Recombinant corynebacterium glutamicum and method for producing L-threonine

A technology of Corynebacterium glutamicum and threonine, which is applied in the field of bioengineering and can solve the problems of few reports on the synthesis of oxaloacetate

Active Publication Date: 2021-08-31
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the research on the production of threonine by Corynebacterium glutamicum mainly focuses on the two aspects of the terminal pathway of threonine synthesis and the export of threonine, while the synthesis of threonine precursor oxaloacetate Less reported

Method used

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  • Recombinant corynebacterium glutamicum and method for producing L-threonine
  • Recombinant corynebacterium glutamicum and method for producing L-threonine
  • Recombinant corynebacterium glutamicum and method for producing L-threonine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Recombinant plasmid pK18mobsacB-P sod -lysC V1M-T311I Construction of and replacement in ATCC13032

[0070] (1) pK18mobsacB-P sod -lysC V1M-T311I Plasmid construction

[0071] Using the ATCC13032 genome as a template, the upstream homology arm up was obtained by PCR amplification with the P21 / P22 primer pair, and the promoter fragment P was obtained by PCR amplification with the P23 / P24 primer pair sod , PCR amplification with P25 / P26 primer pair to obtain lysC V1M-T311I , PCR amplification with 27 / 28 primer pair to obtain the downstream homology arm dn. Use the P21 / P24 primer pair to up, P sod Perform fusion PCR for the template to obtain the fragment up-P sod . Use the P21 / P28 primer pair to up-P sod , lysC V1M-T311I , dn as a template for fusion PCR to obtain the full-length fragment up-P sod -lysC V1M-T311I -dn. The full-length fragment was digested with BamHI, and pK18mobsacB was digested with the same enzyme. The two digested products were...

Embodiment 2

[0074] Example 2: Recombinant plasmid pK18mobsacB-P cspB -hom G378E Construction of and replacement in SMCT018

[0075] (1) pK18mobsacB-P cspB -hom G378E Plasmid construction

[0076] Using the ATCC13032 genome as a template, the upstream homology arm up was obtained by PCR amplification with the P29 / P30 primer pair, and the promoter fragment P was obtained by PCR amplification with the P31 / P32 primer pair cspB , using P33 / P34 primer pair to carry out PCR amplification to obtain hom G378E , using the P35 / P36 primer pair to perform PCR amplification to obtain the downstream homology arm dn. Use the P29 / P32 primer pair to up, P scpB Perform fusion PCR for the template to obtain the fragment up-P cspB . With P29 / P36 primer pair to up-P cspB 、hom G378E , dn as a template for fusion PCR to obtain the full-length fragment up-P cspB -hom G378E-dn. The full-length fragment was digested with BamHI, and pK18mobsacB was digested with the same enzyme. The two digested produc...

Embodiment 3

[0079] Example 3: Recombinant plasmid pK18mobsacB-P sod -thrC V1M Construction of and replacement in SMCT019

[0080] (1) pK18mobsacB-P sod -thrC V1M Plasmid construction

[0081] Using the ATCC13032 genome as a template, the upstream homology arm up was obtained by PCR amplification with the P37 / P38 primer pair, and the promoter fragment P was obtained by PCR amplification with the P39 / P40 primer pair sod -thrC V1M , dn was obtained by PCR amplification with P41 / P42 primer pair. Use the P37 / P42 primer pair to up, P sod -thrC V1M , dn as a template for fusion PCR to obtain the fragment up-P sod -thrC V1M -dn. The full-length fragment was digested with BamHI, and pK18mobsacB was digested with the same enzyme. The two digested products were connected with T4DNA Ligase, transformed into Trans1 T1 competent cells, and the recombinant plasmid pK18mobsacB-P was obtained sod -thrC V1M .

[0082] (2) ThrC replacement in SMCT019

[0083] SMCT019 competent cells were prep...

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Abstract

The invention relates to the technical field of bioengineering, in particular to recombinant corynebacterium glutamicum and a method for producing L-threonine. The corynebacterium glutamicum for producing threonine is constructed by combining and transforming (or enhancing) a hom gene, a lysC gene, a thrC gene and the like which are resistant to feedback inhibition, and (or) a weakening (or knockout) pck gene and (or) a weakening (or knockout) pyk gene, and the acid production effect is very remarkable and far exceeds the highest level which is reported at present and reaches 20g / l.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant Corynebacterium glutamicum and a method for producing L-threonine. Background technique [0002] L-Threonine (L-Threonine), the chemical name is β-hydroxy-α-aminobutyric acid, and the molecular formula is C 4 h 9 NO 3 , the relative molecular mass is 119.12. Threonine is white orthorhombic or crystalline powder. Odorless, slightly sweet taste. It melts and decomposes at 253°C. Soluble in water at high temperature, the solubility at 25°C is 20.5g / 100ml. Isoelectric point 5.6. Insoluble in ethanol, ether and chloroform. [0003] L-threonine is an essential amino acid. Threonine is mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc. In particular, the amount of feed additives is increasing rapidly. It is often added to the feed of immature piglets and poultry. It is the second limiting amino acid in pig feed and the third l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/08C12R1/15
CPCC12N9/2462C12N9/0006C12N9/1205C07K14/721C07K14/34C12P13/08C12Y302/01017C12Y101/01003C12Y207/0104
Inventor 康培刘慧敏吴涛李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD
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