Engineering Mucor circinelloides for enhanced lipid production through homologous overexpression of phosphofructokinase (PFK1 and PFK2) coupled with in-silico modeling analysis

文献类型： 外文期刊

第一作者： Mohamed, Hassan

作者： Mohamed, Hassan;Wang, Xiuwen;Saeed, Tariq;Eltoukhy, Adel;Khalid, Hina;Ramadan, Asmaa S.;Liu, Qing;Song, Yuanda;Mohamed, Hassan;Eltoukhy, Adel;Naz, Tahira;Li, Shaoqi;Saeed, Tariq;Ramadan, Asmaa S.;Awad, Mohamed F.;Song, Yuanda

作者机构：

关键词： Homologous expression; PFK; Lipid accumulation; Gene expression; Fermentation; M. circinelloides

期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：8.5； 五年影响因子：8.7 ）

ISSN： 0141-8130

年卷期： 2025 年 311 卷

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收录情况： SCI

摘要： Phosphofructokinase (PFK) enzyme is considered a key regulatory enzyme of glycolysis, which phosphorylates fructose 6P (F6P) to fructose-1,6-BP, a vital regulatory stage in the glycolytic process. In response to sugar flux, the glycolysis metabolic network of Mucor circinelloides had a significant role in lipid accumulation. To investigate the cytosolic functions of PFK in lipid accumulation, we overexpressed pfk1 and pfk2 via homologous recombination in the oleaginous WJ11 strain of M. circinelloides. Our findings showed that the overexpression of pfk genes increased the biomass by 14.5 % and 28 %, as well as lipid accumulation by 18 % and 28 % in Mc-pfk1 and Mc-pfk2 overexpressing strains, as compared to the Mc-CS control strain. Moreover, the fatty acids (FAs) analysis demonstrated that the overexpression of target genes slightly changed the FAs profile in modified strains. The mRNA expression levels of these two genes in the overexpressing strains using RT-qPCR analysis revealed a noticeable raised 3.7- and 2.8-fold at 24 h in Mc-pfk1 and Mc-pfk2, respectively, compared with the control. Notably, there was also an upregulation of genes that are involved in the production of acetyl-CoA and NADPH, including acl, acc1, acc2, cme1, cme2, fbpase1, and g6pdh2, which suggests an increased metabolic flux toward lipid biosynthesis. This coordinated upregulation indicates that PFK activity may affect lipid metabolism indirectly through pathways that produce necessary lipid precursors and reduce equivalents, in addition to directly influencing glycolytic flux. The in-silico elucidation and computational paradigm of pfk1 and pfk2 in WJ11 imparted the regulatory effect through their corresponding targets and provided evidence for large-scale mechanistic studies. This is the first report to explore the function of pfk genes in WJ11 during its lipid metabolic processes. Therefore, it provides a theoretical basis for further genetic engineering to promote sustainable lipid production for biological industries.

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