Influence of
flocculation on the ethanol production by recombinant Saccharomyces
cerevisiae
expressing amylolytic genes from Lipomyces kononenkoae.
Ricardo Cordero Otero, Nivetha Ramachandran, Sakkie Pretorius
Institute for Wine Biotechnol., Stellenbosch University, Victoria street,
Stellenbosch, 7602, Republic of South Africa (iwbt25@sun.ac.za)
Starch is one of
the most widely occurring renewable resources. Utilization of starch for
production of ethanol is a promising strategy for alternative fuel. Alpha
amylases and glucoamylases enzymes are produced by a variety of microbes in
nature. Among yeasts, Lipomyces kononenkoae secrete amylolytic
enzymes that hydrolyze alpha-1,4 and alpha-1,6 linkages in starch. But L.
kononenkoae
cannot be used in the industrial fermentations because of its low ethanol
tolerance and poorly characterized genetics. In contrast, the conventional use
of Saccharomyces cerevisiae in fermentations, well known genetics and its
GRAS status makes it an ideal candidate for producing commercially important
products from starch. Recombinant S. cerevisiae strains expressing two
genes LKA1
and LKA2
encoding amylolytic enzymes from L. kononenkoae were developed.
Transcriptional expression and secretion have been directed by PGK1pt sequences
and the use of MFalpha
signal peptide sequence. The foreign genes were expressed singly and in
combination to study the synergism of starch degradation in small-scale
fermentations. We propose that a flocculent phenotype might decrease the
interval for the interaction between the enzyme and the substrate. That could
allow the secreted enzymes to act more efficiently on the substrate degradation
during fermentation. Starch fermentation by amylolytic engineered strains
expressing LKA1
and LKA2
genes with flocculating and non-flocculating phenotype will be discussed.