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Construction of threonine production strain and method for producing threonine

A construction method and threonine technology, which is applied in the construction of threonine production strains with high conversion rate and the field of threonine production, can solve the problems of multiple by-products, slow growth of strains, difficulty in obtaining high-yield strains, etc.

Pending Publication Date: 2021-12-28
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, traditional mutation breeding is difficult to obtain high-yield strains due to the slow growth of strains and the production of more by-products due to random mutations

Method used

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  • Construction of threonine production strain and method for producing threonine
  • Construction of threonine production strain and method for producing threonine
  • Construction of threonine production strain and method for producing threonine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Preparation of strain MHZ-0214-3 (promoter strengthening) that strengthens the mqo gene

[0037] (1) pTarget-tac-mqo plasmid construction

[0038] Step1: Using the pTargetT plasmid as a template (from the document Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, JiangY, Chen B, et al. Appl. Environ Microbiol, 2015), select the sgRNAmqotac-F / sgRNA-R primer pair, The sgRNA fragment with N20 was amplified①; Step2: Using the W3110 genome as a template, the mqotac-UF / mqotac-UR primer pair was selected to amplify the upstream homology arm containing the tac promoter②; Step3: Using the W3110 genome as a template As a template, select the mqotac-DF / mqotac-DR primer pair to amplify the downstream homology arm ③ containing the tac promoter; Step4: use ①, ②, ③ as templates, select the sgRNAmqotac-F / mqotac-DR primer pair, and amplify Add the sgRNA-up-Ptac-down fragment, also known as Donor DNA; Step5: Carry out SpeI and PstI double enzyme dig...

Embodiment 2

[0044] Embodiment 2: the bacterial strain MHZ-0214-4 (RBS-mqo) of the mqo gene that prepares RBS strengthening

[0045] (1) pTarget-RBS3-mqo plasmid construction

[0046] Step1: Using the pTargetT plasmid as a template (from the document Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, JiangY, Chen B, et al. Appl. Environ Microbiol, 2015), select the sgRNAmqoRBS-F / sgRNA-R primer pair, The sgRNA fragment with N20 was amplified ①; Step 2: Using the W3110 genome as a template, the mqoRBS-UF / mqoRBS-UR primer pair was selected to amplify the upstream homology arm containing RBS ②; Step 3: Using the fragment ② as a template, Use the mqoRBS-UF / mqoRBS-RBSR primer pair to amplify the upstream homology arm containing RBS③; Step4: Using the W3110 genome as a template, use the mqoRBS-DF / mqoRBS-DR primer pair to amplify the downstream homology arm containing RBS Source arm ④; Step5: ①, ③, ④ as the template, select the sgRNAmqoRBS-F / mqoRBS-DR primer pair, amplif...

Embodiment 3

[0053] Embodiment 3: prepare the bacterial strain MHZ-0214-5 (multiple copy) of strengthening mqo gene

[0054] (1) pTarget-zwf::mqo plasmid construction

[0055] Step1: Using the pTargetT plasmid as a template (from the literature Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, JiangY, Chen B, et al. Appl. Environ Microbiol, 2015), select the sgRNAmqo2-F / sgRNA-R primer pair to amplify Add the sgRNA fragment with N20①; Step2: Use the W3110 genome as a template, select the mqo2-UF / mqo2-UR primer pair, and amplify the upstream homology arm②; Step3: Use the W3110 genome as a template, select mqo-F / mqo-R primer pair to amplify the mqo fragment ③; Step4: using the W3110 genome as a template, select the mqo2-DF / mqo2-DR primer pair to amplify the downstream homology arm containing RBS ④; Step5: use ①, ②, ③, ④ are used as templates, and sgRNAmqo2-F / mqo2-DR primer pair is selected to amplify the sgRNA-up-mqo-down fragment, also called DonorDNA; Step6: Spe...

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Abstract

The invention relates to the field of microorganisms, in particular to construction of a threonine production strain with a high conversion rate and a method for producing threonine. L-threonine yield of novel escherichia coli is higher than that of respective control strains, and the shake-flask conversion rate of strains with enhanced mqo expression activity is 24-30%, which is increased by 6-12 conversion percentages compared with original strains; the shake-flask conversion rate of strains with enhanced cyoABCDE expression activity is 26-30%, which is also increased by 8-12 conversion percentages compared with the original strains. Results of shake flasks modified by the two sites show that the production capacity of threonine can be obviously improved by enhancing the expression of malic acid:quinone oxidoreductase gene mqo or enhancing the activity of oxidase at the tail end of a respiratory chain.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to the construction of high-conversion-rate threonine-producing strains and a method for producing threonine. Background technique [0002] L-threonine is one of the 8 kinds of amino acids necessary for the growth of humans and animals. It is widely used in feed, food additives and preparation of pharmaceutical auxiliary materials. At present, L-threonine is mainly produced by microbial fermentation, and various bacteria can be used for L-threonine production, such as wild-type induced mutants of Escherichia coli, Corynebacterium, Serratia, etc. as production strains. Specific examples include mutants resistant to amino acid analogues or various auxotrophs such as methionine, lysine, and isoleucine. However, traditional mutation breeding is difficult to obtain high-yield strains due to the slow growth of strains and the production of more by-products due to random mutations. [0003]...

Claims

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Application Information

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IPC IPC(8): C12P13/08C12N1/21C12N15/70C12N15/67C12N15/53C12R1/19
CPCC12P13/08C12N15/70C12N15/67C12N9/0006C12N9/0057C12Y101/05004
Inventor 刘慧敏康培尹春筱
Owner MEIHUA BIOTECH LANGFANG CO LTD
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