# US 5,705,371 A · Bacterial strain of escherichia coli BKIIM B-3996 as the producer of L-threonine

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- Number: US 5,705,371 A
- Document ID: US5705371A
- Status: Granted
- Kind: A
- Application number: 08/633,028
- Filed: 1996-04-16
- Granted: 1998-01-06
- Earliest claimed: 1990-06-12
- Assignees: Ajinomoto Co., Inc.
- Inventors: Vladimir Georgievich Debabov; Jury Ivancvich Kozlov; Evgeny Moiseevich Khurges; Vitaly Arkadievich Livshits; Nelli Isaakcvna Zhdanova; Mikhail Markovich Gusyatiner; Alexandr Konstantinovich Sokolov; Tatyana Alexandrovna Bachina; Nikolai Kazimirovich Yankovsky; Jury Dmitrievich Tsygankov; Andrei Jurievich Chistoserdov; Tatyana Grigorievna Plotnikova; Irina Clegovna Shakalis; Alla Valentinovna Belareva; Raisa Alexandrovna Arsatiants; Albert Fedorovich Sholin; Tamara Mikhailcvna Pozdnyakova
- Web page: https://patentgrep.com/patent/US5705371A

## CPC

- C12P 13/08: Lysine; Diaminopimelic acid; Threonine; Valine
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A bacterial strain of Escherichia coli BKIIM B-3996, a producer of L-threonine, containing a recombinant plasmid pVIC40 and deposited on Nov. 19, 1987 in the collection of microorganism cultures at the USSR Antibiotics Research Institute under Reg. No. 1867.

## Claims

### Claim 1

An E. coli strain which produces L-threonine, derived by a process comprising the steps of:
(i) transducing E. coli strain M1 with a first bacteriophage P1 bearing genetic material necessary for saccharose assimilation, and
(ii) isolating spontaneously occuring, threonine resistant a transductant capable of utilizing saccharose or a saccharose-bearing substrate as a source of carbon.

### Claim 2 (depends on 1)

An E. coli strain derived from the E. coli strain of claim 1 by a process comprising the steps of:
(iii) transducing the E. coli strain of claim 1 with a second bacteriophage P1 which bears a transposon which inactivates threonine dehydrogenase activity, and
(iv) isolating a transductant lacking threonine dehydrogenase activity.

### Claim 3 (depends on 2)

The E. coli strain of claim 2, wherein said second bacteriophage P1 was obtained by growing bacteriophage P1 on a mutant E. coli strain devoid of threonine dehydrogenase activity and bearing a mutation in the threonine dehydrogenase-encoding gene.

### Claim 4 (depends on 2)

The E. coli strain of claim 2 without the plasmid pYN7.

### Claim 5

The E. coli strain VNIIgenetika TDH-6.

### Claim 6

A method for deriving an E. coli strain which produces L-threonine, said method comprising the steps of:
(i) transducing E. coli strain M1 with a first bacteriophage P1 bearing genetic material necessary for saccharose assimilation, and
(ii) isolating a spontaneously occurring, threonine resistant transductant capable of utilizing saccharose or a saccharose-bearing substrate as a source of carbon.

### Claim 7 (depends on 6)

The method of claim 6, further comprising the steps of:
(iii) transducing said transductant with a second bacteriophage P1 which bears a transposon which inactivates threonine dehydrogenase activity, and
(iv) isolating a transductant lacking threonine dehydrogenase activity.

### Claim 8 (depends on 7)

The method of claim 7, wherein said second bacteriophage P1 was obtained by growing bacteriophage P1 on a mutant E. coli strain devoid of threonine dehydrogenase activity and bearing a mutation in the threonine dehydrogenase-encoding gene.

### Claim 9 (depends on 7)

The method of claim 7 wherein said transductant lacking threonine dehydrogenase activity does not contain the plasmid pYN7.

### Claim 10 (depends on 7)

The method of claim 7, wherein said transductant lacking threonine dehydrogenase activity is E. coli strain VNIIgenetika TDH-6.

### Claim 11 (depends on 6)

The method of claim 6, wherein said E. coli strain which produces L-threonine is E. coli strain VNIIgenetika 472T23.

### Claim 12 (depends on 6)

The method of claim 6, wherein said E. coli strain which produces L-threonine is an E. coli strain VNIIgenetika 472T23 without the plasmid pYN7.

### Claim 13 (depends on 6)

A process for producing L-threonine in an E coli strain derived by the method of claim 6, said process comprising the steps of culturing said E. coli strain under conditions for production of L-threonine and isolating L-threonine from the culture.

### Claim 14 (depends on 7)

A process for producing L-threonine in an E coli strain derived by the method of claim 7, said process comprising the steps of culturing said E. coli strain under conditions for production of L-threonine and isolating L-threonine from the culture.

### Claim 15 (depends on 8)

A process for producing L-threonine in an E coli strain derived by the method of claim 8, said process comprising the steps of culturing said E. coli strain under conditions for production of L-threonine and isolating L-threonine from the culture.

### Claim 16 (depends on 14)

The process of claim 14 wherein the E. coli strain does not contain the plasmid pYN7.

### Claim 17 (depends on 14)

The process of claim 14, wherein said E. coli strain is E. coli strain VNIIgenetika TDH-6.

### Claim 18 (depends on 13)

The process of claim 13, wherein said E. coli strain is E coli strain VNIIgenetika 472T23.

### Claim 19 (depends on 13)

The process of claim 13, wherein said E. coli strain is E. coli strain VNIIgenetika 472T23 without the plasmid pYN7.

### Claim 20

The plasmid pVIC40.