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Patent Application Titled "Vector with Codon-Optimised Genes for an Arabinose Metabolic Pathway for Arabinose Conversion in Yeast for Ethanol...

August 12, 2014

Patent Application Titled "Vector with Codon-Optimised Genes for an Arabinose Metabolic Pathway for Arabinose Conversion in Yeast for Ethanol Production" Published Online

By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors BOLES, Eckhard (Darmstadt, DE); ROTHER, Beate (Einbeck, DE), filed on March 31, 2014, was made available online on July 31, 2014 (see also Johann Wolfgang Goethe-Universitaet Frankfurt am Main).

The assignee for this patent application is Johann Wolfgang Goethe-Universitaet Frankfurt am Main.

Reporters obtained the following quote from the background information supplied by the inventors: "The beer, wine and baking yeast Saccharomyces cerevisiae has already been used for centuries for the production of bread, wine and beer owing to its characteristic of fermenting sugar to ethanol and carbon dioxide. In biotechnology, S. cerevisiae is used particularly in ethanol production for industrial purposes, in addition to the production of heterologous proteins. Ethanol is used in numerous branches of industry as an initial substrate for syntheses. Ethanol is gaining increasing importance as an alternative fuel, due to the increasingly scarce presence of oil, the rising oil prices and continuously increasing need for petrol worldwide.

"In order to produce bioethanol inexpensively and efficiently, the use of lignocellulose-containing biomass, such as for example straw, waste from the timber industry and agriculture and the organic component of everyday household waste, presents itself as an initial substrate. Firstly, said biomass is very convenient and secondly is present in large quantities. The three major components of lignocellulose are lignin, cellulose and hemicellulose. Hemicellulose, which is the second most frequently occurring polymer after cellulose, is a highly branched heteropolymer. It consists of pentoses (L-arabinose, D-xylose), uronic acids (4-O-methyl-D-glucuronic acid, D-galacturonic acid) and hexoses (D-mannose, D-galactose, L-rhamnose, D-glucose) (see FIG. 1). Although, hemicellulose can be hydrolized more easily than cellulose, but it contains the pentoses L-arabinose and D-xylose, which can normally not be converted by the yeast S. cerevisae.

"In order to be able to use pentoses for fermentations, these must firstly enter the cell through the plasma membrane. Although S. cerevisiae is not able to metabolize D-xylose, it can uptake D-xylose into the cell. However, S. cerevisiae does not have a specific transporter. The transport takes place by means of the numerous hexosetransporters. The affinity of the transporters to D-xylose is, however, distinctly lower than to D-glucose (Kotter and Ciriacy, 1993). In yeasts which are able to metabolize D-xylose, such as for example P. stipitis, C. shehatae or P. tannophilus (Du Preez et al., 1986), there are both unspecific low-affinity transporters, which transport D-glucose, and also specific high-affinity proton symporters only for D-xylose (Hahn-Hagerdal et al., 2001).

"In earlier experiments, some yeasts were found, such as for example Candida tropicalis, Pachysolen tannophilus, Pichia stipitis, Candida shehatae, which by nature ferment L-arabinose or can at least assimilate it. However, these yeast lack entirely the capability of fermenting L-arabinose to ethanol, or they only have a very low ethanol yield (Dien et al., 1996).

"Conversion of L-arabinose

"In order for the pentose L-arabinose to be metabolised by S. cerevisiae, it must enter into the cell via transport proteins and be converted to the metabolite D-xylulose-5-phosphate in three enzymatic steps. These three enzymatic steps may be made available to the yeast by heterologously expressed genes. D-xylulose-5-phosphate functions as an intermediate of the pentose phosphate pathway and can be decomposed further to yield ethanol under anaerobic conditions in the cell (see FIG. 2).

"Becker and Boles (2003) describe the engineering and the selection of a laboratory strain of S. cerevisiae which is able to use L-arabinose for growth and for fermenting it to ethanol. This was possible due to the over-expression of a bacterial L-arabinose metabolic pathway, consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous over-expression of yeast galactose permease transporting L-arabinose in the yeast strain. Molecular analysis of the selected strain showed that the predetermining precondition for a use of L-arabinose is a lower activity of L-ribulokinase. However, inter alia, a very slow growth is reported from this yeast strain (see FIG. 2).

"So far, it was only possible to express the native genes of bacterial arabinose metabolic pathways that are essential for metabolising arabinose in S. cerevisiae on single plasmids or to integrate them individually in the yeast genome, respectively (Karhumaa et al, 2006). This means that each yeast transformant with a functional arabinose metabolic pathway contained at least three plasmids or the genes integrated into the rDNA locus (Becker and Boles, 2003; Karhumaa et al, 2006).

"The presence of the genes on different plasmids is associated with a number of disadvantages. On the one hand, plasmids that are present simultaneously represent additional stress for the yeast cells ('Plasmid stress', Review of E. coli by Bailey (1993)). On the other hand, the plasmids used have strong homologies in their sequences, which can lead to loss of information within the plasmids due to homologous recombination (Wiedemann, 2005). However, the main disadvantages associated with the use of plasmids lie in the fact that they remain unstable in the strains without selection pressure and that they are not suitable for industrial use.

"Moreover, it would be ideal for industrial applications if the microorganism used were able to metabolise all of the sugars present in the medium. Since the yeasts currently used industrially are not capable of metabolising the arabinose in the medium, it would be highly advantageous to provide the strains with this additional capability in a stable manner.

"The object of the present invention is therefore to provide means that overcome the disadvantages known from the prior art of introducing genes of a bacterial L-arabinose metabolic pathway into host cells individually, and which in particular may be usable for industrial yeast strains."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The object is solved according to the invention by the provision of nucleic acid molecules comprising three nucleic acid sequences, each of which codes for a polypeptide of an L-arabinose metabolic pathway, in particular a bacterial L-arabinose metabolic pathway.

"A nucleic acid molecule according to the invention is a recombinant nucleic acid molecule. Furthermore, nucleic acid molecules according to the invention comprise dsDNA, ssDNA, PNA, CNA, RNA or mRNA, or combinations thereof."

For more information, see this patent application: BOLES, Eckhard; ROTHER, Beate. Vector with Codon-Optimised Genes for an Arabinose Metabolic Pathway for Arabinose Conversion in Yeast for Ethanol Production. Filed March 31, 2014 and posted July 31, 2014. Patent URL:

Keywords for this news article include: Alcohols, Ethanolamines, Johann Wolfgang Goethe-Universitaet Frankfurt am Main.

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Source: Life Science Weekly

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