Abstract

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 °C), which increases the cost and fermentation time. Achievement of soluble expression in elevated temperatures (> 30 °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 the E. coli system.

Key points

• Soluble expression of lipoxygenase at 30 °C or higher temperatures is industrially beneficial.

• Strategies comprise plasmid partition and optimized auto-induction medium with glucose, lactose, and glycerol as carbon source.

• Combinatorial strategy further improved LOX soluble expression at 30 °C and 37 °C.

中文翻译：

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高温下在大肠杆菌中有效表达脂氧合酶的组合策略

摘要

脂氧合酶（LOXs）是一类非血红素铁氧化还原酶，可催化向多不饱和脂肪酸中添加氧。它们可用于食品和医疗行业。在大多数研究中，LOX在微生物中的可溶性表达需要低温（<20°C），这会增加成本和发酵时间。在高温（> 30°C）下实现可溶性表达将缩短生产阶段，从而实现具有成本效益的工业应用。在这项研究中，组合策略用于增强可溶性LOXs的表达，包括质粒稳定性系统以及用于自动诱导表达的优化碳源。质粒稳定性分析表明，活性分配系统和依赖质粒的系统对于质粒稳定性都是必不可少的。其中，parBCA中的酶活性增加了2倍（诱导6小时后每克干细胞重量（U / g-DCW）为498±13单位）。此外，由葡萄糖，乳糖和甘油组成的优化碳源可用作自诱导表达培养基，有效提高了LOX的总表达和可溶性表达，使LOX的可溶性表达提高了7倍。最终，采用包括优化的质粒分配和自动诱导培养基的组合策略，LOX的可溶性表达提高了11倍。我们的工作为在高温下高效生产异源蛋白质提供了广泛，可概括和组合的策略。可用作LOX的自动诱导表达培养基，有效提高了LOX的总表达和可溶性表达，使LOX的可溶性表达提高了7倍。最终，采用包括优化的质粒分配和自动诱导培养基的组合策略，LOX的可溶性表达提高了11倍。我们的工作为在高温下高效生产异源蛋白质提供了广泛，可概括和组合的策略。可用作LOX的自动诱导表达培养基，有效提高了LOX的总表达和可溶性表达，使LOX的可溶性表达提高了7倍。最终，采用包括优化的质粒分配和自动诱导培养基的组合策略，LOX的可溶性表达提高了11倍。我们的工作为在高温下高效生产异源蛋白质提供了广泛，可概括和组合的策略。大肠杆菌系统。

关键点

•30°C或更高温度下脂氧合酶的可溶性表达在工业上是有益的。

•策略包括质粒分配和以葡萄糖，乳糖和甘油为碳源的优化自动诱导培养基。

•组合策略可进一步改善30°C和37°C的LOX可溶性表达。