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Method for producing L-ornithine by using mixed sugar raw material in double-bacterium co-culture system

A co-cultivation system, ornithine technology, applied in the field of genetic engineering and biology, to achieve the effect of expanding the substrate spectrum, reducing production costs and optimizing production

Pending Publication Date: 2022-08-05
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on the construction of xylose and arabinose metabolic pathways for Corynebacterium glutamicum to produce L-ornithine by double-bacteria co-cultivation using multiple carbon sources. - The method of ornithine is of great significance to the L-ornithine production industry

Method used

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  • Method for producing L-ornithine by using mixed sugar raw material in double-bacterium co-culture system
  • Method for producing L-ornithine by using mixed sugar raw material in double-bacterium co-culture system
  • Method for producing L-ornithine by using mixed sugar raw material in double-bacterium co-culture system

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Xylose Utilizing Strain Corynebacterium glutamicum Construction of GX01

[0040] (1) Construction of integrated plasmid: Escherichia coli Escherichia coli The MG1655 genome was used as a template and upstream primers were used xyl AB-F: atcgaaaatgcaagcctattttgaccagct (SEQ. No. 1) and downstream primers xyl AB-R: ccaaagatttacgccattaatggcagaagttgc (SEQ.No.2) PCR amplification xyl AB fragment (such as SEQ.No.3 in the Sequence Listing xyl AB sequence shown). The PCR conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 3 min, final extension at 72°C for 5 min, and storage at 4°C for 59 min; the number of cycles was 30 cycles.

[0041] Corynebacterium glutamicum C. glutamicum jp-07092 genome as template, using primers ldh A-up-F1: cggtacccggggatcggaacaccatgcgattaaggtgc (SEQ. No. 4) and ldh A-up-R1: PCR amplification of gcttgcattttcgatcccacttcctgattt cc (SEQ.No.5) ldh...

Embodiment 2

[0050] Example 2: Arabinose Utilizing Strain Corynebacterium glutamicum Construction of GA01

[0051](1) Construction of integrated plasmid: Escherichia coli Escherichia coli The MG1655 genome was used as a template and upstream primers were used ara BAD-F: atcgaaaatggcgattgcaattggcc (SEQ. No. 10) and downstream primers ara BAD-R: ccaaagatttactgcccgtaatatgccttcgc (SEQ.No.11) amplified by PCR ara BAD fragment (such as SEQ.No.12 in the Sequence Listing ara BAD sequence shown). The PCR conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 5 min, final extension at 72°C for 5 min, and storage at 4°C for 59 min; the number of cycles was 30 cycles.

[0052] Corynebacterium glutamicum Corynebacterium glutamicum The jp-07092 genome was used as a template, using ldh A-up-F2: cggtacccgg ggatcggaac accatgcgat taaggtgc (SEQ. No. 13) and ldh A-up-R2: atcgccattt tcgatcccac ttcctgatt (SEQ.No.14) a...

Embodiment 3

[0061] Example 3: A Utilization Corynebacterium glutamicum GX01 and Corynebacterium glutamicum Method for producing L-ornithine by using mixed sugar raw material in GA01 double bacteria co-cultivation system

[0062] (1) Construction of Corynebacterium glutamicum Corynebacterium glutamicum GX01 and Corynebacterium glutamicum Dual bacteria co-culture system of GA01

[0063] Prepare LB solid medium: tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L, sterilize at 121 °C for 20 min.

[0064] Preparation of seed liquid medium: glucose 20 g / L, K 2 HPO 4 ·3H 2 O 1.5 g / L, KH 2 PO 4 0.5 g / L, MgSO 4 ·7H 2 O 0.4 g / L, urea 2.5 g / L, MnSO 4 ·H 2 O 0.02 g / L, FeSO 4 ·7H 2 O 0.02 g / L, Biotin 0.1 mg / L, VB 1 0.1 mg / L, arginine 0.02 g / L, with H 3 PO 4 Adjust pH to 7.0 and sterilize at 110 °C for 10 min.

[0065] The formula for preparing the co-culture fermentation medium is: glucose 20 g / L, arabinose 20 g / L, xylose 10 g / L, K 2 HPO 4 ·3...

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Abstract

The invention discloses a method for producing L-ornithine by using a mixed sugar raw material in a double-bacterium co-culture system. The method comprises the following steps of: constructing a xylose utilizing strain Cornebacterium glutamicum GX01 and an arabinose utilizing strain Cornebacterium glutamicum GA01 by taking an ornithine high-yield strain Cornebacterium glutamicum jp-07092 as an original strain; l-ornithine is produced by fermentation of a double-bacterium co-culture system. According to the method disclosed by the invention, xylose and arabinose can be utilized, so that ornithine can be produced, and meanwhile, the problem of high fermentation cost caused by only utilization of single glucose is solved to a certain extent; through construction of a double-bacterium co-culture system, corynebacterium glutamicum can produce ornithine by using complex carbon sources of xylose, arabinose and glucose, and environmental pollution and resource waste are reduced.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biotechnology, and in particular relates to a method for producing L-ornithine by using a mixed sugar raw material in a double bacteria co-cultivation system. Background technique [0002] L-ornithine is widely present in various tissues and cells of organisms. It is an important intermediate product in the arginine synthesis pathway. It participates in the urea cycle (also known as ornithine cycle), and is also involved in the metabolism of proline and polyamine. key intermediate metabolites. As a non-protein amino acid, L-ornithine plays a key role in regulating cellular nitrogen metabolism. It has been used for the prevention and treatment of liver metabolism-related diseases, and has attracted wide attention in the pharmaceutical and nutritional chemicals market. The preparation methods of L-ornithine are mainly divided into four categories: natural product extraction, chemical synthes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12N15/61C12N15/54C12P13/10C12R1/15
CPCC12P13/10C12N9/92C12N9/1205C12N9/90C12N15/52C12N15/77C12Y503/01005C12Y207/01017C12Y503/01004C12Y207/01015C12Y501/03004C12R2001/15Y02E50/10
Inventor 郝宁史靓刘兆星唐渝舒刘平郭格格李涛
Owner NANJING UNIV OF TECH
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