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Methods for the preparation of a fine chemical by fermentation

A chemical, fine technology, applied in the field of fine chemicals prepared by fermentation, which can solve problems such as consumption

Inactive Publication Date: 2008-07-30
BASF SE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, biosynthesis of 1 molecule of lysine from oxaloacetate consumes 4 molecules of NADPH

Method used

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  • Methods for the preparation of a fine chemical by fermentation
  • Methods for the preparation of a fine chemical by fermentation
  • Methods for the preparation of a fine chemical by fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0170] Example 1: Production of lysine from fructose and glucose by Corynebacterium glutamicum

[0171] Analysis of the metabolic flux of lysine-producing Corynebacterium glutamicum in comparative batch cultures with glucose and fructose. For this, pre-grown cells were transferred to tracking medium and incubated for approximately 5 hours. Analysis of substrates and products at the beginning and end of the chase experiment revealed significant differences between the two carbon sources. A total of 11.1 mM lysine was produced with glucose, while a lower concentration of only 8.6 mM was achieved with fructose. During the incubation period of more than 5 hours, the cell concentration changed from 3.9g L -1 Increased to 6.0g L -1 (glucose) and from 3.5g L -1 increased to 4.4g L -1 (fructose). Due to the fact that threonine and methionine are not present in the medium, internal sources are likely used by the cells for biomass synthesis. The average specific sugar absorption ...

Embodiment 2

[0179] Example 2: In manual inspection and tracking experiments 13 C-marker type

[0180] The relative mass isotopomer fractions of secreted lysine and trehalose were quantified by GC-MS. These mass isotopomer fractions are sensitive to intracellular fluxes and thus show a fingerprint of the fluxome of the biological system under study. As shown in Figure 2, the marker profile of secreted lysine and trehalose was significantly different between glucose and fructose grown cells of C. glutamicum. Differences were found for both the tracking markers applied and the products of the two measurements. This suggests substantial differences in carbon flux patterns depending on the carbon source applied. As previously shown, from Corynebacterium glutamicum to [1- 13 C] and [ 13 C 6 ] The mass isotopologue fractions of two parallel cultures of a glucose mixture were almost identical (Wittmann, C.H.M. Kim and E. Heinzle. 2003. Metabolicflux analysis at miniaturized scale. submitted...

Embodiment 3

[0181] Example 3: Estimation of Intracellular Flux

[0182] The central question of the studies carried out was the comparative study of the intracellular flux of C. glutamicum during the production of lysine using glucose and fructose as carbon sources, respectively. To this end, the experimental data obtained from the tracking experiments were used to calculate the metabolic flux distribution for each substrate, using flux estimation software as described above. Parameter estimation was performed by minimizing the deviation between experimental and theoretical mass isotopomer fractions. The method performed utilizes an optimized metabolite balance during each step. This included (i) stoichiometric data on product secretion (Table 2) and (ii) stoichiometric data on anabolic requirements for biomass precursors (Table 3). The set of intracellular fluxes that gave the smallest deviation between experimental and simulated marker configurations was considered to be the best esti...

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PUM

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Abstract

The invention features methods of increasing the production of a fine chemical, e.g., lysine from a microorganism, e.g., corynebacterium by way of deregulating an enzyme encoding gene, i.e., fructose- l,6-bisphosphatase. In a preferred embodiment, the invention provides methods of increasing the production of lysine in corynebacterium glutamicum by way of increasing the expression of fructose-1, 6-bisphosphatase activity. The invention also provides a novel process for the production of lysine by way of regulating carbon flux towards oxaloacetate (OAA). In a preferred embodiment, the invention provides methods for the production of lysine by way of utilizing fructose or sucrose as a carbon source.

Description

Background of the invention [0001] The industrial production of the amino acid lysine has become an economically important industrial process. Lysine is used commercially as an animal feed additive due to its ability to improve feed quality by increasing the absorption of other amino acids, in human medicine, especially as a component of perfusion solutions, and in the pharmaceutical industry. [0002] The commercial production of this lysine is mainly carried out with Gram-positive Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum (Kleemann, A.et.al." Amino Acids," in ULLMANN'S ENCYCLOPEDIA OF INDUSTRIALCHEMISTRY, vol. A2, pp. 57-97, Weinham: VCH-Verlagsgesellschaft (1985)). These organisms are used to produce about 250,000 tons of lysine per year. Numerous studies have been undertaken to isolate mutated bacterial strains that produce greater amounts of lysine. Microorganisms used in microbial methods for amino acid production are divided ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/77C12N9/14C12N1/21C12P13/08C12R1/15C12N9/16
CPCC12P13/08C12N9/16A61K35/66C12N15/52A61K35/76
Inventor O·策尔德尔C·克洛普罗格H·施罗德S·哈夫纳B·克勒格尔P·基弗E·海因茨勒C·维特曼
Owner BASF SE
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