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Lipid biosynthesis is a significant metabolic response to nitrogen starvation in oleaginous fungi. The oleaginous fungus Mucor circinelloides copes with nitrogen stress by degrading AMP through AMP deaminase (AMPD). However, the mechanism of AMPD in regulating lipogenesis remains largely unclear. To elucidate the mechanism of AMPD in lipid synthesis in this M. circinelloides, we identified two genes (ampd1 and ampd2) encoding AMPD and constructed an ampd double knockout mutant. The engineered M. circinelloides strain elevated cell growth and lipid accumulation, as well as the content of oleic acid (OA) and gamma-linolenic acid (GLA). In addition to the expected increase in transcription levels of genes associated with lipid and TAG synthesis, we observed suppression of lipid degradation and reduced amino acid biosynthesis. This suggested that the deletion of AMPD genes induces the redirection of carbon towards lipid synthesis pathways. Moreover, the pathways related to nitrogen metabolism, including nitrogen assimilation and purine metabolism (especially energy level), were also affected in order to maintain homeostasis. Further analysis discovered that the transcription factors (TFs) related to lipid accumulation were also regulated. This study provides new insights into lipid biosynthesis in M. circinelloides, indicating that the trigger for lipid accumulation is not entirely AMPD-dependent and suggest that there may be additional mechanisms involved in the initiation of lipogenesis., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Mucor circinelloides WJ11, an oleaginous filamentous fungus, produces 36% lipid of its cell dry weight when cultured in a high C/N ratio medium, however, the yield of γ-linolenic acid (GLA) is insufficient to make it competitive with other plant sources. To increase the GLA content in M. circinelloides WJ11, this fungus was engineered by overexpression of its key genes such as Δ6-, Δ12-, and Δ9-desaturases involved in GLA production. Firstly, we tried to overexpress two Δ6-desaturase isozymes to determine which one played important role in GLA synthesis. Secondly, Δ6-and Δ12-desaturase were co-overexpressed to check whether linoleic acid (LA), the precursor for GLA synthesis, is a limiting factor or not. Moreover, we tried to explore the effects of simultaneous overexpression of Δ6-, Δ12-, and Δ9-desaturases on GLA production. Our results showed that overexpression (1 gene) of DES61 promoted higher GLA content (21% of total fatty acids) while co-overexpressing (2 genes) DES61 and DES12 and simultaneous overexpressing (3 genes) DES61 , DES12 , and DES91 increased the GLA production of engineered strains by 1.5 folds and 1.9 folds compared to the control strain, respectively. This study provided more insights into GLA biosynthesis in oleaginous fungi and laid a foundation for further increase in GLA production into fungus such as M. circinelloides ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wang, Yang, Mohamed, Shah, Li, Pang, Wu, Xue, Shi, Sadaqat and Song.)

Linolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides . This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications.