High-Level Production of Bacteriotoxic Phospholipase A1 in Bacterial Host Pseudomonas fluorescens Via ABC Transporter-Mediated Secretion and Inducible Expression.

: Bacterial phospholipase A1 (PLA1) is used in various industrial fields because it can catalyze the hydrolysis, esterification, and transesterification of phospholipids to their functional derivatives. It also has a role in the degumming process of crude plant oils. However, bacterial expression of the foreign PLA1-encoding gene was generally hampered because intracellularly expressed PLA1 is inherently toxic and damages the phospholipid membrane. In this study, we report that secretion-based production of recombinant PlaA, a bacterial PLA1 gene, or co-expression of PlaS, an accessory gene, minimizes this harmful effect. We were able to achieve high-level PlaA production via secretion-based protein production. Here, TliD/TliE/TliF, an ABC transporter complex of Pseudomonas fluorescens SIK-W1, was used to secrete recombinant proteins to the extracellular medium. In order to control the protein expression with induction, a new strain of P. fluorescens, which had the lac operon repressor gene lacI, was constructed and named ZYAI strain. The bacteriotoxic PlaA protein was successfully produced in a bacterial host, with help from ABC transporter-mediated secretion, induction-controlled protein expression, and fermentation. The final protein product is capable of degumming oil efficiently, signifying its application potential.

Cell-free synthesis of functional phospholipase A1 from Serratia sp.

BACKGROUND: Phospholipase A1 is an enzyme that hydrolyzes phospholipids at the sn-1 position. It has potential applications across diverse fields including food, pharmaceutical, and biofuel industries. Although there has been increasing interest in the use of phospholipase A1 for degumming of plant oils during biodiesel production, production of recombinant phospholipase A1 has been hampered by low efficiency of gene expression and its toxicity to the host cell. RESULTS: While expression of phospholipase A1 in Escherichia coli resulted in extremely low productivity associated with inhibition of transformed cell growth, drastically higher production of functional phospholipase A1 was achieved in a cell-free protein synthesis system where enzyme expression is decoupled from cell physiology. Compared with expression in E. coli, cell-free synthesis resulted in an over 1000-fold higher titer of functional phospholipase A1. Cell-free produced phospholipase A1 was also used for successfully degumming crude plant oil. CONCLUSIONS: We demonstrate successful production of Serratia sp. phospholipase A1 in a cell-free protein synthesis system. Including the phospholipase A1 investigated in this study, many industrial enzymes can interfere with the regular physiology of cells, making cellular production of them problematic. With the experimental results presented herewith, we believe that cell-free protein synthesis will provide a viable option for rapid production of important industrial biocatalysts.