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Methods for the production of products in host cells

Inactive Publication Date: 2006-03-30
DODGE TIMOTHY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] The present invention provides methods to produce ascorbic acid intermediates comprising host cells genetically engineered to reduce carbon substrate diverted to metabolic pathways, thus increasing the productivity of the host cell.
[0014] c) allowing the production of an ascorbic acid intermediate from the carbon source, wherein the production of said ascorbic acid intermediate is enhanced compared to the production of the ascorbic acid intermediate in the unaltered bacterial host strain.
[0016] In one embodiment, such host cells have a modification in a polynucleotide encoding an enzymatic activity that phosphorylates D-glucose at its 6th carbon and / or a modification in a polynucleotide encoding an enzymatic activity that phosphorylates D-gluconate at its 6th carbon. Such host cells when cultured in the presence of a carbon source demonstrated increased yield of product as measured directly and / or indirectly. In some embodiments of the invention, the methods comprise host cells having a modification in a polynucleotide encoding an enzymatic activity that phosphorylates D-glucose at its 6th carbon and a modification in a polynucleotide encoding an enzymatic activity that phosphorylates D-gluconate at its 6th carbon.

Problems solved by technology

However, most of the current methodologies utilized to produce compounds over-express the products.
There are still problems remaining concerning the diversion of substrates used to produce the desired end-product by the cell for metabolic (catabolic) purposes, reducing the efficiency and overall production of thereof.

Method used

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  • Methods for the production of products in host cells
  • Methods for the production of products in host cells
  • Methods for the production of products in host cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0198] Construction of a Genomic Library from P. citrea 1 39-2a

[0199]P.citrea genomic DNA was prepared using the DNA-Pure™ genomic DNA Isolation Kit (CPG, Lincoln Park, N.J.). 50 micrograms of this DNA was partially digested with the restriction enzyme Sau3A accordingly the manufacturer recommendations (Roche Molecular Biochemicals, Indianapolis, Id.). The products of the digestion were separated on a 1% agarose gel and the DNA fragments of 3-5 kilobases were purified from the gel using the Qiaquick Gel extraction kit (Qiagen Inc. Valencia, Calif.). The resulting DNA was ligated with BamHI-linearized plasmid pBK-CMV (Stratagene, La Jolla, Calif.). A library of around 10xx different plasmids was obtained in this way.

example 2

[0200] Isolation of the Structural Gene for the Glucokinase Enzyme.

[0201] To select for a plasmid that carries the glucokinase gene from P. citrea, the genomic library (see above) was transformed into a E. coli strain devoid of the glucokinase gene (glkA) and the PTS transport system, strain NF9, glk-, (Flores et al., Nat. Biotech. 14, 620-623). After transformation, the cells were selected for growth on M9 media with glucose as the only carbon source. With this strategy, plasmids able to complement the glk− or pts− mutations were selected.

[0202] After 48 hrs. of incubation at 37° C., many colonies were visible. Several of these colonies were further purified and their plasmids isolated and characterized by restriction analysis. It was found that all the plasmids contained a common DNA fragment.

[0203] After re-transforming these plasmids back into NF9, glk−, all of them allowed growth on M9-glucose media, corroborating that they were able to complement at least one of the mutatio...

example 3

[0205] Inactivation of the Glucokinase Gene by Homologous Recombination.

[0206] The general strategy to inactivate genes by homologous recombination with a a suicide vector has been delineated before ( Miller and Mekalanos., J. Bacteriol. 170 (1988) 2575-2583). To inactivate the glk gene from P.citrea by this approach two plasmids were constructed: pMD5 and pMD6.

[0207] To construct pMD5, plasmid pMD4 was digested with the Ncol and SnaB1 restriction enzymes accordingly manufacturer specifications. (Roche Molecular Biochemicals, Indianapolis, Id.). The cohesive ends generated by these enzymes were blunt-ended with T4 polymerase using standard techniques. This DNA was ligated with a loxP-Cat-loxP cassette isolated from pLoxCat2 as a SpeI-EcoRV DNA fragment. (Palmeros et al., Gene (2000) 247, 255-264.). This cassette codes for Chloramphenicol resistance. The ligation mixture was transformed into TOP10 competent cell (Invitrogen, Carlsbard Calif.). selecting for growth on Chloramphenico...

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Abstract

The invention provides methods and host cells for the production of ascorbic acid intermediates. The invention also provides host cells having a modification in a polynucleotide that uncouples the catabolic pathway from the oxidative pathway by deleting the encoding for an endogenous enzymatic activity that phosphorylates D-glucose at its 6th carbon and / or a polynucleotide that has deleted the encoding for endogenous enzymatic activity that phosphorylates D-gluconate at its 6th carbon. Such host cells are used for the production of products, such as, ascorbic acid intermediates. Nucleic acid and amino acid sequences with inactivated enzymatic activity which phosphorylates D-glucose at its 6th carbon and inactivated enzymatic activity which phosphorylates D-gluconate at its 6th carbon are provided.

Description

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH [0001] This invention was made with the United States Government support under Award No. 70 NANB 5H1138 awarded by the United States Department of Commerce. The Government has certain rights in this invention.TECHNICAL FIELD [0002] The present invention relates to engineering of metabolic pathways in host cells and provides methods and systems for the production of products in host cells. In particular, the present invention provides methods and systems for the production of ascorbic acid intermediates in host cells. BACKGROUND ART [0003] Numerous products of commercial interest, such as intermediates of L-ascorbic acid, have been produced biocatalytically in genetically engineered host cells. L-Ascorbic acid (vitamin C, ASA) finds use in the pharmaceutical and food industry as a vitamin and antioxidant. The synthesis of ASA has received considerable attention over many years due to its relatively large market...

Claims

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Application Information

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IPC IPC(8): C12P17/04C12P7/60C12N15/09C12N15/52C12P7/40C12P7/42C12P7/46C12P7/58C12P19/02
CPCC12N15/52C12P19/02C12P7/60C12P7/46C12P17/04
Inventor DODGE, TIMOTHYVALLE, FERNANDO
Owner DODGE TIMOTHY