Crude vegetable oils are refined to remove impurities that adversely impact in their stability, color and flavor. In recent years, enzymatic degumming methods using phospholipase C (PLC) enzymes provide an environmentally friendly process for phospholipids removal with improved oil recovery yields.
In this study, Pichia pastoris was used as the expression system for the production of PLC-Y, a modified PLC enzyme derived from Bacillus cereus. Production of secreted PLC-Y driven by the methanol inducible AOX1 promoter was optimized by genetic strain engineering which included gene codon optimization, generation of multi-copy chromosomal integrations and the co-expression of helper factors supporting protein f... More
Crude vegetable oils are refined to remove impurities that adversely impact in their stability, color and flavor. In recent years, enzymatic degumming methods using phospholipase C (PLC) enzymes provide an environmentally friendly process for phospholipids removal with improved oil recovery yields.
In this study, Pichia pastoris was used as the expression system for the production of PLC-Y, a modified PLC enzyme derived from Bacillus cereus. Production of secreted PLC-Y driven by the methanol inducible AOX1 promoter was optimized by genetic strain engineering which included gene codon optimization, generation of multi-copy chromosomal integrations and the co-expression of helper factors supporting protein folding, processing and secretion processes. In addition, tunable promoters directing helper factor expression were tested.
In batch cultures, a strain harboring seven integrated copies of the PLC-Y expression cassette and co-expressing the HAC1 transcription factor under an attenuated AOX1 promoter showed a 6.2 fold increase in the production titers compared to the strain harboring a single gene copy. A fed-batch fermentation process developed using this engineered strain produced 4.5 g/l of this enzyme. The results presented in this work show the viability of using PLC-Y for oil degumming and provide a manufacturing process for its cost effective production.
