Study on the Mechanism of Efficient Extracellular Expression of Toxic Streptomyces Phospholipase D in Brevibacillus Choshinensis Under Mg2+ Stress

BackgroundPhospholipase D (PLD) has significant advantages in the food and medicine industries due to its unique transphosphatidylation. However, the high heterologous expression of PLD is limited by its cytotoxicity. The present study sought to express the strong extracellular protein of PLD in the non-pathogenic Brevibacillus choshinensis (B. choshinensis).ResultsThe extracellular PLD was effectively expressed by the strong promoter (P2) under Mg2+ stress, with the highest activity of 1.0×104 U·L-1. The inductively coupled plasma–mass spectrometry (ICP-MS) results elucidated that the fast expression of PLD by P2 promoter without Mg2+ stress induced the ionic homeostasis perturbation caused by the highly enhanced Ca2+ influx, leading to cell injury or death. Under Mg2+ stress, Ca2+ influx was significantly inhibited, and the strengths of P2 promoter and HWP gene expression were weakened. The study results revealed that the mechanism of Mg2+ induced cell growth protection and PLD expression might be related to the lowered strength of PLD expression by P2 promoter repression to meet with the secretion efficiency of B. choshinensis, and the redistribution of intracellular ions accompanied by decreased Ca2+ influx.ConclusionsThe PLD production was highly improved under Mg2+ stress. By ICP-MS and qPCR analysis combined with other results, the mechanism of the efficient extracellular PLD expression under Mg2+ stress was demonstrated. The relatively low-speed PLD expression during cell growth alleviated cell growth inhibition and profoundly improved PLD production. These results provided a potential approach for the large-scale production of extracellular PLD and novel insights into PLD function.

An Off-the-Shelf Approach for the Production of Fc Fusion Proteins by Protein Trans-Splicing towards Generating a Lectibody In Vitro

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a “lectibody”. Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

Obtaining and purification of lysostaphin secreted in the culture liquid by Brevibacillus choshinensis/pNCMO2/lsf12 recombinant strain

Here, «host-vector» expression system of Brevibacillus choshinensis was developed and used for producing a recombinant lysostaphin with high-output. The recombinant plasmid pNCMO2/lsf12 was constructed, and its expression in Brevibacillus choshinensis (strain Brevibacillus choshinensis/pNCMO2/lsf12) provided a synthesis of the 27-kDa protein, which was secreted into the culture medium. Its specific staphylolitic activity being 557 U/mg at optimal pH (7.5-8.0) and temperature (50-55 °C) values was comparable with the natural and recombinant analogs. We hope that developed methods of a deep cultivation of the recombinant Brevibacillus choshinensis/pNCMO2/lsf12 strain for a high-yield production (up to 90 mg/L) and a single-stage purification of lysostaphin (up to 90% homogeneity) become the basis for the production of the enzyme on an industrial scale. Brevibacillus choshinensis, ion-exchange chromatography, lysostaphin The work was financially supported by the Grant No. 050 of Rospotrebnadzor «Monitoring of borreliosis pathogens circulation in regions of the Russian Federation and improvement of diagnostic tools for borreliosis»