High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

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.

Phospholipase D and Pathogenic Microorganisms Invasion / 微生物学通报

Phospholipase D(PLD) is ubiquitous in bacteria,fungi,and mammal.In pathogenic microorganisms,PLD can be pathogenic determinant and play a role in spore generation.In mammalian cells,PLD functions in several signal transduction pathways,such as membrane transportation,mitosis regulation,and actin cytoskeleton regulation.In the process of pathogens invasion host cells,both of the pathogen and host cells’ PLD will be activated and a series of cascade reaction will be generated.During this process,pathogen’s PLD can regulate the polymerization and reorganization of its own actin filaments and induce the polymerization or reorganization of the host cell actin filaments near the foci,thus to promote the phagocytosis of the pathogen by host cell.Investigating the role of PLD activation in the infection will be significance for further understanding the molecular mechanism of pathogen-host cell interaction.