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KMID : 0545120180280122046
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 12 p.2046 ~ p.2056
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Combinatorial Fine-tuning of Phospholipase DExpression by Bacillus subtilis WB600for the Production of Phosphatidylserine
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Huang Tingting
Lv Xueqin
Li Jianghua
Shin Hyun-Dong
Du Guocheng
Liu Long
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Abstract
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Phospholipase Dhasgreat commercial value due to its transphosphatidylation products can use in the foodand medicine industries.In order to construct a strain that can be used for the production of PLD, we employed a series of combinatorial strategies to increase PLD expression in Bacillus subtilis WB600. These strategies included screening of signal peptides, selection of different plasmids, and optimization of the sequences of the ribosome-binding site (RBS) and the spacer region.We found that using the signal peptide amyEresults in the highest extracellular PLD activity (11.3 U/ml) andin a PLD expression level 5.27-fold higherthan when the endogenous signal peptide is used. Furthermore, the strain harboringthe recombinant expression plasmid pMA0911-PLD-amyE-his produced PLD with activity enhanced by69.03% (19.1 U/ml).We then used the online tool \RBS Calculator v2.0 to optimize the sequences of the RBS and the spacer. Using the optimized sequences resulted in an increase in the enzyme activityby about 26.7% (24.2 U/ml). In addition,we found through a transfer experiment that the retention rate of the recombinant plasmid after 5 generations was still 100%.The final product,phosphatidylserine (PS), was successfully detected,withtransphosphatidylation selectivity at 74.6%.This is similar to the values for the original producer.
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KEYWORD
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Phospholipase D, phosphatidylserine, ribosome-binding site calculator, Bacillus subtilis, secretory expression
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