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Method for the production of 1,3-propanediol by recombinant organisms compising for vitamin B12 transport

A production method, B12 technology, applied in the field of molecular biology, capable of solving problems affecting transcription and translation, etc.

Inactive Publication Date: 2001-06-20
EI DU PONT DE NEMOURS & CO +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Transcriptional regions have been reported to be involved in the attenuation regulation of amino acid biosynthetic pathways, but btuB regulation is unusual in that important regulatory sites are located in the coding sequence of btuB and the regulation affects both transcription and translation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example General Method

[0104] Methods of phosphorylation, ligation and conversion are well known in the art. Techniques suitable for use in the following examples can be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0105]Materials and methods suitable for maintaining and growing bacterial cultures are well known in the art. Techniques applicable to the following examples can be found in Manual of Methods for General Bacteriology (Philipp Gerhardt, R.G.E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G,. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989). Unless otherwise noted, all reagents and materials used for the maintenance and grow...

Embodiment 1

[0145] B 12 Construction of the transport gene expression cassette

[0146] built B 12 Expression plasmids for the transfer genes, btuB, btuC, btuD and btuE can exist as replication elements. All expression plasmids use the trc promoter for transcription. Each plasmid also contains the gene for β-lactamase selection in E. coli in ampicillin-containing media, or the chloramphenicol acetyltransferase gene for selection in chloramphenicol-containing media . The origin of replication of the plasmid is ColE or p15A.

[0147] The btuB gene was amplified from E. coli chromosomal DNA by PCR using primers (SEQ ID NO 18 and SEQ ID NO: 19) with an added NcoI site at the 5' end and BamHI at the 3' end. The reaction mixture contained 10mM Tris, pH8.3, 50mM KCl, 1.5mM MgCl 2 , 0.0001% gelatin, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 1 μM various primers, 1-10ng target DNA, 25 units / mL Amplitaq TM DNA polymerase (Perkin-Elmer Cetus, Norwalk CT). The PCR paramete...

Embodiment 2

[0152] have B 12 Transformants of transgene and DhaB activity

[0153] E. coli strain FM5 was transformed with dha plasmid pDT24(specR), btuB plasmid pBtuB1(ampR) or pBtuB2(chlR), or btuBCED plasmid pBCED(chlR). Screening was performed on LB plates containing 50 mg / ml spectinomycin, 50 mg / ml ampicillin or 50 mg / ml chloramphenicol. Colonies resistant to appropriate antibiotics were used for 1,3-propanediol production and vitamin B 12 ingest.

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PUM

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Abstract

Recombinant organisms are provided comprising genes encoding genes encoding glycerol dehydratase, 1,3-propanediol, 1,3-propanediol oxidoreductase, a gene encoding vitamin B12 receptor precursor (BtuB), a gene encoding vitamin B12 transport system permease protein (BtuC) and a gene encoding vitamin B12 transport ATP-binding protein (BtuD). The recombinant microorganism is contacted with a carbon substrate and 1,3-propanediol is isolated from the growth media.

Description

field of invention [0001] The present invention relates to the field of molecular biology and the production of 1,3-propanediol using recombinant organisms. More specifically it describes the effects of intracellular vitamin B 12 transport and clonal expression of genes that efficiently convert carbon substrates to 1,3-propanediol. Background of the invention [0002] 1,3-Propanediol is a monomer used in the production of polyester fibers and in the manufacture of polyurethanes and cyclic compounds. [0003] Many chemical routes for the synthesis of 1,3-propanediol are known. For example, 1,3-propanediol can be prepared 1) from ethylene oxide over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and acid; 2) by catalytic solution-phase hydrogenation of acrolein followed by Reduction; or 3) reacting by hydrocarbons such as glycerol in the presence of carbon monoxide and hydrogen on a catalyst containing Group VIII atoms on the periodic table. Alth...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N1/21C12N5/10C12N15/31C12N15/52C12P7/18C12R1/19C12N15/09
CPCC12P7/18C12N15/52C12N15/11
Inventor G·M·怀特德B·布尔图伊斯D·E·特林布尔A·A·加滕拜
Owner EI DU PONT DE NEMOURS & CO
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