Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid
Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid
复制标题
嗜热丝状真菌嗜热毁丝霉的代谢工程产生富马酸
DOI：
10.1186/s13068-018-1319-1
复制
发表时间：
2018-12-03
影响因子：
6.3
通讯作者：
Tian, Chaoguang
中科院分区：
文献类型：
--
作者：
Gu, Shuying;Li, Jingen;Tian, Chaoguang
富马酸广泛应用于食品和制药行业，是公认的用途广泛的工业化工原料。对能源和环境问题的日益关注导致了对利用可再生原料进行生物转化的微生物发酵生产富马酸的关注。丝状真菌是用于生产有机酸（包括富马酸）的主要微生物，迄今为止大多数研究都集中在根霉上。嗜热丝状真菌在工业发酵生产化合物方面具有许多优势。然而，以前没有研究集中在富马酸生产的嗜热真菌。结果利用CRISPR/Cas9系统对嗜热丝霉菌进行代谢工程制备富马酸盐的可行性进行了探索。富马酸酶的筛选表明，产自克鲁西念珠菌的富马酸酶最适合在嗜热分枝杆菌中高效生产富马酸。在嗜热m.c rorusei富马酸酶中引入富马酸酶使富马酸滴度提高了3倍。为了进一步增加富马酸的产生，细胞内的富马酸消化途径被破坏。在删除嗜热分枝杆菌的两个富马酸还原酶和线粒体富马酸酶基因后，得到的菌株富马酸滴度增加了2.33倍。增加富马酸的前体苹果酸的池大小，显著提高富马酸的最终效价。最后，与亲本菌株相比，苹果酸-天冬氨酸穿梭体的破坏使细胞内苹果酸含量提高了2.16倍，细胞外富马酸滴度提高了42%。通过对多个基因的战略性代谢工程，最终菌株在补料间歇发酵过程中可以从葡萄糖中产生高达17g/L的富马酸。结论首次对嗜热丝状真菌M. thermophila产富马酸进行了代谢工程研究。这种纤维素水解真菌平台为未来从木质纤维素来源的碳源中可持续、高效地生产富马酸提供了一种有前途的方法。
BackgroundFumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi.ResultsWe explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate-aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17g/L fumaric acid from glucose in a fed-batch fermentation process.ConclusionsThis is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.