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Xylose isomerases and their uses

A technology of xylose isomerase and structural domain, applied in the field of XI polypeptide, can solve the problems of no enzyme activity and low enzyme activity

Inactive Publication Date: 2015-06-17
BP CORP NORTH AMERICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several XI genes have been identified, but the common problem is little or no enzymatic activity after expression in S. cerevisiae

Method used

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  • Xylose isomerases and their uses
  • Xylose isomerases and their uses
  • Xylose isomerases and their uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0120] Materials and methods

[0121] yeast culture

[0122]Unless otherwise stated for a particular example, yeast transformants were grown in SC-ura medium containing about 2% glucose at 30°C for about 24 hours. The medium contains about 20 g of agar, about 134 g of BD Difco TM Amino Acid Free Yeast Nitrogen Base (BD, Franklin Lakes, New Jersey) and about 2 g SC Amino Acid Blend containing about 85 mg of the following amino acid unless otherwise noted (amounts are listed in parentheses): L-Adenine (21.0), L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, glutamine, L-glutamic acid, glycine, L- Histidine, Inositol, L-Isoleucine, L-Leucine (173.4), L-Lysine, L-Methionine, P-Aminoic Acid (8.6), L-Phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine.

[0123] Xylose isomerase activity

[0124] XI activity in cell lysates was assayed using a method based on the method of Kersters-Hilderson et al., 1986, Enzyme Microb. Technol...

Embodiment 2

[0127] Example 2: Activity-Based Discovery Screen of Xylose Isomerase

[0128] Libraries for activity-based discovery ("ABD") screening were in the form of ex vivo phagemids. These libraries were constructed as described in US Patent 6,280,926. The source of these libraries was environmental rumen samples collected from the foregut of dead herbivores.

[0129] E. coli screening strains were constructed to identify genes encoding xylose isomerase activity from environmental libraries. Specifically, the E. coli strain SEL700 was complemented with the pBR322-derived plasmid pJC859 (Kokjohn et al., 1987, J. Bacteriol. 169:1499-1508) containing the E. coli recA gene to generate a wild-type recA expression. type, the SEL700 is recA - , MG1655 derivatives resistant to bacteriophage lambda and containing the F' plasmid.

[0130] The entire coding sequence of the endogenous xylA xylose isomerase gene was then deleted using a two-step marker exchange procedure. Briefly, plasmid p...

Embodiment 3

[0138] Embodiment 3: XI sequence analysis

[0139] Plasmids from both the ABD and SBD screens were purified using the ABI3730xl DNA Analyzer and the ABI v3.1 cycle sequencing chemistry for sequencing vector inserts. Identification of putative ORFs was accomplished by identifying the start and stop codons of the longest protein coding region, followed by manual curation based on homology to published xylose isomerase DNA sequences. The identified XI ORFs are listed in Table 2 below, which indicates the sequence and source organism classification for each XI determined from the ABD or SBD library, as well as their assigned sequence identifiers. The putative catalytic domain (based on sequence alignment with other XIs) is underlined.

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Abstract

This disclosure relates to novel xylose isomerases and their uses, particularly in fermentation processes that employ xylose-containing media.

Description

Background technique [0001] Efficient, commercial production of biofuels from plant material such as sugar cane requires the fermentation of pentose sugars such as xylose. Xylose in plant material usually comes from lignocellulose, which is a matrix composed of cellulose, hemicellulose, and lignin. Lignocellulose is broken down by acid hydrolysis or enzymatic reactions, yielding xylose as well as other monosaccharides such as glucose (Maki et al., 2009, Int. J. Biol. Sci. 5:500-516). [0002] Fungi, particularly Saccharomyces cerevisiae, are commercially relevant microorganisms that ferment sugars into biofuels such as ethanol. However, S. cerevisiae does not metabolize xylose endogenously and requires a genetic modification that allows it to convert xylose to xylulose. Other organisms of limited usefulness in ethanol production are able to metabolize xylose (Nevigot, 2008, Micobiol. Mol. Biol. Rev. 72:379-412). [0003] Two pathways for the metabolism of xylose to xylulose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12P7/10C12N15/10
CPCC12N9/92C12P5/026C12P7/06C12P7/16C12P7/18C12P7/20C12P7/40C12P13/04C12P35/06C12P37/02C12Y503/01005C12P2203/00C12P17/184Y02E50/10Y02E50/30C12N9/90C12P7/10C12P7/42C12P7/46C12P7/48C12P7/54C12P7/56C12P13/02
Inventor 小大卫·尼尔·努恩彼得·卢京比尔李陵亚当·马丁·布尔扎约恩·彼得·弗拉施·巴特内克
Owner BP CORP NORTH AMERICA INC
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