Muthusamy Saraladevi, Vetukuri Ramesh R, Lundgren Anneli, Ganji Suresh, Zhu Li-Hua, Brodelius Peter E, Kanagarajan Selvaraju
Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar, Sweden.
Department of Plant Breeding, Swedish University of Agricultural Sciences, Alnarp, Sweden.
PeerJ. 2020 Apr 28;8:e8904. doi: 10.7717/peerj.8904. eCollection 2020.
The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from () was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the was cloned into a plant viral-based vector pEAQ-. Subsequently, the plasmid was transferred into the and agroinfiltrated into leaves. The expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.
倍半萜β-石竹烯是许多植物中普遍存在的成分，在商业上已被用作化妆品和香水的香料。最近的研究表明它有作为治疗剂和生物燃料的潜在用途。目前，β-石竹烯是从大量植物材料中分离出来的。基于瞬时表达系统的分子农业可用于更可持续地生产β-石竹烯。在本研究中，从（）中分离出一种新的重复β-石竹烯合酶的全长cDNA并进行了功能表征。为了在体外生产β-石竹烯，将其克隆到基于植物病毒的载体pEAQ-中。随后，将质粒转入（）并通过农杆菌渗入法导入（）叶片。在农杆菌渗入后的不同时间点通过定量PCR分析（）的表达。在渗入后9天（dpi）观察到最高水平的转录本。在9 dpi时从叶片中提取AaCPS1蛋白，并使用带有组氨酸标签的钴-亚氨基三乙酸（Co-NTA）亲和色谱法进行纯化，产量为每千克鲜叶89 mg。AaCPS1的蛋白表达也通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析得到证实。AaCPS1蛋白以法尼基二磷酸（FPP）为底物产生β-石竹烯。通过气相色谱-质谱联用（GC-MS）对纯化后的AaCPS1进行产物鉴定和活性测定。GC-MS结果显示，在用FPP测定6小时后，AaCPS1每千克鲜叶最多可产生26.5±1 mg的β-石竹烯。以AaCPS1作为概念验证，我们证明（）可被视为一种表达系统，用于生产催化形成具有工业应用价值化学品的植物蛋白。
