Phospholipase D (PLD, EC 3.1.4.4) catalyzes hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup. In addition to its hydrolytic activity, PLD can also catalyze the transfer of acyl groups to directly synthesize valuable phospholipid derivatives, such as phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylglycerol (PG). These phospholipids have wide applications in the food, cosmetics and pharmaceutical industries [1]. PLD was first reported in 1947 and due to its special catalytic activity, research on PLD has recently increased [2]. PLD has been identified from plants [3], mammals [4], and bacteria [5]. However, these natural sources produce low levels of PLD that cannot meet the industrial demand [6]. Therefore, the production of PLD by microbial fermentation has attracted great attention due to its advantages of high unit activity and low cost.
PLD has been characterized in many microorganisms and is most commonly found in Streptomyces strains, such as Streptomyces PMF [7], S. lividans [8], and Streptomyces sp. YU100 [9]. For microbial production of PLD, Streptomyces strains are most widely used due to the high transphosphatidylation activity of native PLD and natural PLD secretion activity during fermentation. For example, Saovanee et al. isolated Streptomyces sp. SC734 from soil-contaminated palm oil, and the PLD it produced exhibited high activity with a conversion rate of phosphatidylcholine (PC) to PS of up to 94.7% in 100 min [10]. Ogino et al. constructed an overexpression system for secretory production of PLD in S. lividans and the amount of PLD secreted reached a maximum level of 118 mg/L [11]. However, the genetic-transfer systems for Streptomyces remain largely inefficient, which limits efficient production of PLD. Thus, the heterologous expression of Streptomyces PLDs in other model microorganisms, such as yeast or E. coli is highly desired.
Using Pichia pastoris as the host, Liu et al. developed a yeast cell surface display system to express PLD from S. chromofuscus, and the displayed PLD converted 67.5% of PC to PS within 10 h [12]. PLDs from different sources have also been successfully expressed with E. coli used as the host. For example, Carlo et al. expressed the PLD from Streptomyces PMF in E. coli BL21(DE3)pLysS, and 5 mg/L PLD was finally obtained with an enzyme activity of 15 mU/mL [13]. For the high-level and stable production of PLD, several engineering strategies were carried out in E. coli, including optimizing and tightly regulating promoter strength, optimizing codon usage and amino acid supplementation, and maintaining the best cellular state by supplementing nutrition. Finally, a large amount of PLD (81.5 mg/L) was obtained in batch culture [14]. Although there has been considerable progress in heterologous production of PLD, it is still not enough for industrial applications of PLD. Developing an efficient expression system for PLD production is urgently needed.
One of the biggest obstacles to efficient PLD production is that overexpressed PLD is toxic to the host, which may cause plasmid instability, cell lysis and PLD leakage [14]. Secretory production of heterologous proteins has great advantages compared with conventional cytosolic protein production, especially when the heterologous proteins are toxic. In addition, the secretory production of heterologous proteins could simplify the purification processes and reduce cost since cell disruption is not required. Many reports have proposed strategies for improving the secretory production of heterologous proteins, such as optimizing the environmental conditions [15], constructing leaky strains [16] or co-expressing the secretory pathway [17]. The production of PLD in the secretory form seems to be a promising approach to address this issue.
In this study, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately overexpressed in E. coli to compare their transphosphatidylation activity based on synthesis of PS. Recombinant PLDPMF exhibited higher activity. To further improve the synthesis of PLDPMF, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLDPMF gene was expressed together with its native signal peptides (Nat) and PelB. After optimizing induction conditions including induction temperature, induction pH, IPTG concentration, induction time and addition of metal ions, 10.5 U/ml PLD was detected in the fermentation medium. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after bioconversion for 24 h when 50 g/L PC was added.