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An nadh dependent l-xylulose reductase

A xylulose and reductase technology, applied in the field of sucrose or its derivative conversion, can solve the problem of no L-xylulose reductase activity and the like

Inactive Publication Date: 2006-10-18
VALTION TEKNILLINEN TUTKIMUSKESKUS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To our knowledge, there are no reports related to NADH-coupled L-xylulose reductase activity

Method used

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  • An nadh dependent l-xylulose reductase

Examples

Experimental program
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Embodiment approach

[0064] According to a preferred embodiment of the present invention, the shortcoming that said fungus cannot effectively utilize L-arabinose is solved by the genetic modification of the fungus, and the method is characterized in that the gene transformed with NADH-dependent L-xylulose reductase described fungi.

[0065] According to another embodiment, a microorganism (preferably a fungus) is transformed with all or some of the genes encoding the enzymes of the L-arabinose pathway, i.e. at least with aldose reductase, L-arabitol 4-dehydrogenase and the present L- Transformation with a xylulose reductase gene, and optionally with D-xylulose reductase and / or xylulokinase. Preferably, the microorganism is transformed with all genes in the L-arabinose pathway. The resulting microorganisms such as fungi are then able to utilize L-arabinose more efficiently.

[0066] In other embodiments, fungi that utilize D-xylose but cannot utilize L-arabinose, such as genetically engineered S...

Embodiment 1

[0081] Example 1: Screening for enhanced growth in arabinose

[0082] Improved growth in L-arabinose using Saccharomyces cerevisiae strain H2651 (Richard et al.: "The missing link in the fungal L-arabinose catabolic pathway, identification of the L-xylulose reductase gene", Biochemistry, 41, 2002, 6432-7) The Ambrosiozyma monospora cDNA library was screened. H2651 contains all genes of the fungal L-arabinose pathway. The Pichia stipitis XYL1 and XYL2 genes encoding aldose reductase and xylitol dehydrogenase, respectively, were integrated into the URA3 locus. The strain also expresses the endogenous XKS1 gene encoding xylulokinase. The lad1 and lxr1 genes encoding L-arabitol dehydrogenase and L-xylulose reductase from Hypocrea jecorina (Trichoderma reesei) were present on multi-copy expression vectors with LEU2 and URA3 marker genes, respectively.

[0083] Construction of cDNA library of Ambrosiozyma monospora

[0084] Yeast Ambrosiozyma monospora (NRRL Y-1484) was cultured...

Embodiment 2

[0088] Example 2: Expression of L-xylulose reductase in Saccharomyces cerevisiae

[0089] The ALX1 gene was isolated after digestion with SalI-NotI and ligated into a multi-copy expression vector with uracil selectivity and the PGK1 promoter. The expression vector was generated from pFL60 by introducing SalI and NotI restriction sites into the multiple cloning site. The resulting plasmid was designated p2178. This was then transformed into S. cerevisiae strain CEN.PK2. This strain was called H2986 and was deposited under accession number DSM 15821 as described above.

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Abstract

The present invention relates to isolated DNA molecules comprising a gene encoding an enzymatic protein having NADH-dependent L-xylulose reductase activity. The DNA sequence encoding the enzyme protein was identified. The present invention further relates to microorganisms transformed with said DNA molecule of the present invention and NADH-dependent L-xylulose reductase. The invention can be used to convert carbohydrate-containing biological material, such as industrial waste, into useful end products.

Description

field of invention [0001] The present invention relates to isolated DNA molecules comprising genes encoding enzymes capable of utilizing carbohydrates such as sucrose or derivatives thereof in vivo and in vitro, and microorganisms transformed with said DNA molecules. The present invention further relates to the enzyme protein encoded by said DNA molecule, and its use for the conversion of sucrose or its derivatives. Background of the invention [0002] Biological waste from industry, including agriculture, contains eg carbohydrates such as sugars. For a long time, converting these waste materials into useful products has been a hot spot and challenge in the field of biotechnology. [0003] As a specific example of carbohydrates, mention may be made of L-arabinose, which is the main constituent of plant material. Therefore, L-arabinose fermentation also has potential bioengineering significance. [0004] Fungi that can utilize L-arabinose are not necessarily suitable for i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P7/00
CPCC12N9/0006Y02E50/10
Inventor R·维霍R·理查德M·潘迪拉
Owner VALTION TEKNILLINEN TUTKIMUSKESKUS