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Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

a technology of pyruvate decarboxylase and pdc, which is applied in the field of cloning and sequencing of pdc genes from bacteria and uses therefor, can solve the problem that organisms rarely produce all of the necessarily enzymes at the most desirable levels, and achieve the effect of improving the efficiency of substrate utilization and the economics of the fermentation process

Inactive Publication Date: 2008-01-10
UNIV OF FLORIDA RES FOUNDATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes the development of enzymes and biocatalysts that can efficiently break down complex sugars and convert them into ethanol, which can then be fermented to produce alcohol. This can improve the efficiency of substrate utilization and the economics of the fermentation process. The invention provides genes that can be expressed in a range of organisms to achieve high levels of activity and can be used in a biocatalyst to simultaneously saccharify and ferment complex sugars. The use of these enzymes can lead to higher levels of ethanol production and can be done in a cost-effective manner.

Problems solved by technology

However, such organisms rarely produce all of the necessarily enzymes at the most desirable levels.

Method used

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  • Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor
  • Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor
  • Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

Examples

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example 1

Identification and Characterization of PDC Gene from Gram-Negative Bacterium Zymobacter palmae

[0155] In this example, the identification and characterization of a PDC gene from the Gram-negative bacterium Z. palmae is described.

[0156] Briefly, a pyruvate decarboxylase (pdc) operon was isolated from a genomic library of Zymobacter palmae using a degenerate oligonucleotide probe based on the N-terminal amino acid sequence of the purified protein (FIG. 1). A 1.7-kb PstI to BamHI subclone of this fragment (pJAM3440) was found to be required for PDC activity in recombinant E. coli. Based on DNA sequence, a 1668-bp ORF (55 mol % G+C content) was identified in this region that encoded a putative PDC polypeptide (FIG. 1). A canonical Shine-Dalgarno sequence (GGAGG) was 10 bp upstream of the translation start codon (ATG). In addition, a putative −35 and −10 promoter (TTcACt-N17-atTAAT, where N is any nucleotide and uppercase letters match the bacterial promoter consensus) was located 16 to...

example 2

Identification and Characterization of PDC Gene from Gram-Negative Aerobic Bacterium Acetobacter pasteurianus

[0158] In this example, the identification and characterization of a PDC gene from the Gram-negative bacterium Acetobacter pasteurianus is described.

[0159] Briefly, a chromosomal DNA was isolated from cells of A. pasteurianus using the method described by Harwood et al. (17). For Southern analysis, the genomic DNA (1 to 2 μg per lane) was cleaved with restriction enzymes (AatII, BamHI, ClaI, EcoRI, and HincII), separated by gel electrophoresis (0.8% agarose), and transferred by downward capillary action to positively charged nylon membranes. Membranes were equilibrated at 60° C. for 2 h in 5×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 1% blocking reagent, 0.1% N-lauroylsarcosine, and 0.02% sodium dodecyl sulfate (SDS). Random primers were used to label a probe with dixoigenin-11-dUTP using a 0.7-kb KpnI fragment of the Z. mobilis pdc gene as the templa...

example 3

Identification and Characterization of PDC Gene from Gram-Positive Bacterium Sarcina ventriculi

[0167] In this example, the identification and characterization of a PDC gene from the Gram-positive bacterium Sarcina ventriculi is described.

[0168] Briefly, a degenerate oligonucleotide 5′-AARGARGTNAAYGTNGARCAYATGTTYGGNGT-3′ (SEQ ID NO: 11) was synthesized based on the N-terminal amino acid sequence of PDC purified from S. ventriculi (Lowe and Zeikus, 1992) (where, R is A or G; N is A, C, G, or T; Y is C or T). This oligonucleotide was labeled at the 3′-end using terminal transferase with digoxigenin-11-dUTP and dATP as recommended by the supplier (Roche Molecular Biochemicals) and was used to screen genomic DNA from S. ventriculi.

[0169] For Southern analysis, genomic DNA was digested with BglI, EcoRI, or HincII, separated by 0.8% agarose electrophoresis, and transferred to positively charged nylon membranes (Southern, 1975). Membranes were equilibrated at 58° C. for 2 h in 5×SSC (1×S...

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Abstract

The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

Description

RELATED INFORMATION [0001] This application claims priority to U.S. provisional application No. 60 / 288,638, entitled “High-Level Production of Active Sarcina ventriculi Pyruvate Decarboxylase in Recombinant Bacillus megaterium”; U.S. provisional application No. 60 / 288,671, entitled “Cloning, Expression, and Characterization of Pyruvate Decarboxylase from the Acid-Tolerant, Anaerobic Gram-Positive Bacterium Sarcina ventriculi Goodsir”; U.S. provisional application No. 60 / 288,698, entitled “Acetobacter pasteurianus Pyruvate Decarboxylase: Biochemical, Genetic, and Physiological Properties”; U.S. provisional application No. 60 / 288,622, entitled “Biochemical and Biophysical Characterization of Pyruvate Decarboxylase from the Acetic Acid Bacterium Acetobacter pasteurianus”; and U.S. provisional application No. 60 / 288,699, entitled “Pyruvate Decarboxylase: A Key Enzyme for the Oxidative Metabolism of Lactic Acid by Acetobacter pasteurianus”; all of which were filed on May 4, 2001 and are ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/12C07K14/195C12N9/00C12Q1/527C12Q1/68C07H21/04G01N33/53C07K16/40C12N1/15C12N1/19C12N1/21C12N5/10C12N9/10C12N9/88C12N15/09C12N15/74C12P7/06C12P7/24C12P21/02G01N33/566
CPCY02E50/17C12N9/88Y02E50/10C12N15/52
Inventor MAUPIN-FURLOW, JULIE A.TALARICO, LEE ANNRAJ, KRISHNAN CHANDRAINGRAM, LONNIE O.
Owner UNIV OF FLORIDA RES FOUNDATION INC
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