Impact of heterologous expression of xylose reductases and xylitol dehydrogenases from different yeasts for ethanol production by Saccharomyces cerevisiae

Author(s)

Camila Utsunomia

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2014-03-25

Examining board members

Gustavo Henrique Goldman; Nádia Skorupa Parachin; Jaciane Lutz Ienczak

• Advisor: Gustavo Henrique Goldman; Juliana Velasco de Castro Oliveira

Abstract

The ethanol production from agricultural residues, called Second Generation Ethanol or Lignocellulosic Ethanol, arises as an alternative to increase the ethanol production without necessarily expanding the areas for raw material cultivation. The biomass conversion to ethanol is not economically feasible unless hemicellulose is used in addition to cellulose. However, the yeast Saccharomyces cerevisiae is unable to ferment pentoses, released sugars from hemicellulose hydrolysis, mainly xylose. An alternative to S. cerevisiae produces ethanol from pentose is the genetic engineering with genes from microorganisms which naturally perform this conversion. In order to find new yeasts capable to use xylose, we prospected the gut of sugarcane parasitic insects larvae and alcoholic fermentation tanks, identifying 49 isolates, among them the yeasts Rhodotorula mucilaginosa UC11 e Ogataea polymorpha FT212L. In this work were made the isolation and characterization of xyl1 and xyl2 genes, which encode xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, from these two yeasts, and the cloning and overexpression of these genes in the industrial S. cerevisiae strain Pedra-2 (PE-2). Rhodotorula mucilaginosa UC11 and O. polymorpha FT212L were evaluated over the ability to grow on xylose, xyl1 and xyl2 expression analysis and XR and XDH activity. And for the first time XR and XDH from both yeasts were heterologous expressed in S. cerevisiae PE-2 generating PE-2 Rm, PE-2 Op 1 and PE-2 Op 2 strains, which were tested by the capability to grow on xylose. Additionally, in this work was constructed also for the first time a S. cerevisiae PE-2 strain with genes from Scheffersomyces stipitis, since this is one of the best known yeasts capable of utilizing xylose. For this, PE-2 was engineered with pRH 274 plasmid, containing xyl1 and xyl2 from S. stipitis and xks1 from S. cerevisiae, which encodes xylulokinase (XK), generating the PE-2 274 strain. This recombinant strain was characterized through xylose growing assays, XR and XDH activity and xylose/glucose co-fermentation to ethanol in bioreactor. PE-2 274 showed a high potential to produce ethanol from xylose and glucose, and could be the basis for the generation of industrially competitive yeast aiming the second generation ethanol production (AU)

• FAPESP's process: 11/23867-4 - Molecular characterization of the yeasts Rodothorula mucilaginosa e Cryptococcus podzoliccus and other fungi isolated from the intestinal tract of insects that parasitize sugarcane
• Grantee: Camila Utsunomia
• Support Opportunities: Scholarships in Brazil - Master