Ernst Philipp, Zlati Felicia, Kever Larissa, Wirtz Astrid, Goldbaum Rainer, Pietruszka Jörg, Wynands Benedikt, Frunzke Julia, Wierckx Nick
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428 Jülich, Germany.
Institute of Bioorganic Chemistry, Heinrich-Heine University Düsseldorf in Forschungszentrum Jülich GmbH, Wilhelm-Johnen-Straße, 52428, Jülich, Germany.
Metab Eng Commun. 2024 Nov 16;19:e00252. doi: 10.1016/j.mec.2024.e00252. eCollection 2024 Dec.
There is a strong interest in itaconic acid in the medical and pharmaceutical sectors, both as an anti-bacterial compound and as an immunoregulator in mammalian macrophages. Fungal hosts also produce itaconic acid, and in addition they can produce two derivatives 2-hydroxyparaconic and itatartaric acid. Not much is known about these two derivatives, while their structural analogy to itaconate could open up several applications. In this study, we report the production of these two itaconate-derived compounds. By overexpressing the itaconate P450 monooxygenase Cyp3 in a previously engineered itaconate-overproducing strain, itaconate was converted to its lactone 2-hydroxyparaconate. The second product itatartarate is most likely the result of the subsequent lactone hydrolysis. A major challenge in the production of 2-hydroxyparaconate and itatartarate is their co-production with itaconate, leading to difficulties in their purification. Achieving high derivatives specificity was therefore the paramount objective. Different strategies were evaluated including process parameters such as substrate and pH, as well as strain engineering focusing on Cyp3 expression and product export. 2-hydroxyparaconate and itatartarate were successfully produced from glucose and glycerol, with the latter resulting in a higher derivatives specificity due to an overall slower metabolism on this non-preferred carbon source. The derivatives specificity could be further increased by metabolic engineering approaches including the exchange of the native itaconate transporter Itp1 with the itaconate transporter MfsA. Both 2-hydroxyparaconate and itatartarate were recovered from fermentation supernatants following a pre-existing protocol. 2-hydroxyparaconate was recovered first through a process of evaporation, lactonization, and extraction with ethyl acetate. Subsequently, itatartarate could be obtained in the form of its sodium salt by saponification of the purified 2-hydroxyparaconate. Finally, several analytical methods were used to characterize the resulting products and their structures were confirmed by nuclear magnetic resonance spectroscopy. This work provides a promising foundation for obtaining 2-hydroxyparaconate and itatartarate in high purity and quantity. This will allow to unravel the full spectrum of potential applications of these novel compounds.
衣康酸在医学和制药领域备受关注，它既作为一种抗菌化合物，又作为哺乳动物巨噬细胞中的免疫调节剂。真菌宿主也能产生衣康酸，此外，它们还能产生两种衍生物，即2-羟基异衣康酸和衣康酒石酸。人们对这两种衍生物了解不多，不过它们与衣康酸盐的结构相似性可能会带来一些应用。在本研究中，我们报告了这两种衣康酸盐衍生化合物的生产情况。通过在先前构建的高产衣康酸菌株中过表达衣康酸P450单加氧酶Cyp3，衣康酸被转化为其内酯2-羟基异衣康酸。第二种产物衣康酒石酸盐很可能是随后内酯水解的结果。生产2-羟基异衣康酸和衣康酒石酸盐的一个主要挑战是它们与衣康酸共同产生，这给它们的纯化带来了困难。因此，实现高衍生物特异性是首要目标。我们评估了不同的策略，包括底物和pH等工艺参数，以及侧重于Cyp3表达和产物输出的菌株工程。2-羟基异衣康酸和衣康酒石酸盐成功地从葡萄糖和甘油中生产出来，由于在这种非首选碳源上的整体代谢较慢，后者产生了更高的衍生物特异性。通过代谢工程方法，包括用衣康酸转运体MfsA替换天然衣康酸转运体Itp1，可以进一步提高衍生物特异性。按照现有方案从发酵上清液中回收了2-羟基异衣康酸和衣康酒石酸盐。首先通过蒸发、内酯化和用乙酸乙酯萃取的过程回收2-羟基异衣康酸。随后，通过纯化的2-羟基异衣康酸的皂化反应可以得到衣康酒石酸钠盐形式的衣康酒石酸盐。最后，使用了几种分析方法来表征所得产物，其结构通过核磁共振光谱得到证实。这项工作为高纯度、大量获得2-羟基异衣康酸和衣康酒石酸盐提供了有前景的基础。这将有助于揭示这些新型化合物潜在应用的全貌。
