Pichia pastoris engineering for the production of a modified phospholipase C

CLAUDIA ELENA; PABLO RAVASI; SEBASTIAN CERMINATI; SALVADOR PEIRU; MARIA CASTELLI; HUGO MENZELLA

PROCESS BIOCHEMISTRY - (Print)

ELSEVIER SCI LTD

Lugar: Amsterdam; Año: 2016 vol. 51 p. 1935 - 1944

1359-5113

Crude vegetable oils are refined to remove impurities that adversely impact in their stability, color andflavor. In recent years, enzymatic degumming methods using phospholipase C (PLC) enzymes provide anenvironmentally 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 modifiedPLC enzyme derived from Bacillus cereus. Production of secreted PLC-Y driven by the methanol inducibleAOX1 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 supportingprotein folding, processing and secretion processes. In addition,tunable promoters directing helper factorexpression were tested.In batch cultures, a strain harboring seven integrated copies of the PLC-Y expression cassette and coexpressingthe HAC1 transcription factor under an attenuated AOX1 promoter showed a 6.2 fold increasein the production titers compared to the strain harboring a single gene copy. A fed-batch fermentationprocess developed using this engineered strain produced 4.5 g/l of this enzyme. The results presented inthis work show the viability of using PLC-Y for oil degumming and provide a manufacturing process forits cost effective production.