ABSTRACT
Nervonic acid, a natural fatty acid compound and also a core component of nerve fibers and nerve cells, has been widely used to prevent and treat related diseases of the brain nervous system. At present, fatty acids and their derivatives are mainly obtained by natural extraction or chemical synthesis which are limited by natural resources and production costs. In this study, the de novo synthetic pathway of nervonic acid was constructed in Yarrowia lipolytica by means of synthetic biology, and the yield of nervonic acid was further improved by metabolic engineering and fermentation optimization. Specially, heterologous elongases and desaturases derived from different organism were successfully expressed and evaluated for their potential for the production of nervonic acid in Y. lipolytica. Meanwhile, we overexpressed the genes involved in the lipid metabolism to increase the nervonic acid titer to 111.6 mg/L. In addition, the potential of adding oil as auxiliary carbon sources for nervonic acid production by the engineered Y. lipolytica was analyzed. The results indicated that supplementation with colleseed oil as an auxiliary carbon source can be beneficial for the nervonic acid productivity, which led to the highest concentration of 185.0 mg/L in this work. To summarize, this study describes that the Y. lipolytica can be used as a promising platform for the production of nervonic acid and other very long-chain fatty acids.
ABSTRACT
Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.
ABSTRACT
Background: Asthma is a complex respiratory disease characterized by airway inflammation and remodeling. MicroRNAs (miRNAs) mediate various cellular processes including macrophage polarization and play an important role in the pathogenesis of asthma. In present study, we aimed to screen miRNA profiling involved in macrophage polarization and investigate its possible functions and mechanisms. Methods: An OVA-sensitized mouse model was established and 2-chloroadenosine (2-CA) was used to interfere with macrophages. The airway inflammation and remodeling were assessed. The identification and function of M2 alveolar macrophages were assessed by flow cytometry, RT-qPCR, arginase activity and co-culture experiment. Microarray screening was used to select miRNAs which were related to macrophage polarization and RNA interference (RNAi) technique was performed to confirm the function of the selected miRNA and its target gene. Results: Alveolar macrophages of asthmatic mice showed significant M2 polarization. 2-CA alleviated airway inflammation and remodeling as well as M2 polarization. In vitro, IL-4-induced M2 macrophages promoted the proliferation of α-SMA-positive cells. And miRNA profiling showed a remarkable increased expression of miR-378a-3p in IL-4 induced M2 macrophages. Dual luciferase reporter assay confirmed growth factor receptor binding protein 2 (GRB2) was a target gene of miR-378a-3p. A miR-378a-3p inhibitor and knockdown of GRB2 repolarized alveolar macrophages from M1 to M2 phenotype. Conclusion: Our findings suggest that miR-378a-3p/GRB2 pathway regulates the polarization of alveolar macrophages which acts as a potential therapeutic target for airway inflammation and remodeling in asthma.