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Objectives: To investigate the effects of low‐intensity pulsed ultrasound (LIPUS) on the proliferation, differentiation, and tumor necrosis factor‐α (TNF‐α)‐induced lipolysis of 3T3‐L1 cells, and to explore the feasibility of regulating the release of free fatty acids (FFA) to prevent lipotoxicity. Methods: Different intensities (30, 60, 90, and 120 mW/cm2) of LIPUS were applied to 3T3‐L1 preadipocytes for different durations (5, 10, 15, 20, 25, and 30 minutes). Appropriate parameters for subsequent experiments were selected by assessing cell viability. The effect of LIPUS on the proliferation and differentiation of 3T3‐L1 cells was evaluated by microscope observation, flow cytometry, and lipid content determination. After treated with LIPUS and TNF‐α (50 ng/mL), the degree of lipolysis was assessed by measuring the extracellular FFA content. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used to detect the mRNA expression of relevant genes. Results: Different parameters of LIPUS significantly enhance the viability of 3T3‐L1 cells (P <.05), with 20 minutes and 30 mW/cm2 as the most suitable settings. After LIPUS treatment, 3T3‐L1 cell proliferation accelerated, apoptosis rate and G1 phase cell proportion decreased, the content of lipid droplets and TG was increased in differentiated cells, while FFA release decreased (P <.05). The expression of PCNA, PPARγ, C/EBPα, Perilipin A mRNA increased, and the expression of TNF‐α, ATGL, HSL mRNA decreased (P <.05). Conclusions: LIPUS could promote the proliferation and differentiation of 3T3‐L1 cells and inhibit TNF‐α‐induced lipolysis, indicating its potential as a therapy for mitigating lipotoxicity caused by decompensated adipocytes. [ABSTRACT FROM AUTHOR]

Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and insufficient precursor supply. Compartmentalization in cellular organelles can isolate biosynthetic pathways from competing pathways, and thus provide a more compact and conducive environment for biosynthesis. Herein, the farnesyl diphosphate biosynthetic pathway and α-bisabolene synthase were compartmentalized in the yeast Yarrowia lipolytica peroxisome to enable high-level α-bisabolene production. Along with compartmentalization of the α-bisabolene biosynthesis pathway, a systems metabolic engineering approach that comprises mediating product export with an efflux pump, optimizing the gene copy numbers of rate-limiting enzymes, balancing the distribution of the common precursor acetyl-CoA between native lipid biosynthesis and heterologous α-bisabolene production, improving ATP and acetyl-CoA supply, and dynamic regulation of peroxisomes was then employed to further enhance the bisabolene production. Consequently, the optimized engineered strain produced 3028.9 mg L−1 α-bisabolene from waste cooking oil as the sole carbon source through shake flask cultivation. Finally, fed-batch fermentation was performed to accomplish α-bisabolene production at 15.5 g L−1, the highest ever reported for α-bisabolene from an engineered microbe. Taken together, this work lays the foundation for peroxisome engineering of Y. lipolytica, towards the production of α-bisabolene in a highly sustainable manner. [ABSTRACT FROM AUTHOR]