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Enhanced 2-keto-L-gulonic acid production

A 2-KLG, biosynthetic technology, applied in the field of industrial production enhancement of 2-KL, can solve the problems of increased production of final products, difficult to ensure enhanced production of final products or intermediates, etc.

Inactive Publication Date: 2004-09-08
GENENCOR INT INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Furthermore, minimal knowledge about the transport systems involved is hardly guaranteed to enable enhanced production of desired end products or intermediates
Synthetic machinery may already be saturated and thus increased presence of substrate does not necessarily lead to increased production of desired end product
In addition, increased transport of substrates that are used directly or indirectly as metabolites in addition to their use as production substrates may not result in the desired increased production

Method used

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  • Enhanced 2-keto-L-gulonic acid production
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  • Enhanced 2-keto-L-gulonic acid production

Examples

Experimental program
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Effect test

Embodiment I

[0147] Materials and Methods

[0148] a. Plasmids, bacterial strains and culture medium: plasmid pBCL1920, Klebsiella oxytoca, Pantoea citrea 1392A. P. citrea strain 1392A is a variant of 39140 P. citrea. pD92 is described representing a vector containing the DKG reductase gene. US Patent No. 5,376,544. Murphy III medium contains fructose 0.5%, phosphate 1.6%, MgSO4.7H2O 0.2%, Soytone 0.2%, citrate 0.01%, (NH4)2SO4 1%, trace salts such as Fe, Co, Mn, Zn in the ppm range and vitamins such as niacin, folic acid and B12; M9 medium contains 0.9% phosphate, 0.1% NaCl, 0.1% NH4Cl, MgSO4 0.0005%, CaCl2 0.025%; fermentation medium contains potassium phosphate (Potassium Phosphate) 1% , MgSO4 0.15%, Glutamate 1.5%, Fructose 2.5%, Ammonium Sulfate 0.1% and a vitamin blend containing Biotin, Thiamine, Pyridoxine, Riboflavin, Niacin, Folic Acid and B12, and Trace metal mixture solution of iron, Zn and Mn ions at 10-100 ppm level; transport assay medium containing 100 mM potassium phos...

Embodiment II

[0156] Example II provides that transporters of substrates may be the basis for rate-limiting factors of whole-cell biotransformation

[0157] This example illustrates the key steps in the formation of 2KLG from glucose that can be compartmentalized into 4 fractions (Figure 5). The key intermediate 2,5-DKG is produced in Pantoea citrea at a rate of 14-15 g / l / hr by three periplasmic enzymes (Sonoyama et al., Appl. Environ. Microbiol., 1982, 43: 1064- 1069). The second component is the rate at which DKG is required to be transported within the cytoplasmic space of the cell. The third is the rate of conversion of DKG to 2KLG by DKG reductase (US Patent No. 5,032,514). Conversion of DKG to 2KLG is not rate-limiting when DKG reductase is overexpressed. In our current 2KLG production fermentation, inducible plasmids are used to increase or decrease the specific reductase activity relative to our general fermentation (currently using pD92), where the DKG reductase is under the con...

Embodiment III

[0164] Example III provides evidence that substrate transport into cells for biotransformation is indeed the rate-limiting step

[0165] Cell pellets from three different times of the fermentation process, seed-flask stage, fructose-fed stage and glucose-fed stage, were collected and processed as described in the experiments. Using DKG uptake assays for these cell types, the rates of DKG uptake (introduction of DKG for conversion to KLG) were determined to be 0.5 g / l / hr, 2.7 g / l / hr and 2.75 g / l / hr (Figure 9). The rate of DKG input is equal to the rate of KLG output. This result therefore demonstrates that the rate of biotransformation of DKG to KLG is dependent on the rate of import of 2,5-DKG into cells. The present invention will thus demonstrate that by increasing the DKG uptake transporter by overexpression, the rate of 2,5-DKG import will be enhanced, and thus the rate of 2KLG production will be enhanced. An expert in the field can imagine that the key limiting factor i...

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Abstract

A method for enhancing a host cell's biosynthetic production of 2-KLG is described. Such method comprises selecting a host cell that has an at least partially intracellular synthetic pathway which utilizes 2,5-DKG to produce 2-KLG; increasing the transport of said 2,5-DKG into said host cell while maintaining the integrity of the host cell; culturing the host cell to produce said 2,5-DKG; and producing 2-KLG. The transport of the 2,5-DKG is increased by transforming into the host cell DNA encoding for one or more enzymes transporting the 2,5-DKG into the host cell.

Description

field of invention [0001] The present invention generally relates to the enhancement of industrial production of 2-KL, and in particular to the overexpression of the genome encoding the protein that transports 2,5-diketoglutarate from the periplasm to the cytoplasmic region of the cell. The present invention provides expression vectors, methods and systems for enhancing 2-KLG production in microorganisms. Background of the invention [0002] It is generally known that 2-keto-L-gulonic acid (2-KLG) can be easily converted to L-ascorbic acid (vitamin c) in a one-step chemical process by the method of Reichstein (Reichstein, T. et al., Helv. Chim. Acta, 1934, 17, 311-328; Reichstein, T. et al., Helv. Chim. Acta, 1933, 16, 561, 1019). There are recombinant microorganisms that express heterologous enzymes to convert the starting substrate to 2-KLG. Recombinant DNA technology has been used to bioconvert D-glucose to 2-KLG in a single fermentative step in Erwinia herbicola (Ander...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K14/24C07K14/26C07K16/40C12N1/21C12N9/14C12N15/31C12P7/60
CPCC07K14/24C07K14/26C12P7/60
Inventor M·库玛F·瓦勒V·A·达特沃斯J·A·赫啻
Owner GENENCOR INT INC
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