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Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same

a technology of o-phosphoserine and microorganisms, which is applied in the direction of microorganisms, enzymology, transferases, etc., can solve the problems of difficult industrial application of the conversion process, strong user aversion, and environmental pollution, and achieves high yield and higher production efficiency

Inactive Publication Date: 2012-07-26
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0010]The method of the present invention in which O-phosphoserine is produced at high yield by a recombinant microorganism and is used for conversion into cysteine, as it is, is more friendly to the environment and ensures higher efficiency in the production of cysteine than do chemical synthesis methods. The cysteine and its derivatives produced by the fermentation and bioconversion of the present invention can be widely used in the production of animal and human foods and food additives.

Problems solved by technology

However, not only does the production of cysteine from hairs or feathers ensure a yield of as low as 7˜8%, but also the use of hydrochloric acid or sulfuric acid produces a lot of waste resulting in environmental pollution.
Further, extraction from hairs or feathers may induce the user to have a strong aversion thereto.
This conversion process is, however, difficult to apply industrially due to the low solubility of the precursor D,L-ATC.
Excessive accumulation of L-cysteine within microorganisms incurs intracellular toxicity, exhibiting a limitation in the production of L-cysteine at a high concentration.
To overcome this drawback, L-cysteine-exporting proteins are employed, but there have been no significant improvements in productivity.

Method used

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  • Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same
  • Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same
  • Microorganism producing o-phosphoserine and method of producing l-cysteine or derivatives thereof from o-phosphoserine using the same

Examples

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example 1

Preparation of Phosphoserine Phosphatase (serB) Deficient Corynebacterium Strain

[0048]Corynebacterium glutamicum 13032 was modified by deleting the serB gene (SEQ ID NO: 7, EC 3.1.3.3) encoding phosphoserine phosphatase, which catalyses the synthesis of L-serine from O-phosphoserine, therefrom. To this end, a fragment for inactivation of serB was constructed. In this regard, primers were designed for the preparation of the recombinant strain 13032-ΔserB of the present invention. First, the serB sequence of Corynebacterium glutamicum 13032 was obtained with reference to the data of the NIH GenBank, and primers SEQ ID NOS: 9 to 14 were synthesized on the basis of the serB sequence. For the site-specific gene disruption, a pDC vector which cannot replicate in Corynebacterium glutamicum was employed. A pDC-ΔserB plasmid in which the open reading frame of serB was internally disrupted was constructed and adopted for the preparation of a site-specific serB gene deletion in Corynebacterium...

example 2

Assay for O-Phosphoserine Productivity in the Phosphoserine Phosphatase Deficient Corynebacterium Strain

[0050]The mutant strain CB01-0047, resulting from the deletion of serB from Corynebacterium glutamicum 13032, which was anticipated to accumulate O-phosphoserine, was spread over BHIS plates and incubated overnight in a 30° C. incubator.

[0051]Afterwards, the colonies appearing on the BHIS plates were inoculated in 25 mL of a titer medium shown in Table 1 using a platinum loop and then incubated at 30° C. for 48 hours with shaking at 200 rpm. The results are summarized in Table 2, below.

TABLE 1CompositionAmount (per liter)Glucose100gKH2PO41.1g(NH4)2SO445gMgSO4•7H2O1.2gHSM20gTrace elements20mlCalcium carbonate30gpH7.2Trace elementsBiotin0.09gThiamine0.45gCa-Panthenate0.45gNCA3gFeSO4•7H2O9gMnSO4•4H2O9gZnSO4•7H2O0.045gCuSO4•5H2O0.045g

TABLE 2SugarO-phosphoserineStrainOD 562 nmconsumed (g / L)(g / L)C. glutamicum251000.0213032CB01-00476.5230.07

[0052]The CB01-0047 strain was observed to grow...

example 3

Preparation of E. coli Strain Having the Reduced Activity of Phosphoserine Phosphatase (SerB)

[0053]E. coli was modified by deleting the serB gene (SEQ ID NO: 8) encoding phosphoserine phosphatase, which catalyses the synthesis of L-serine from O-phosphoserine, therefrom. The deletion mutant E. coli K12 was prepared using the one-step inactivation method (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000) to delete an antibiotic-resistant maker gene. To prepare the serB deletion strain, first, PCR was performed on a pKD3 plasmid (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000; GenBank No. AY048742) using a pair of primers of SEQ ID NOS: 15 and 16. The PCR product was (introduced into competent cells of pKD46 containing E. coli K12 (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000; GenBank No. AY048746) by electroporation. Thereafter, strains that showed resistance to chloramphenicol were subjected to PCR to conf...

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Abstract

The present invention provides methods for the production of cysteine or derivates thereof by culturing a microorganism having reduced activity of endogenous phosphoserine phosphatase. The O-phosphoserine produced by such an organism can then be reacted with a sulfide in the presence of a sulfydrylase or a microorganism expressing a sulfhydrylase to produce cysteine or a derivative thereof. Microorganisms having the properties noted above are also provided herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the priority benefit of Korean Patent Application Nos. 10-2011-0086081, filed Aug. 26, 2011 and 10-2010-0102664, filed Oct. 20, 2010. The contents of these patent applications are incorporated herein by reference in their entireties.STATEMENT REGARDING SEQUENCE LISTING[0002]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is HANO—004—00US_ST25.txt. The text file is 28 KB, was created on Oct. 20, 2011, and is being submitted electronically via EFS-Web.TECHNICAL FIELD[0003]The present invention relates to a method for production of cysteine or its derivatives using O-phosphoserine as an intermediate and recombinant microorganism for use in production of O-phosphoserine.BACKGROUND ART[0004]L-cysteine is an amino acid that plays an important r...

Claims

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Application Information

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IPC IPC(8): C12P13/12C12N1/21
CPCC12N9/1085C12N9/16C12Y301/03003C12P13/12C12Y205/01065C12P13/06C12N1/20C12N15/09C12N15/52C12N15/63C12N15/70C12N15/77
Inventor CHANG, JIN SOOKJO, JAE HYUNBAE, HYUN AESONG, BYEONG CHEOLKIM, SOLKIM, HYE WON
Owner CJ CHEILJEDANG CORP
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