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Beta-alanine producing strain and preparation method and application thereof

A technique for producing alanine and bacteria, applied in the field of genetic engineering, can solve problems such as plasmid loss, limit product application, increase cost, etc., and achieve the effect of reducing production cost, increasing yield and sugar conversion rate

Active Publication Date: 2019-09-24
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the transformation of this strain with a plasmid, it is necessary to add corresponding antibiotics, otherwise it is easy to cause loss of the plasmid
This increases the cost of production to a certain extent and limits the application of the product

Method used

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  • Beta-alanine producing strain and preparation method and application thereof
  • Beta-alanine producing strain and preparation method and application thereof
  • Beta-alanine producing strain and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Preparation of the promoter mutant pET28am of pET28a(+)

[0040] The Escherichia coli competent cells used in this example are commercially purchased Escherichia coli DH5α, and the one-step directional cloning seamless cloning kit used in the recombination reaction was purchased from Novozyme, but not limited to this company. The sequencing reaction was sequenced at BGI, but not limited to this company.

[0041] (1) PCR amplification: using PpET28am-F / PpET28am-R as primers and plasmid pET28a (+) as template, PCR amplification obtained pET28am fragment, about 5300bp, and gel recovery.

[0042] (2) Recombination reaction: Use DpnI to process the fragments recovered from the gel to eliminate the original pET28a(+) plasmid that may be mixed in, and then use the one-step directional cloning seamless cloning kit to carry out the recombination reaction, and the reaction product is transformed into E. coli competent cells , spread on LB plates containing kanamyci...

Embodiment 2

[0044] Embodiment 2: Preparation of pET28am-gene plasmid

[0045]The Escherichia coli competent cells used in this example are commercially purchased Escherichia coli DH5α, and the one-step directional cloning seamless cloning kit used in the recombination reaction was purchased from Novozyme, but not limited to this company. The sequencing reaction was sequenced at BGI, but not limited to this company. The genes referred to here are exogenous genes ADC, AspDH and PC.

[0046] (1) Whole gene synthesis: According to the sequence (NO: 3) of the gene ADC (KY123117) derived from Bacillus tequilensis, the gene was synthesized and named BtADC, and added at both ends of the sequence Restriction sites of NdeI and HindIII; according to the sequence (NO: 4) of the gene PC (CP025534.1) derived from Corynebacterium glutamicum (Corynebacterium glutamicum), the gene was synthesized and named as CgPC, and in NdeI and HindIII restriction sites were added to both ends of the sequence; accord...

Embodiment 3

[0048] Embodiment 3: Preparation of pET28am-gene-Kana plasmid

[0049] The Escherichia coli competent cells used in this example are commercially purchased Escherichia coli DH5α, and the one-step directional cloning seamless cloning kit used in the recombination reaction was purchased from Novozyme, but not limited to this company. The sequencing reaction was sequenced at BGI, but not limited to this company. In this example, Kana fragments derived from plasmid pKD4 were respectively inserted downstream of the T7 terminator of the pET28am-gene vector obtained in Example 2. The genes referred to here are exogenous genes BtADC, PaeAspDH and CgPC.

[0050] (1) PCR amplification: with 28amF1 / 28amR1 as primers, respectively with the three plasmid vectors pET28am-BtADC, pET28am-CgPC, pET28am-PadAspDH obtained in Example 2 as templates, PCR amplification obtains the pET28am-BtADC fragment (about 5800bp), pET28am-CgPC fragment (about 8806bp), pET28am-PadAspDH fragment (about 6184bp)...

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Abstract

The invention discloses a beta-alanine producing strain and a preparation method and application thereof. The beta-alanine producing strain (Escherichia coli) has a preservation number of CGMCC NO: 17830. The beta-alanine producing strain constructed by the invention can realize the effective accumulation of the beta-alanine in the fermentation liquor in the fermentation process without adding aspartic acid in the fermentation process, and improves the yield of the beta-alanine and the sugar conversion rate. The yield of the beta-alanine is up to 50g / L, the sugar-acid conversion rate is up to 40%, and the producing strain has the application potential of large-scale production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a β-alanine genetic engineering production bacterium. Background technique [0002] Beta-alanine is the only beta-amino acid found so far in nature. It is a natural non-protein amino acid and is an important precursor of pantothenic acid in living organisms. β-alanine and its derivatives are widely used in medicine, food, chemical industry and other industries. In pharmaceutical applications, it is an intermediate in many B vitamin drugs. It can be used in the preparation of electroplating corrosion inhibitors in the chemical industry. Because of its ability to enhance muscle endurance, β-alanine is often added to some functional drinks. [0003] The main production methods of β-alanine are chemical method, enzymatic method and fermentation method. Chemical methods include acrylonitrile method, β-aminopropionitrile method, succinimide degradation metho...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/06C12R1/19
CPCC12N9/0006C12N9/88C12N9/1029C12N9/1217C12N9/0016C12N9/93C12N9/0022C12P13/06C12Y101/01001C12Y203/01008C12Y207/02004C12Y104/01002C12Y401/01031C12Y403/01001C12Y604/01001C12Y104/03015
Inventor 张娟冯志彬
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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