Background

Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD.

Results

Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20°C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105.81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression.

Conclusions

After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

How to cite: Wu R, Cao J, Liu F, et al. High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization. Electron J Biotechnol 2021;50.https://doi.org/10.1016/j.ejbt.2020.12.002

中文翻译：

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组合优化优化蓝藻链霉菌磷脂酶D在大肠杆菌中的高水平可溶性表达

背景

磷脂酶D（PLD）用作生产磷脂酰丝氨酸（PS）的生物催化剂。通常，PLD在大肠杆菌中以不溶形式表达。在大肠杆菌中高效表达PLD的可溶性表达对于PLD的工业生产非常重要。

结果

染色体链霉菌PLD编码基因经过密码子优化，克隆后没有信号肽，并在大肠杆菌中表达。最佳重组大肠杆菌用没有His标签的pET-28a构建pET-28a + PLD / BL21（DE3）。经过系统优化后，在250 mL摇瓶中，最高PLD活性达到104.28±2.67 U / mL。通过饲喂乳糖并在放大至5.0 L发酵罐后于20°C诱导，最高PLD活性提高至122.94±1.49 U / mL。在5.0升的发酵罐中，用1.0％（w / v）的廉价糊精代替混合碳源并添加进料培养基仍可实现105.81±2.72 U / mL的PLD活性。来自鱼加工废料的鱼蛋白ept和来自淀粉的糊精都非常便宜，它们被发现有益于可溶性PLD表达。

结论

组合优化后，在大肠杆菌中实现了PLD的高水平可溶性表达。高PLD活性以及在发酵罐中获得的廉价培养基可以完全满足PLD工业生产的要求。

引用方法：吴荣，曹健，刘芳，等。磷脂酶d的从高级别可溶性表达链霉菌chromofuscus在大肠杆菌通过组合优化。电子生物技术杂志2021; 50.https：//doi.org/10.1016/j.ejbt.2020.12.002