A combinatorial stress-alleviation strategy for efficient β-carotene production in Escherichia coli

β-carotene is widely used in food processing and animal feed industries. However, metabolic stress is a primary factor limiting cell growth and β-carotene production in Escherichia coli. To alleviate cellular growth stress, we improved cell membrane tolerance to β-carotene with the cell growth rate increased by 41.22 %, and enhanced β-carotene production (286.57 mg/L). To further enhance the supply of β-carotene precursors, 16 genetic targets were modified for strengthening the metabolic flux of the acetyl-CoA, isopentenyl pyrophosphate/dimethylallyl diphosphate (IPP/DMAPP), farnesyl diphosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) nodes, which led to 619.79 mg/L β-carotene. Thereafter, dynamic regulation of mvaE enabled simultaneous control of acetyl-CoA entry into the MVA pathway and mevalonate synthesis, which raised β-carotene titer and cell growth by 45.75 % and 11.43 %, respectively. Finally, a novel "Transient-state" continuous fermentation was developed, which effectively increased the β-carotene production to 3.93 g/L (54.5 mg/g DCW), with an average productivity of 81.9 mg/L/h. This study presents an effective strategy for improving the biosynthesis of hydrophobic natural compounds, offering a scalable approach for industrial production and wider applications in microbial metabolic engineering.