Romsuk Jutapat, Yasumoto Shuhei, Fukushima Ery Odette, Miura Kenji, Muranaka Toshiya, Seki Hikaru
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
Industrial Biotechnology Initiative Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan.
Front Plant Sci. 2022 Aug 18;13:991909. doi: 10.3389/fpls.2022.991909. eCollection 2022.
Oleanolic acid is a pentacyclic triterpenoid found in numerous plant species and is a precursor to several bioactive triterpenoids with commercial potential. However, oleanolic acid accumulates at low levels in plants, and its chemical synthesis is challenging. Here, we established a method for producing oleanolic acid in substantial quantities heterologous expression of pathway enzymes in . The "Tsukuba system" is one of the most efficient agroinfiltration-based transient protein expression systems using the vector pBYR2HS, which contains geminiviral replication machinery and a double terminator for boosting expression. Additionally, the pBYR2HS vector contains an expression cassette for the gene-silencing suppressor p19 protein from tomato bushy stunt virus, which can also contribute to enhancing the expression of target proteins. In this study, we evaluated the applicability of this system to heterologous triterpenoid production in . cytochrome P450 monooxygenase (CYP) 716A12 is the first enzyme to be functionally characterized as β-amyrin C-28 oxidase producing oleanolic acid. A mutant CYP716A12 (D122Q) with improved catalytic activity engineered in our previous study was co-expressed with other enzymes in . leaves. Using pBYR2HS, oleanolic acid yield was increased 13.1-fold compared with that using the conventional binary vector, indicating the advantage of the Tsukuba system. We also demonstrated the efficacy of co-expressing a mutant HMGR1 catalytic domain, additional NADPH-cytochrome P450 reductase (CPR) transferring electrons to heterologous CYPs, and application of ascorbic acid for preventing leaf necrosis after agroinfiltration, to improve product yield. As a result, the product yields of both simple (β-amyrin) and oxidized (oleanolic acid and maslinic acid) triterpenoids were significantly improved compared with the previously reported yield in heterologous triterpenoid production in . leaves.
齐墩果酸是一种五环三萜类化合物，存在于多种植物物种中，是几种具有商业潜力的生物活性三萜类化合物的前体。然而，齐墩果酸在植物中的积累水平较低，其化学合成具有挑战性。在此，我们建立了一种通过在烟草叶片中异源表达途径酶大量生产齐墩果酸的方法。“筑波系统”是基于农杆菌浸润的最有效的瞬时蛋白表达系统之一，使用载体pBYR2HS，该载体包含双生病毒复制机制和用于增强表达的双终止子。此外，pBYR2HS载体包含来自番茄丛矮病毒的基因沉默抑制蛋白p19的表达盒，这也有助于增强目标蛋白的表达。在本研究中，我们评估了该系统在烟草中异源三萜类化合物生产中的适用性。细胞色素P450单加氧酶（CYP）716A12是第一个被功能鉴定为产生齐墩果酸的β-香树脂醇C-28氧化酶的酶。我们在之前的研究中改造的具有更高催化活性的突变体CYP716A12（D122Q）与其他酶在烟草叶片中共同表达。使用pBYR2HS，齐墩果酸产量比使用传统二元载体时提高了13.1倍，表明了筑波系统的优势。我们还证明了共表达突变体HMGR1催化结构域、额外的将电子转移到异源CYP的NADPH-细胞色素P450还原酶（CPR）以及在农杆菌浸润后应用抗坏血酸预防叶片坏死以提高产物产量的有效性。结果，与之前报道的烟草叶片异源三萜类化合物生产中的产量相比，简单（β-香树脂醇）和氧化（齐墩果酸和山楂酸）三萜类化合物的产物产量均显著提高。
