Introduction of FLO1 to and characterization of a xylose fermenting Saccharomyces cerevisiae

Examensarbete för masterexamen
Master Thesis
Henriksson, Oskar
The interest for bioethanol as a fuel is increasing rapidly all over the world. This is due to a number of factors including declining oil reserves and a will to use carbon neutral fuels. Ethanol production today is associated with many problems. The dominating feedstocks are sugar and starch-based agriculture crops. These can be used as food and feedstuff for domesticated animals. Agriculture today is very oil dependent and it is therefore both an ethical as well as an economical issue whether it is reasonable to continue bioethanol production in this way. The ideal feedstock appears to be hydrolyzates from lignocellulosic biomass. Lignocellulosic materials are composed of the three polymers cellulose, hemicellulose and lignin which combined are the structural elements in the plant cell wall. Lignocellulose is available in abundance from a variety of sources all over the world and is currently very cheap. The common baker’s yeast Saccharomyces cerevisiae is a good organism for ethanol production and is widely used today. However, it does not perform satisfactory when fermenting lignocellulosic hydrolyzatesbecause 1) The hydrolyzates have a high content of xylose, which S. cerevisiae can not utilize as substrate for fermentation; 2) During the hydrolysis process toxic compounds are produced which inhibit growth and decrease ethanol production. Xylose fermenting S. cerevisiae can be obtained through genetic engineering and higher tolerance to inhibitors can be achieved by high cell densities. In this study, we focused on generating high local cell densities through the expression of the FLO1 gene, a gene coding for a protein called flocculin which enables the yeast to flocculate, meaning that the cells clump together in flocs of several thousand cells. We tried to activate FLO1 in a xylose fermenting strain of S. cerevisiae named CEN.PK XXX, based on the CEN.PK113-7D strain. A number of different approaches were conducted in order to achieve this. Among these, the endogenous promoter of FLO1 was intended to be replaced by a strong constitutive promoter. However it was found that the strain lacks FLO1 making the attempt impossible. A gene disruption cassette was intended to be constructed including the FLO1 gene under the control of the TDH3 promoter. However, the attempt was not successful. When FLO1 was cloned into a shuttle vector and transformed to competent, recombinase negative Escherichia coli cells, a fragment of approximately 2000 bp was lost. This behavior is likely due to the large regions of highly repetitive sequences in the FLO1 gene which causes some recombination mechanism to occur in the host bacteria. The CEN.PK XXX strain was characterized under anaerobic conditions in Erlenmayer flasks in media supplemented with 50 g/l glucose, 50 g/l xylose or a mixture of 25 g/l, each of xylose and glucose, respectively. It could be concluded that CEN.PK XXX utilizes xyloseat rates that are among the highest reported in the litterature, however, the strain accumulates xylitol, which is an intermediate of xylose fermentation and this accumulation is likely due to an imbalance of cofactors in the metabolic pathway
Industriell bioteknik , Energi , Livsvetenskaper , Industrial Biotechnology , Energy , Life Science
Arkitekt (konstruktör)
Geografisk plats
Byggnad (typ)
Teknik / material