Serrani-Yarce Juan Carlos, Escamilla-Trevino Luis, Barros Jaime, Gallego-Giraldo Lina, Pu Yunqiao, Ragauskas Art, Dixon Richard A
BioDiscovery Institute and Department of Biological Sciences, University of North Texas, Denton, 76203 TX, USA.
BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Biotechnol Biofuels. 2021 Feb 27;14(1):50. doi: 10.1186/s13068-021-01905-1.
Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called "esters" pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the "reverse" direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an "incomplete" shikimate shunt.
The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight.
Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.
羟基肉桂酰辅酶A：莽草酸羟基肉桂酰转移酶（HCT）是所谓通往单木质醇的“酯类”途径的核心酶。最初的设想是，HCT在该途径中发挥两次作用，形成香豆酰莽草酸，然后以“反向”方式将咖啡酰莽草酸转化为咖啡酰辅酶A。一种能直接从咖啡酰莽草酸形成咖啡酸的咖啡酰莽草酸酯酶（CSE）的发现，让人质疑木质素生物合成中是否需要HCT的反向反应。HCT功能丧失在几种双子叶植物中会导致严重的生长表型，但在一些单子叶植物中影响较小，这使人怀疑这种酶以及莽草酸分流途径在单子叶植物和双子叶植物中是否发挥相同作用。模式禾本科植物短柄草有两个HCT基因，但缺乏经典的CSE基因。因此，本研究旨在评估在具有“不完整”莽草酸分流途径的物种中，HCT作为木质素修饰靶点的效用。
比较了重组短柄草HCT与拟南芥、蒺藜苜蓿和柳枝稷（柳枝稷）HCT在正向和反向反应中的动力学特性。与两种蒺藜苜蓿HCT一样，短柄草HCT2的反向HCT反应在动力学上最有利，并且当HCT1下调时该酶被诱导。通过RNA干扰下调短柄草HCT1或同时下调HCT1和HCT2会导致木质素水平降低，木质素组成和分子量仅有适度变化。
与双子叶植物中HCT下调后观察到的情况相比，短柄草中HCT1下调或两个HCT基因同时下调导致木质素含量/组成和细胞壁结构的变化较小，对生物量产量几乎没有负面影响。然而，HCT下调会显著提高生物量糖化效率，使该基因成为通过生物技术改良禾本科植物用于生物加工的首选靶点。
