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, can solve problems affecting transcription and translation

Inactive Publication Date: 2007-01-31
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

[0162] B 12 Construction of the transport gene expression cassette

[0163] built B 12 The expression plasmids for the transgenes, 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.

[0164] The btuB gene was amplified from E. coli chromosomal DNA by PCR using primers (SEQ ID NO 18 and SEQ ID NO: 19) with added NcoI site at the 5' end and BamHI at the 3' end. The reaction mixture contained 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM 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 parameters are 9...

Embodiment 2

[0169] have B 12 Transformants of transgene and DhaB activity

[0170] 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.

Embodiment 3

[0172] Coenzyme B in FM5 transformed with pBCED 12 increased intake of

[0173] In containing 25mL medium (medium with NH 4 OH titrated to pH 6.8 with 0.2MKH 2 OH, 2.0g / L citric acid, 2.0g / L mgSO 4 ·7H 2 O, 1.2 mL 98% H 2 SO 4 , 0.3g / L ferric ammonium citrate, 0.2g / L CaCl 2 2H 2 O, 5 mL trace metal mix, 5 g / L yeast extract, 10 g / L LD-glucose, and appropriate antibiotics. Trace metal mixture contains (g / L): Na 2 SO 4 (4.0), MnSO 4 ·H 2 O(0.80), ZnSO 4 ·7H 2 O(1.6), CoSO 4 (0.52), CuSO 4 ·7H 2 O(0.12) and FeSO 4 ·7H 2 O(4.0)) in a 250ml Erlenmeyer flask grown at 37°C overnight with a rotation speed of 250rpm. Dilute the overnight culture (1 / 100) with 25ml of M9 medium and continue to grow until the OD is reached 660 ~1.0. When IPTG was added, the IPTG concentration was added to 0.2 mM at this time point and incubation was continued for 1 hr.

[0174] Add cyanocobalamin (cyanocobalamin, CNCbl) or coenzyme B to the M9 culture at this concentration 12 . All ...

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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) reaction by hydrocarbons such as glycerol in the presence of carbon monoxide and hydrogen over a catalyst containing atoms of group VIII of the periodic table. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/53C12N15/55C12N15/60C12P17/18C12N9/04C12N9/16C12N9/88C12N1/21C07K14/24C12N15/09C12N1/15C12N5/10C12N15/52C12P7/18C12R1/19
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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