Boecker Simon, Grätz Stefan, Kerwat Dennis, Adam Lutz, Schirmer David, Richter Lennart, Schütze Tabea, Petras Daniel, Süssmuth Roderich D, Meyer Vera
1Department Biological Chemistry, Institute of Chemistry, Technische Universität Berlin, Straße des 17. Juni 124, 10623 Berlin, Germany.
2Department Applied and Molecular Microbiology, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.
Fungal Biol Biotechnol. 2018 Mar 2;5:4. doi: 10.1186/s40694-018-0048-3. eCollection 2018.
Fungal cyclodepsipeptides (CDPs) are non-ribosomally synthesized peptides produced by a variety of filamentous fungi and are of interest to the pharmaceutical industry due to their anticancer, antimicrobial and anthelmintic bioactivities. However, both chemical synthesis and isolation of CDPs from their natural producers are limited due to high costs and comparatively low yields. These challenges might be overcome by heterologous expression of the respective CDP-synthesizing genes in a suitable fungal host. The well-established industrial fungus was recently genetically reprogrammed to overproduce the cyclodepsipeptide enniatin B in g/L scale, suggesting that it can generally serve as a high production strain for natural products such as CDPs. In this study, we thus aimed to determine whether other CDPs such as beauvericin and bassianolide can be produced with high titres in , and whether the generated expression strains can be used to synthesize new-to-nature CDP derivatives.
The beauvericin and bassianolide synthetases were expressed under control of the tuneable Tet-on promoter, and titres of about 350-600 mg/L for bassianolide and beauvericin were achieved when using optimized feeding conditions, respectively. These are the highest concentrations ever reported for both compounds, whether isolated from natural or heterologous expression systems. We also show that the newly established Tet-on based expression strains can be used to produce new-to-nature beauvericin derivatives by precursor directed biosynthesis, including the compounds 12-hydroxyvalerate-beauvericin and bromo-beauvericin. By feeding deuterated variants of one of the necessary precursors (d-hydroxyisovalerate), we were able to purify deuterated analogues of beauvericin and bassianolide from the respective expression strains. These deuterated compounds could potentially be used as internal standards in stable isotope dilution analyses to evaluate and quantify fungal spoilage of food and feed products.
In this study, we show that the product portfolio of can be expanded from enniatin to other CDPs such as beauvericin and bassianolide, as well as derivatives thereof. This illustrates the capability of to produce a range of different peptide natural products in titres high enough to become industrially relevant.
真菌环缩肽（CDPs）是由多种丝状真菌非核糖体合成的肽，因其抗癌、抗菌和驱虫生物活性而受到制药行业的关注。然而，由于成本高和产量相对较低，CDPs的化学合成和从天然生产者中分离都受到限制。通过在合适的真菌宿主中异源表达各自的CDP合成基因，这些挑战可能会被克服。最近，这种成熟的工业真菌经过基因重编程，能够以克/升的规模过量生产环缩肽恩镰孢菌素B，这表明它通常可以作为CDPs等天然产物的高产菌株。因此，在本研究中，我们旨在确定是否可以在该真菌中高产量生产其他CDPs，如白僵菌素和球孢交酯，以及所产生的表达菌株是否可用于合成新型CDP衍生物。
白僵菌素和球孢交酯合成酶在可调节的Tet-on启动子控制下表达，在使用优化的补料条件时，球孢交酯和白僵菌素的产量分别达到约350 - 600毫克/升。这是这两种化合物无论是从天然还是异源表达系统中分离出来所报道的最高浓度。我们还表明，新建立的基于Tet-on的表达菌株可用于通过前体定向生物合成生产新型白僵菌素衍生物，包括化合物12 - 羟基戊酸 - 白僵菌素和溴代 - 白僵菌素。通过添加一种必需前体（d - 羟基异戊酸）的氘代变体，我们能够从各自的表达菌株中纯化白僵菌素和球孢交酯的氘代类似物。这些氘代化合物有可能用作稳定同位素稀释分析中的内标，以评估和量化食品和饲料产品中的真菌腐败情况。
在本研究中，我们表明该真菌的产品组合可以从恩镰孢菌素扩展到其他CDPs，如白僵菌素和球孢交酯及其衍生物。这说明了该真菌有能力生产一系列不同的肽类天然产物，其产量高到足以具有工业相关性。
