LuÃs E. Trujillo
Universidad de las Fuerzas Armadas , Ecuador
Title: A genetically modified Pichia pastoris-based biocatalyst for short-chain FOS production from sucrose
Biography
Biography: LuÃs E. Trujillo
Abstract
The methylotrophic yeast Pichia pastoris is not a saccharolityc yeast so, it has become in an important host organism for the high level production of recombinant enzymes that use sucrose as substrates. We have developed a yeast based- biocatalyst for massive conversion of sucrose into fructooligosaccharides (FOS), particularly the commercially most attractive 1-kestose (GF2). These compounds are highly demanded as prebiotics in the functional food market so, the gene encoding a native plant sucrose:sucrose 1-fructosyltransferase (1-SST) able to produce vast amounts of FOS from sucrose, was expressed constitutively in Pichia pastoris. Secretion driven by the S. cerevisiae alpha-factor signal peptide resulted in periplasmic retention (30%) and extracellular release (70%) of the recombinant enzyme. The gradual increase of the transgene dosage from one to nine integrated copies proportionally enhanced 1-SST yield to an overall activity of 140 U/mL without causing toxicity to the yeast cells. The acquired ability to utilize sucrose allowed the transgenic yeast to reach high biomass values (above 370 g wet weight / L) at relatively low costs in fed-batch fermentations for 72 h. Intact cells were entrapped in calcium alginate beads and shown to yield high FOS levels (55-60% of carbohydrates in the reaction mixture) in concentrated reactions (initial sucrose 50ºBx) at pH 5.5-6.0 and 30-40ºC. Sucrose hydrolysis was negligible and the 1-kestose content (90% of total FOS) was three-fold higher than the values reported with current fungal systems. In terms of productivity and stability, the immobilized cells (1 kg) produced 2.5 kg FOS per day and retained 90% of its original activity after 20 cycles of continuous reuse at 30ºC. The biocatalyst offers flexibility of operation in packed-bed, batch or continuous-flow stirred tanks reactors. This genetically modified yeast allow the industrial production of FOS in a cost effective manner.