Lipoxygenases (LOXs) are a family of non-heme iron oxidoreductases, which catalyze the addition of oxygen into polyunsaturated fatty acids. They have applications in the food and medical industries. In most studies, the soluble expression of LOXs in microbes requires low temperature (< 20 degrees C), which increases the cost and fermentation time. Achievement of soluble expression in elevated temperatures (> 30 degrees C) would shorten the production phase, leading to cost-efficient industrial applications. In this study, a combinatorial strategy was used to enhance the expression of soluble LOXs, comprising plasmid stability systems plus optimized carbon source used for auto-induction expression. Plasmid stability analysis suggested that both active partition systems and plasmid-dependent systems were essential for plasmid stability. Among them, the parBCA in it resulted in the enzyme activity increasing by a factor of 2 (498 +/- 13 units per gram dry cell weight (U/g-DCW) after 6-h induction). Furthermore, the optimized carbon source, composed of glucose, lactose, and glycerol, could be used as an auto-induction expression medium and effectively improve the total and soluble expression of LOX, which resulted in the soluble expression of LOX increased by 7 times. Finally, the soluble expression of LOX was 11 times higher with a combinatorial strategy that included both optimized plasmid partition and auto-induction medium. Our work provides a broad, generalizable, and combinatorial strategy for the efficient production of heterologous proteins at elevated temperatures in theE. colisystem.