Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A(2) of Streptomyces violaceoruber

Abstract

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA(2) in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA(2) was produced to an activity of 0.517 +/- 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA(2) (P-PLA(2)), showed activity of 17.0 +/- 1.3 U/ml in the intracellular fraction and 21.7 +/- 0.7 U/ml in the culture broth. The extracellular PLA(2) activity obtained with the recombinant E. coli system was 3.2- fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA(2). Finally, we observed that the extracellular PLA(2) from the recombinant E. coli P-PLA(2) culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA(2) expression system led to extracellular production of PLA(2) to a productivity of 678 U/l.h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA(2).