磷脂酶D(phospholipase D,PLD;EC 3.1.4.4)可催化卵磷脂转化为功能性磷脂,在食品和医药领域具有重要应用价值。该研究首次在茂原链霉菌(Streptomyces mobaraensis)中建立PLD高效异源表达系统,并解析其酶学特性及催化性能。基于CRISPR-Cas9基因编辑系统,将来源于Streptomyces racemochromogenes 10-3的PLD基因整合至S.mobaraensis DSM40587的谷氨酰胺转氨酶(transglutaminase,TGase)基因位点,成功构建重组菌株SM-PLD1。摇瓶发酵36 h后胞外PLD酶活力达131.20 U/mL。通过启动子工程策略,采用甘油醛-3-磷酸脱氢酶强启动子Pgapdh替换SM-PLD1中的TGase启动子,得到的重组菌株SM-PLD2的酶活力较SM-PLD1提高48%至194.29 U/mL。在此基础上,在壳聚糖酶基因座插入第二拷贝PLD基因表达盒,得到重组SM-PLD2d酶活力提升至244.66 U/mL,较初始菌株SM-PLD1提高了86%。纯化后的重组PLD比酶活力达726.31 U/mg,最适反应条件为50 ℃和pH 7.5,且在30~50 ℃下保温2 h后仍保留50%以上活性,表现出优异的稳定性。此外,以2-甲基四氢呋喃为反应介质,PLD成功催化磷脂酰丝氨酸合成。研究结果为PLD的规模化制备及功能磷脂绿色合成提供了理论依据和技术支撑。
Phospholipase D (PLD;EC 3.1.4.4), a key biocatalyst that mediates the hydrolysis of lecithin into functional phospholipids, has significant industrial applications in the food and pharmaceutical sectors.In this study, we successfully developed a novel and highly efficient heterologous expression system for PLD in Streptomyces mobaraensis for the first time.The PLD gene from S.racemochromogenes 10-3 was integrated into the transglutaminase (TGase) gene locus of S.mobaraensis DSM40587 using CRISPR-Cas9 genome editing, generating recombinant strain SM-PLD1.Fermentation experiments demonstrated that after 36 h of shake-flask fermentation, the extracellular PLD activity of SM-PLD1 reached 131.20 U/mL.To further enhance enzyme production, promoter engineering was employed.The strong glyceraldehyde-3-phosphate dehydrogenase promoter (Pgapdh) replaced the native TGase promoter in SM-PLD1, leading to the creation of SM-PLD2, which exhibited a remarkable 48% increase in enzyme activity, reaching 194.29 U/mL.Subsequently, a second PLD gene expression cassette was introduced into the chitosanase gene locus of SM-PLD2, generating the recombinant strain SM-PLD2d.This strain demonstrated an impressive enzyme activity of 244.66 U/mL, representing an 86% increase compared to the initial strain SM-PLD1.The purified recombinant PLD showed a specific activity of 726.31 U/mg, with optimal reaction conditions at 50 ℃ and pH 7.5.Moreover, the enzyme exhibited excellent stability, retaining over 50% of its activity after incubation at 30-50 ℃ for 2 hours.Additionally, the recombinant PLD successfully catalyzed the synthesis of phosphatidylserine using 2-methyltetrahydrofuran as the reaction medium.These findings not only provide valuable insights into the enzymatic properties and catalytic performance of PLD but also offer a robust theoretical foundation and technical framework for the large-scale production of PLD and the green synthesis of functional phospholipids, paving the way for future industrial applications and further research in this promising field.
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