Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1999-04-20
2002-06-25
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S320100
Reexamination Certificate
active
06410302
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to recombinant integration vectors. More specifically, this invention provides recombinant integration vectors containing sequences of a gene encoding an aldose reductase (AR), but not the entire AR gene. The recombinant vector can be used to specifically delete or disrupt the AR-encoding gene of a host cell. The recombinant vector also permits any heterologous sequence to be integrated into the host genomic AR sequence. Integration into the AR sequence of, for example a yeast strain, renders the recombinant strain less efficient at producing, or even unable to produce, xylitol from xylose. The recombinant vector can further be used to insert genes coding for xylose utilizing enzymes, which provides a recombinant strain which not only can utilize xylose but is simultaneously prevented from xylitol formation through the action of the AR.
2. Description of the Related Art
Lignocellulose is the main component of forest product residues and agricultural waste. Lignocellulosic raw materials are mainly composed of cellulose, hemicellulose, and lignin. The cellulose fraction is made up of glucose polymers, whereas the hemicellulose fraction is made up of a mixture of glucose, galactose, mannose, xylose, and arabinose polymers. The lignin fraction is a polymer of phenolic compounds.
The cellulose and hemicellulose fractions can be hydrolyzed to monomeric sugars, which can be fermented to ethanol. Ethanol can serve as an environmentally friendly liquid fuel for transportation, since carbon dioxide released in the fermentation and combustion processes will be taken up by growing plants in forests and fields.
The price for lignocellulose-derived ethanol has been estimated by von Sivers et al. (“Cost analysis of ethanol production from willow using recombinant
Escherichia coli”, Biotechnol. Prog.
10:555-560, 1994). The calculations are based on the fermentation of all hexose sugars (glucose, galactose, and mannose) to ethanol. It was estimated that the fermentation of pentose sugars (xylose and arabinose) to ethanol will reduce the price of ethanol by approximately 25%. Xylose is found in hardwood hemicellulose, whereas arabinose is a component in hemicellulose in certain agricultural crops, such as corn. In order to make the price more competitive, the price must be reduced.
The release of monomeric sugars from lignocellulosic raw materials also releases by-products, such as weak acids, furans, and phenolic compounds, which are inhibitory to the fermentation process. Numerous studies have shown that the commonly used Baker's yeast,
Saccharomyces cerevisiae
, is the only ethanol producing microorganism that is capable of efficiently fermenting non-detoxified lignocellulose hydrolysates (Olsson and Hahn-Hägerdal, “Fermentation of lignocellulosic hydrolysates for ethanol production”,
Enzyme Microbial Technol.
18:312-331, 1996). Particularly efficient fermenting strains of
S. cerevisiae
have been isolated from the fermentation plant at a pulp and paper mill (Linden et al., “Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of
Saccharomyces cerevisiae
from a spent sulfite liquor fermentation plant”,
Appl. Environ.Microbiol.
58:1661-1669, 1992).
S. cerevisiae
ferments the hexose sugars glucose, galactose and mannose, but is unable to ferment the pentose sugars xylose and arabinose due to the lack of one or more enzymatic steps.
S. cerevisiae
can ferment xylulose, an isomerization product of xylose, to ethanol (Wang et al., “Fermentation of a pentose by yeasts”,
Biochem. Biophys. Res. Commun.
94:248-254,1980; Chiang et al., “D-Xylulose fermentation to ethanol by
Saccharomyces cerevisiae”, Appl. Environ. Microbiol.
42:284-289,1981; Senac and Hahn-Hägerdal, “Intermediary metabolite concentrations in xylulose- and glucose-fermenting
Saccharomyces cerevisiae cells”, Appl. Environ. Microbiol.
56:120-126, 1990).
In eukaryotic cells, the initial metabolism of xylose is catalyzed by a xylose reductase (XR), which reduces xylose to xylitol, and a xylitol dehydrogenase (XDH), which oxidizes xylitol to xylulose. Xylulose is phosphorylated to xylulose 5-phosphate by a xylulose kinase (XK) and further metabolized through the pentose phosphate pathway and glycolysis to ethanol.
S. cerevisiae
has been genetically engineered to metabolize and ferment xylose. The genes for XR and XDH from the xylose fermenting yeast
Pichia stipitis
have been expressed in
S. cerevisiae
(European Patent to C. Hollenberg, 1991; Hallborn et al., “Recombinant yeasts containing the DNA sequences coding for xylose reductase and xylitol dehydrogenase enzymes”, WO91/15588; Kötter and Ciriacy, “Xylose fermentation by
Saccharomyces cerevisiae”, Appl. Microbiol. Biotechnol.
38:776-783, 1993). The transformants metabolize xylose but do not ferment the pentose sugar to ;ethanol.
When the gene for the enzyme transaldolase (TAL) is overexpressed in xylose- metabolizing transformants, the new recombinant strains grow better on xylose but still do not produce any ethanol from xylose (Walfridsson et al., “Xylose-metabolizing
Saccharomyces cerevisiae
strains overexpressing the TKL1 and TAL1genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase”,
Appl. Environ. Microbiol.
61:4184-4190, 1995). In these strains, the major metabolic by-product, in addition to cell mass, is xylitol formed from xylose through the action of the enzyme XR. When the expression of XDH is ten times higher than the expression of XR, xylitol formation is reduced to zero (Walfridsson et al., “Expression of different levels of enzymes from
Pichia stipitis
XYL1 and XYL2 genes in s and its effect on product formation during xylose utilization”,
Appl. Microbiol. Biotechnol.
48:218-224, 1997). However, xylose is still not fermented to ethanol.
The gene for xylulose kinase (XK) from
S. cerevisiae
has been cloned and overexpressed in XR-XDH-expressing transformants of
S. cerevisiae
(Deng and Ho, “Xylulokinase activity in various yeasts including
Saccharomyces cerevisiae
containing the cloned xylulokinase gene”,
Appl. Biochem. Biotechnol.
24/25:193-199, 1990; Ho and Tsao, “Recombinant yeasts for effective fermentation of glucose and xylose”, WO95/13362, 1995; Moniruzzaman et al., “Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces strain”,
World J. Microbiol. Biotechnol.
13:341-346,1997). These strains have been shown to produce net quantities of ethanol in fermentations of mixtures of xylose and glucose. Using the well established ribosomal integration protocol, the genes have been chromosomally integrated to generate strains that can be used in complex media without selection pressure (Ho and Chen, “Stable recombinant yeasts for fermenting xylose to ethanol”, WO97/42307; Toon et al., “Enhanced cofermentation of glucose and xylose by recombinant Saccharomyces yeast strains in batch and continuous operating modes”,
Appl. Biochem. Biotechnol.
63/65:243-255, 1997).
In prokaryotic cells, xylose is isomerized to xylulose by a xylose isomerase (Xl). Xylulose is further metabolized in the same manner as in the eukaryotic cells. Xl from the thermophilic bacterium
Thermus thermophilus
was expressed in
S. cerevisiae
, and the recombinant strain fermented xylose to ethanol (Walfridsson et al., “Ethanolic fermentation of xylose with
Saccharomyces cerevisiae
harboring the
Thermus thermophilus
xylA gene which expresses an active xylose (glucose) isomerase”,
Appl. Environ. Microbiol.
62:4648-4651, 1996). The low level of ethanol produced was assumed to be due to the fact that the temperature optimum of the enzyme is 85° C., whereas the optimum temperature for a yeast fermentation is 30° C.
Recently, the gene for Xl from a mesophilic bacterium,
Streptomyces diastaticus
, has been cloned and transformed into
S. cerevisiae
. When xylose is fermented by an Xl expressing transformant of
S. cerevisiae
, a considerable amount of xylitol is formed in addition to ethanol. The
Cordero Otero R. R.
Hahn-Hägerdal B.
Träff K. L.
Van Zyl W. H.
Finnegan Henderson Farabow Garrett & Dunner LLP
Forskarpatent I Syd AB
Monshipouri Maryam
Prouty Rebecca E.
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