Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A₂ 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 .

Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli

“…2 C). The results of this study and earlier studies (Jung & Park, 2008 ; Lee et al, 2020 ) suggested that more rapid expression of target proteins via supplementation of rare-codon tRNAs may lead to a higher accumulation of target proteins, which in turn accelerates misfolding and/or degradation of the proteins. We noted that the values of specific activities associated with CMC were much lower than those associated with Avicel, which is consistent with a previous study which reported that the main catalytic activity of CelA was attributed to the GH9 module, a major characteristic of which is endoglucanase activity (Yi et al, 2013 ).…”

Simultaneous production of 2′‐fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency

“…We previously observed that engineered E. coli BL21 star (DE3) strains expressing PLA 2 from S. violaceoruber [ 23 ] or a truncational mutant (TM1) of CelA from Caldicellulosiruptor bescii [ 20 ] with the aid of signal peptides could secrete endogenous proteins into the culture medium in a non‐specific manner. This observation suggests that PLA 2 with the N‐terminal PelB signal peptide (P‐PLA 2 ) and TM1 with the N‐terminal DsbA signal peptide (D‐TM1) could serve as co‐expression partners that help FKP and FucT2 to be secreted into the culture medium (Figure 1B).…”

“…[ 26 ] Therefore, instead of searching for a signal peptide suitable for the secretion of FKP and FucT2, we decided to exploit heterologous proteins capable of inducing non‐specific secretion of cytoplasmic proteins into the culture medium. We previously observed the non‐specific secretion of numerous endogenous proteins into the culture medium when P‐PLA 2 [ 23 ] and D‐TM1 [ 20 ] were expressed in E. coli . Therefore, these two heterologous proteins were selected as co‐expression partners to mediate the extracellular secretion of FKP and FucT2.…”

Protein Engineering of a Germacrene A Synthase From Lactuca sativa and Its Application in High Productivity of Germacrene A in Escherichia coli

“…DCW). E. coli BL21 Star (DE3) is characterized by containing a mutated rne131 gene encoding ribonuclease E (RNaseE), which can enhance the stability of messenger RNA, which in turn elevates the expression level of target proteins ( Lee et al, 2020 ). Hence, the E. coli BL21 Star (DE3) carrying the plasmid pMM was used as a chassis and cultivated in SBMSN medium to evaluate the yield of GA.…”