背景回顾：Lignins are cell-wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. 

主要研究：Here, we explored the role of the two CSE homologs in poplar (Populus tremula x P. alba). 

结果1-表达模式鉴定：Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. 

结果2-单突：CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. 

结果3-双突：In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced lignin content translated into a four-fold increase in cellulose-to-glucose conversion upon limited saccharification. 

结果4-双突代谢谱：Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. 

结果5-体外酶活验证：This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. 

结论：Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

 摘 要 

木质素是定位于细胞壁的芳香族聚合物，为木质组织提供了机械强度和疏水性能。木质素单体通过苯丙烷途径合成，其中咖啡酰莽草酸酯酶CSE的作用是将咖啡酰莽草酸转化为咖啡酸。本文中，作者研究了两个CSE同源蛋白在杂交杨（Populus tremula x P. alba）中的作用。通过构建报告株系，作者发现CSE1和CSE2启动子的表达模式比较相近。CRISPR-Cas9产生的cse1和cse2单突变体具有类似于野生型的木质素水平。然而，CSE1和CSE2基因并不是完全冗余的，因为两个单突植株都积累了咖啡酰莽草酸。相反，cse1 cse2双突植株的木质素含量降低了35%，并伴随着生长受限。在受限的糖化过程中，木质素含量的降低转化为四倍的纤维素-to-葡萄糖转化。对双突植株的酚类物质代谢谱研究显示，除了咖啡酰莽草酸外，还存在大量代谢变化，包括p-coumaroyl、5-hydroxyferuloyl、feruloyl和sinapoyl shikimate的积累。这说明CSEs具有广泛的底物特异性，体外的酶动力学也证实了这一推测。综上，本文的研究结果在羟基肉桂酰莽草酸的水平上提出了苯丙烷途径中的一条替代途径，并且表明CSE是一个很有前途的、可用于培育生物质提炼植物而进行改良的潜在靶标。