Extracellular glucoamylase activity in the Yarrowia lipolytica engineered strains

Production of the rProtein (glucoamylase) in the new platform strains. Strains were cultivated in the presence or absence of erythritol to induce expression. Glucoamylase activity was measured after 24 and 48 hours of incubation.Values show Volumetric productivity [μM/(mL*h)], and Specific productivity normalized to biomass [μM/(mL*h)/OD600], by different Background strain and number of glucoamylase copies. Methodology: Glucoamylase enzymatic activity was assayed in supernatants using the microDNS (3,5-dinitrosalicylic acid) method.The substrate was 0.2% soluble starch in 0.1 M acetate buffer (pH 5.0). One unit of enzymatic activity corresponds to the release of 1 µmol glucose equivalent per mL per hour at 40 °C. Reference prototrophic strains (JMY9438, JMY9451) were assayed in parallel to establish background levels. Readings were normalized to blanks containing distilled water. Measurements were taken at 540 nm using a Tecan Spark plate reader (Tecan Group Ltd., Switzerland).GA-glucoamylase; 1x, 2x, 3x - one, two or three copies of the gene encoding glucosamylase; +Klf1: additional co-expression of the gene encoding the transcription factor Klf1; ERY +: induction with erythritol conditions, ERY -: no induction, control culture; JMY9438, JMY9451 - recombinant strains developed in the BimLip laboratory.