Cappetta Elisa, De Palma Monica, D'Alessandro Rosa, Aiello Alessandra, Romano Raffaele, Graziani Giulia, Ritieni Alberto, Paolo Dario, Locatelli Franca, Sparvoli Francesca, Docimo Teresa, Tucci Marina
National Research Council, Institute of Bioscience and Bioresources, Portici, Italy.
Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy.
Front Plant Sci. 2022 Jun 20;13:913374. doi: 10.3389/fpls.2022.913374. eCollection 2022.
The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon ( L. var. ) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through -mediated metabolic engineering. Bioinformatic data mining in the transcriptome allowed the selection and molecular characterization of (stearic acid desaturase) and (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent overexpressing (OE) and RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the OE6 line compared with 30.4% in the wild type (WT), whereas silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the KO2 line with respect to 17.7% in the WT. Moreover, OE6 and KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts.
开发具有较小环境足迹的可持续供应植物成分和天然物质的有效工具，有助于缓解气候变化的严峻形势。基于植物细胞培养的生物精炼厂可能是应对这一挑战的技术进步，并能提供标准化成分且不受栽培植物季节性变化影响的潜在无限量天然物质。单不饱和（MUFA）脂肪酸作为可生物降解塑料的补充剂、化妆品行业的生物添加剂和生物润滑剂，正受到广泛关注。刺菜蓟（L. var.）愈伤组织培养物积累脂肪酸和多酚，因此适合大规模生产生物化学品、有价值的化合物以及生物燃料前体。为了提高其潜在用途，我们设计了一种生物技术方法，通过介导的代谢工程来增加油酸含量。对转录组进行生物信息学数据挖掘，使得能够选择并对编码油酸和亚油酸形成关键酶的（硬脂酸去饱和酶）和（脂肪酸去饱和酶）基因进行分子表征，将其作为代谢工程的靶点。总共获得了22个快速生长的独立过表达（OE）和27个RNAi敲除（KO）转基因株系。对每个构建体的五个独立转基因株系的进一步表征表明，成功地，过表达增加了亚油酸含量，例如在OE6株系中，相对于野生型（WT）的30.4%，亚油酸含量增加到相对脂肪酸含量的42.5%，而沉默则减少了亚油酸，有利于其前体油酸的积累，例如在KO2株系中，相对于WT的17.7%，油酸含量几乎增加到相对脂肪酸含量的57%。此外，与WT中的2.7 mg/g DW相比，OE6和KO2的总多酚含量也显著增加，分别高达约4.7和4.1 mg/g DW，这主要是由于二咖啡酰奎宁酸和阿魏酰奎宁酸的积累。这些结果为有效创建基于工程化刺菜蓟细胞的生物精炼厂奠定了基础，该生物精炼厂可积累来自初级和特殊代谢的高水平有价值化合物，以满足工业对可再生和可持续创新生物产品来源的需求。
