Source: http://www.google.com/patents/US7803587?dq=U.S.+Patent+No.+4,528,643
Timestamp: 2016-05-06 11:27:17
Document Index: 770330926

Matched Legal Cases: ['Application No. 10', 'Application No. 10', 'art 1', 'art 2', 'art 1', 'art 2']

Patent US7803587 - Method for developing culture medium using genome information and in silico ... - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsA method for developing a culture medium using genome information and in silico analysis. In one implementation, the method involves developing a minimal synthetic medium, including the steps of constructing a metabolic network using genome information of prokaryotic cell or eukaryotic cell, selecting...http://www.google.com/patents/US7803587?utm_source=gb-gplus-sharePatent US7803587 - Method for developing culture medium using genome information and in silico analysisAdvanced Patent SearchPublication numberUS7803587 B2Publication typeGrantApplication numberUS 11/743,668Publication dateSep 28, 2010Priority dateMay 4, 2006Fee statusPaidAlso published asUS20080003661Publication number11743668, 743668, US 7803587 B2, US 7803587B2, US-B2-7803587, US7803587 B2, US7803587B2InventorsSang Yup Lee, Ho Nam Chang, Hyohak Song, Tae Yong Kim, Bo-Kyung ChoiOriginal AssigneeKorea Advanced Institute Of Science And TechnologyExport CitationBiBTeX, EndNote, RefManPatent Citations (11), Non-Patent Citations (34), Referenced by (3), Classifications (22), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetMethod for developing culture medium using genome information and in silico analysis
US 7803587 B2Abstract
1. A method for developing a culture medium using in silico analysis and genome information, comprising the steps of:
(a) constructing a metabolic network using genome information of target eukaryotic cell or prokaryotic cell;
(b) removing any one among external metabolites from the constructed metabolic network and conducting metabolic flux analysis using in silico simulation;
(c) determining the removed external metabolite as a component of a minimal synthetic medium when a objective function value is 0 after the in silico simulation, wherein the objective function value is 0 if any one of the components required by eukaryotic or prokaryotic cell cannot be produced, or returning to step (b) when the objective function value is not 0;
(d) confirming whether a relevant metabolite can be produced internally and used without providing it externally when relevant metabolic reaction formula exists after examining the relevant reaction formula and deficient enzymes through comparison with other organism species synthesizing the metabolite by itself;
(e) determining components of a minimal synthetic medium determined by repeating steps (b) to (d) as a minimal synthetic medium for in silico; and
(f) determining a final culture medium after culturing target eukaryotic or prokaryotic cells in the determined minimal synthetic medium for in silico.
5. A method for developing a culture medium for producing succinic acid using in silico analysis and genome information, comprising the steps of:
(a) constructing a metabolic network using genome information of eukaryotic cell or prokaryotic cell having succinic acid-producing ability;
(d) confirming whether a relevant metabolite can be produced internally and used without providing it externally when relevant metabolic reaction formula exists after examining the relevant reaction formula and deficient enzymes through the comparison with other organism species synthesizing the metabolite by itself;
(f) determining a final culture medium after culturing target eukaryotic or prokaryotic cells having the ability to produce succinic acid in the determined the minimal synthetic medium for in silico.
9. The method according to claim 5, wherein said prokaryotic cell having succinic acid-producing ability is the genus Mannheimia. 10. The method according to claim 9, wherein said genus Mannheimia is Mannheimia succiniciproducens MBEL55E (KCTC0697BP).
This application claims priority under 35 USC 119 of Korean Patent Application No. 10-2006-0040581 filed May 4, 2006. The disclosure of Korean Patent Application No. 10-2006-0040581 is hereby incorporated herein in its entirety, for all purposes.
FIG. 2 shows metabolic networks for synthesizing deficient amino acid (A: methionine; B: cysteine). Rectangular filled with grey represents the gene present in the metabolic network of M. succiniciproducens. On the other hand, rectangular filled with white indicates the gene absent in the metabolic network of M. succiniciproducens. FIG. 3 shows metabolic networks for synthesizing deficient vitamin (A: nicotinic acid; B: pantothenate; C: pyridoxine; and D: thiamine). Rectangular filled with grey represents the gene present in the metabolic network of M. succiniciproducens. On the other hand, rectangular filled with white indicates the gene absent in the metabolic network of M. succiniciproducens. FIG. 4 is a graph showing the growth of Mannheimia cultured in a culture medium according to the present invention.
To predict components of a synthetic medium for growth of Mannheimia and the production of succinic acid, metabolic flux analysis method was used based on the constructed whole metabolic network of Mannheimia. If all metabolites, metabolic pathways and the stoichiometric matrix in the pathways (Sij T, metabolite i in the j reaction) are known, the metabolic flux vector (vj, flux of j pathway) can be calculated, in which a change in the metabolite X with time can be expressed as the sum of all metabolic fluxes. Assuming that a change in X with time is constant i.e., under the assumption of the quasi-steady state, the following equation is defined.
wherein ci is weighted value, and vi is metabolic flow. Generally, the maximization of biomass formation rate (i.e., specific growth rate), the maximization of metabolite production and the minimization of byproduct production, and the like, are used as the objective functions. αmax,i and αmin,i are limit values which each metabolic flux can have, and they can assign the maximum and minimum values permissible in each metabolic flux. Particularly, it has been reported that the case of the maximization of biomass formation is most similar to physiological phenomena which actual cell shows (Ibarra, R. U., Edwards, J. S., and Palsson, B. {acute over (�)}., 2002).
Synthesis through metabolic flux
through metabolic flux
through metabolic flow
Nicotinic acid metabloism
K2HPO4 8.709
g/L (50 mM)
g/L (100 mM)
A limited medium, a complex medium, a minimal synthetic medium and an optimal synthetic medium were prepared as follows: nutrients of the limited medium and the complex culture medium, which have the compositions and concentrations shown in Table 3 and Table 4 were dissolved in deionized distilled water, respectively. pH of the culture medium was adjusted to 7 using 5N NaOH solution. The prepared culture medium was put into glass Erlenmeyer flask and CO2 gas was injected into the flask for ten minutes and then, the culture medium was gas-tightly sealed with a cork. The limited medium and the complex medium into which CO2 gas was injected were pressurized and sterilized (121� C., 15 min), and they were cooled to room temperature and then used as culture media. The yeast extract added to the complex medium was Bacto™ Yeast Extract manufactured and sold by BacBecton, Dickinson and Company (Sparks, Md., USA).
The bacteria were cultivated in a test tub, a flask, and a fermentor. Test tube culture was performed as follows: 20 ml of the culture medium was transferred to 80 ml of test tube and then, 1 ml of Mannheimia succiniciproducens MBEL55E kept under −70� C. was inoculated into the test tube. In case of Mannheimia succiniciproducens MBEL55E kept in temperature below −70� C., the culture was performed in a complex medium. Accordingly, since yeast extracts used in the complex medium remained, washing was performed at the time of primary inoculation to remove them from Mannheimia succiniciproducens MBEL55E. Specifically, the inoculated solution was centrifuged at 5000 rpm, 4� C., for 5 min and the supernatant was removed and then washed using relevant medium. The washing was repeated three times. The culture was performed up to optical density (OD600) of 1.5 in a 39� C. incubator after CO2 gas was injected into the inoculated test tube. To enhance an adaptation applicable ability of cells to a new relevant culture medium and promote activities thereof, subculture was repeated ten times. After the culture broth was mixed with 30% (w/v) glycerol solution in a ratio of 1:1 (v/v) and kept below −70� C., it was used as inoculum in a further experiment.
Final succinic
acid conc. (g/L)
Optimal synthetic
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Biotechnol.", Apr. 1999, pp. 407-421, vol. 51, No. 4.Referenced byCiting PatentFiling datePublication dateApplicantTitleUS9037445 *Jul 10, 2008May 19, 2015University of Pittsburgh—of the Commonwealth System of Higher EducationFlux balance analysis with molecular crowdingUS20090061445 *Jul 10, 2008Mar 5, 2009Oltvai Zoltan NFlux balance analysis with molecular crowdingUS20130095566 *Apr 18, 2013University Of Pittsburgh - Of The Commonwealth System Of Higher EducationFlux Balance Analysis With Molecular Crowding* Cited by examinerClassifications U.S. Classification435/142, 703/11, 435/11, 435/253.6, 435/4, 435/161International ClassificationG06G7/48, C12N1/20, C12Q1/00, C12P7/44, C12Q1/68Cooperative ClassificationG06F19/12, C12N2500/38, C12N1/20, C12N5/0018, C12N2500/40, C12P7/46, C12N2500/32European ClassificationG06F19/12, C12N5/00M, C12N1/20, C12P7/46Legal EventsDateCodeEventDescriptionMar 5, 2014FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services