Zhang Guixiang, Wu Yougen, Haq Muhammad Zeeshan Ul, Yang Yuzhang, Yu Jing, Zhang Junfeng, Yang Dongmei
College of Horticulture, Hainan University, Haikou, 570228, PR China.
College of Horticulture, Hainan University, Haikou, 570228, PR China.
Protein Expr Purif. 2019 Nov;163:105454. doi: 10.1016/j.pep.2019.105454. Epub 2019 Jul 10.
Pogostemon cablin is an important commercial source of patchouli oil, whose main active ingredient is patchouli alcohol. This sesquiterpene is a product of the mevalonate pathway, in which 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) is the rate-limiting enzyme. In this study, P. cablin HMGCR cDNA, comprising 2209 nucleotides encoding 425 amino acid residues was isolated, and bioinformatics analysis was used to analyze the protein sequence. Based on this analysis, a C-terminal truncated variant was engineered for recombinant expression in E. coli. The 38 kDa recombinant protein was identified by SDS-PAGE, and assayed for mevalonolactone production. According to the PcHMGCR1 gene sequence alignment with other species, the HMGCR protein had obvious resemblance with other plants HMG coenzyme A reductase and had homology with other species including plants, fungi, archaebacteria and animals. The prokaryotic expression vector was constructed by restriction enzyme digestion to be transformed into E. coli to express the recombinant protein, and 38 kDa recombinant protein was identified by the SDS-PAGE. Enzymatic activity was detected using GC-MS and, as a result, mevalonolactone was detected in the in vitro reaction mixture. Differential expression analysis showed that PcHMGCR1 expressed the highest amount in roots. The research results are of great significance for further research on the molecular biosynthesis mechanism of Patchouli alcohol in P. cablin.
广藿香是广藿香油的重要商业来源，其主要活性成分是广藿香醇。这种倍半萜是甲羟戊酸途径的产物，其中3-羟基-3-甲基戊二酰辅酶A还原酶（HMGCR）是限速酶。在本研究中，分离出了包含2209个核苷酸、编码425个氨基酸残基的广藿香HMGCR cDNA，并利用生物信息学分析对该蛋白质序列进行了分析。基于此分析，构建了一个C端截短的变体用于在大肠杆菌中进行重组表达。通过SDS-PAGE鉴定出了38 kDa的重组蛋白，并对其甲羟戊酸内酯的产生进行了测定。根据PcHMGCR1基因序列与其他物种的比对，HMGCR蛋白与其他植物的HMG辅酶A还原酶有明显的相似性，并且与包括植物、真菌、古细菌和动物在内的其他物种具有同源性。通过限制性酶切构建原核表达载体，将其转化到大肠杆菌中表达重组蛋白，并通过SDS-PAGE鉴定出38 kDa的重组蛋白。使用GC-MS检测酶活性，结果在体外反应混合物中检测到了甲羟戊酸内酯。差异表达分析表明，PcHMGCR1在根中的表达量最高。该研究结果对于进一步研究广藿香中广藿香醇的分子生物合成机制具有重要意义。
