Ketogulonigenium vulgare engineering strain, preparation method and application thereof

A technology of Bacillus ketoglobulina and engineering strains, applied in the field of bioengineering, can solve the problems of time-consuming and labor costs, and achieve the effects of saving time and cost, reducing consumption, reducing cost and pollution

Active Publication Date: 2016-10-12
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the time-consuming and labor cost of adding substances from external sources, and the concentration of the added substances is directly related to the cost of the enterprise, it has limitations in enterprise application

Method used

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  • Ketogulonigenium vulgare engineering strain, preparation method and application thereof
  • Ketogulonigenium vulgare engineering strain, preparation method and application thereof
  • Ketogulonigenium vulgare engineering strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Construction of strains

[0071] After genomic sequencing, it was found that many genes in the amino acid synthesis pathway of Ketogulonaccus were missing, including homoserine kinase, pyrroline-5-carboxylic acid reductase, histamine phosphatase, and K.Vulgare amino acid synthesis pathway gene deletion diagrams. figure 1 . In this example, the homoserine kinase gene, pyrroline-5-carboxylic acid reductase, and histamine phosphatase were introduced into Bacillus ketogulonium. See the construction ideas of strains expressing missing amino acid pathways figure 2 .

[0072] 1. Construction of pMCS2-Thr.g strain

[0073] Homoserine kinase (EC No. 2.7.1.39) is a key gene deletion in the Threonine pathway. When synthesizing the modified gene, select the source of Gluconobacter oxydans, and then pass the target gene shown in SEQ ID NO:1 through KpnI and HindIII Connected to the plasmid pBBR1MCS2, transferred into the large intestine for cloning and amplification, and obtai...

Embodiment 2

[0089] Example 2 Single bacteria shake flask fermentation test

[0090] The ketoguronic acid bacilli (pMCS2-Thr.g, pMCS2-Thr.s, pMCS2-Pro.g, pMCS2-Pro.s, pMCS2-His.b) containing the amino acid-encoding vector obtained in Example 1 were seed cultured , And then inoculated into the fermentation medium for fermentation, the specific operation is as follows:

[0091] 1) Method for cultivating seeds of ketoguronic acid bacteria:

[0092] Take 200 μL of the preserved glycerol bacteria on the resistant solid plate. The plate contains kanamycin with a concentration of 50 μg / mL. Slightly tilt and shake to cover the bacterial liquid evenly and completely on the solid plate, and incubate at 30°C for 24h. The original strain was cultured on a solid plate without resistance for 24 hours.

[0093] 2) Single bacteria shake flask fermentation

[0094] Add 200 μL of the original ketoguronic acid bacillus and different recombinant bacteria introduced with different amino acid modules to solid seed med...

Embodiment 3

[0099] Example 3 Fermentation test of mixed bacteria (modified bacteria with different amino acids and endophytic bacillus)

[0100] Add 200 μL of the original ketoguronic acid bacillus and the different recombinant bacteria with different amino acid modules obtained in Example 1 to the solid seed medium, culture at 30°C for 24 hours, then wash off the plate, and inoculate endophytic bacillus glycerol. 450μL in seed culture medium, cultivated at 30℃ for 12h, different amino acid modified bacteria were inserted into the fermentation medium with the same initial OD, and the initial OD600 of the initial inoculation of Bacillus was 1 / 2 of that of ketogulonobacter into the fermentation medium, 30 ℃, 250r / min, shake flask fermentation, sampling and analysis at different time points such as 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, etc., to detect the production and yield of ketogulonic acid (2-keto-L-gulonic acid) . The results are shown in Table 2, Table 3, Figure 5 , 6 .

[0101] Table...

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Abstract

The invention relates to the technical field of biological engineering, and especially relates to a ketogulonigenium vulgare engineering strain, a preparation method and an application thereof. The ketogulonigenium vulgare engineering strain comprises a key gene during an amino acid synthesis process; and the key gene in the amino acid synthesis process is in the key gene during a threonine synthesis process, the key gene during a proline synthesis process and the key gene during a histidine synthesis process. By constructing the deleted gene during the amino acid synthesis process in the bacterial strain, when individual fermentation of the ketogulonigenium vulgare engineering strain is carried out, sorbose can better conversed to ketogulonigenium vulgare; when ketogulonigenium vulgare engineering strain and the endophytic bacillus are subjected to mixed fermentation, the fermentation period is reduced, compared with the original bacterial strain, the period is shortened by about 10 h, the time cost is saved; from the yield aspect, the yield is increased by about 20% by comparing with the original strain; effect of companion fungus dependence is reduced when mixed fermentation is carried out, amount of the companion fungus which is Bacillus spp is reduced, and the cost and pollution are reduced.

Description

Technical field [0001] The invention relates to the technical field of bioengineering, in particular to an engineered strain of ketoguronic acid bacillus and a preparation method and application thereof. Background technique [0002] Vitamin C (English: Vitamin C, also known as L-ascorbic acid) is an acidic hexose derivative. It is a dilute hexose lactone. It is mainly sourced from fresh fruits and vegetables. It is a water-soluble substance necessary for the growth and metabolism of the body. Sex vitamins. Vitamin C has two isomers, L-type and D-type. Only the L-type has physiological functions. Both the reduced and oxidized types have physiological activities. In the organism, vitamin C forms antibodies and collagen, tissue repair (including some redox effects), metabolism of phenylalanine, tyrosine, and folic acid, utilization of iron and carbohydrates, synthesis of fats and proteins, and maintenance Necessary for immune function, hydroxylation and serotonin, to maintain the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P39/00C12P7/60C12P17/04C12R1/01
CPCC12N9/0028C12N9/1205C12N9/16C12P7/60C12P17/04C12P39/00C12Y105/01002C12Y207/01039C12Y301/03015
Inventor 潘才惠丁明珠王瑞钊元英进
Owner TIANJIN UNIV
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