Optimization of Pichia pastoris fermentation process for PLC production

MARCHISIO, FIORELA; VAL, DIEGO; CERMINATI, SEBASTIAN; AGUIRRE, ANDRES; PEIRÚ, SALVADOR; MENZELLA HUGO; CASTELLI, MARÍA E.

Congreso; Pichia 2021; 2021

BioGrammatics

Crude vegetable oils are a complex mixture of triacylglycerols, phospholipid (PLs) or gums, sterols, tocopherols, free fatty acids, metal traces and other minor compounds. Vegetable oils must be refined before being suitable for consumption or for applications such as biodiesel. Oil degumming (or phospholipid removal) is the first step in the industrial process of oil refining and it is associated with major losses. Over the last decades, physical and chemical methods for PLs removal have been replaced for enzymatic degumming, with several advantages over the formers. This procedure takes advantage of the conversion catalyzed by phospholipases to reduce the gums volume and increase the overall oil yield with significant economic benefit for industry. Our group has developed an enzymatic mixture of two phospholipases C (PC-PLC, PI-PLC) and a lisoacyltransferase GCAT (L-GCAT) that efficiently hydrolyzes phospholipids and can be employed in the conditions presently used in industry for crude oil treatment. Each of these enzymes is obtained by heterologous production in fed-batch fermentation processes using different microbial hosts, Escherichia coli for PI-PLC and L-GCAT and Pichia pastoris for PC-PLC. Here we describe the optimization of the Pichia pastoris fermentation process to obtain PC-PLC. The optimized process included the design of the culture medium, an optimization of the adaptation phase length and the feeding strategy. We confirmed that a continuous strategy using a protease deficient strain is the best option, with remarkably high productivity. This optimized fermentation strategy together with an efficient downstream processing provides a manufacturing alternative for the cost effective production of the enzyme.