟基肉桂酸衍生物，其中最突出的是通过提纯和核磁共振鉴定为紫丁香基乳酸己糖苷的化合物。作者的试验数据显示当CAD1基因的表达下调时，松柏醛会转化为阿魏酸及其衍生物，而芥子醛也会经氧化耦合到S′(8-8)S′与木质素，亦或转化为芥子酸及其衍生物。最主要转变为羟基肉桂酸的化合物是紫丁香基乳酸己糖苷。另外，不同预处理条件下的低程度糖化试验显示葡萄糖与木糖释放的增加，表明下调CAD1基因是一个比较有前途的策略，用以提高木质素纤维的生物量。

doi: https://doi.org/10.1104/pp.17.00834

Silencing CAFFEOYL SHIKIMATE ESTERASE affects lignification and improves saccharification

First author:Marina de Lyra Soriano Saleme; Affiliations: Ghent University (根特大学): Gent, Belgium

Corresponding author: Wout A Boerjan

Caffeoyl shikimate esterase (CSE; 咖啡酰莽草酸酯酶) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis thaliana and later in Medicago truncately (苜蓿). However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues (木质化组织) of different plant species. Here, we show that downregulation of CSE in hybrid poplar (Populus tremula × P. alba) resulted in up to 25% reduced lignin deposition (木质素沉积), increased levels of p-hydroxyphenyl units (对羟基苯基结构单元) in the lignin polymer (木质素多聚体), and a relatively higher cellulose content. The transgenic trees were morphologically (形态) indistinguishable from the WT. Ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS; 超高效液相色谱-质谱法)-based phenolic (酚类) profiling revealed a reduced abundance of several oligolignols (寡木素酚) containing guaiacyl (愈创木基) and syringyl (紫丁香基) units and their corresponding hydroxycinnamaldehyde (羟基肉桂醛) units, in agreement with the reduced flux towards coniferyl and sinapyl alcohol (松柏醇与芥子醇). These trees accumulated the CSE substrate caffeoyl shikimate (咖啡酰莽草酸) along with other compounds belonging to the metabolic classes of benzenoids (苯型烃类) and hydroxycinnamates (羟基肉桂酸). Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE downregulated lines resulted in up to 62% more glucose released per plant upon limited saccharification (糖化) when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments (酸和碱预处理) were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic (木质纤维素的) biomass crops for sugar platform biorefineries (生物炼制).

咖啡酰莽草酸酯酶CSE是最近发现对于拟南芥中的木质素生物合成非常重要，之后在苜蓿中也有类似发现。然而，在不同植物木质化组织中有些会明显缺失CSE的活性，因此该酶的功能还需进一步验证。本文作者在杂种杨树中下调表达了CSE基因，结果显示木质素沉积下降了25%，木质素多聚体的对羟基苯基结构单元有所提升，同时纤维素的含量有了很高的提升。转基因的树木在形态学上可以明显的跟野生型区分开。基于超高效液相色谱-质谱法的酚类谱研究发现含愈创木基和紫丁香基的寡木素酚的含量有所下降，同样与之对应的羟基肉桂醛也呈现下降趋势，这与转基因植株体内化合物向松柏醇与芥子醇转变的结果相一致。这些树会积累CSE酶的底物咖啡酰莽草酸，同时也会积累一些苯型烃类与羟基肉桂酸类似的代谢产物。此外，在CSE下调表达的株系中减少的木质素总量结合相对提升的纤维素含量最终导致了在没有预处理的高达的62%的更多的葡萄糖释放，而在用酸和碱预处理后这个数值分别达到了86%和91%。本文的结果显示CSE酶不仅仅是木质化进程的重要参与者之一，同时也是未来潜在的提升木质纤维素生物通量的研究方向。

doi: https://doi.org/10.1104/pp.17.00920

通讯：Wout A Boerjan (http://www.vib.be/en/research/scientists/pages/wout-boerjan-lab.aspx)