Application of metabolic flux analysis to the heterologous production of a polygalacturonase from Aspergillus kawachii.

ORTIZ, G E; ROJAS, N L; GRAMISCI, B R; CHESINI, M; CAVALITTO, S

Curitiba, Brasil.

Congreso; 4th International Congress on Bioprocess in Food Industries; 2010

Pectinases catalyze the hydrolysis of pectin and/or pectic acid. Among pectinases, endopolygalacturonases (PGase; E.C. 3.2.1.15.) are the most important biocatalysts. A. kawachii expresses a PGase, namely PG1, active at acidic pH values. Because A. kawachii produces very little amount of PG1, it was decided to clone pg1 gene and express it in Saccharomyces cerevisiae. The gene pg1, coding for the acidic PGase, was amplified by PCR through specific primers and cloned into the pYES2 expression vector, generating the pYES2:PG1 plasmid. An intron, not removable by splicing during maturation of the primary transcript in yeast, was deleted from pYES2:PG1 through the use of PCR and restriction-enzyme digestion; giving rise to the pYES2:PG1ΔI construct. The gene expressing PG1 was placed under the control of GAL1. GAL1 promoter is induced by galactose but only in absence of glucose because the last one acts as a strong inhibitor of the promoter. The pYES2:PG1ΔI plasmid is functional in S. cerevisiae and that the pre-PG1 protein codified by it is correctly processed and delivered into the extracellular space. Heterologous protein was produced in batch cultures using a 1.5 l New Brunswick BioFlo 310 bioreactor. To control the repression and induction of GAL1 promoter, batch culture was divided in two stages. In the first one, yeasts were cultured with glucose as carbon and energy source (CES). Due to the glucose repression over GAL1, no protein was produced in this stage. During the growth phase, a yield for biomass and ethanol of 0.195 cmol X/cmol S and 0.472 cmol P/cmol S, respectively were obtained. These values and a respiratory quotient above 1 during the whole process are typical of an overflow metabolism (i. e., from the Crabtree effect). Once glucose was depleted, galactose was added as inductor and CES (induction phase). During this phase, 10 mg of protein (2 U/ml) was produced, biomass and ethanol yield was reduced up to 0.049 cmol X/cmosS and 0.344 cmol P/cmol S respectively, and O2 consumption was increased. The carbon and energy balances were found to be closed to unity; indicating that only biomass, ethanol, and CO2 are produced during cultivation under these conditions. Protein concentration (in mass) was negligible and was no considered for balance calculation. In order to elucidate the reason for this decrease in biomass and ethanol yield during induction phase, a metabolic model was developed and a metabolic flux analysis was carried out. From this analysis, a large increase (ten times) in maintenance energy was found, indicating the great level of stress caused by the heterologous protein production (metabolic burden). From these results, it can be concluded that it is necessary a continuous adding of CES in order to improve the production of the recombinant enzyme. This condition can be achieved using a fed-batch culture system.