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A kind of engineering strain of ketoglobulina and its preparation method and application

A technology of ketogulonic acid bacteria and engineering strains, applied in the field of bioengineering, can solve problems such as time-consuming and labor costs, and achieve the effects of saving time costs, reducing fermentation cycles, and reducing costs and pollution

Active Publication Date: 2020-03-17
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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  • A kind of engineering strain of ketoglobulina and its preparation method and application
  • A kind of engineering strain of ketoglobulina and its preparation method and application
  • A kind of engineering strain of ketoglobulina and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0070] The construction of embodiment 1 bacterial classification

[0071] After genome sequencing, it was found that many genes in the amino acid synthesis pathway of Bacillus ketoglobulina were missing, including homoserine kinase, pyrroline-5-carboxylate reductase, histidinol phosphatase, K.Vulgare amino acid synthesis pathway gene deletion diagram figure 1 . In this example, homoserine kinase gene, pyrroline-5-carboxylate reductase, and histidinol phosphatase were introduced into Bacillus ketoglobulina. See figure 2 .

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

[0073] Homoserine kinase (EC number is 2.7.1.39) is the key gene for threonine (Threonine) path deletion. When synthesizing and transforming the gene, select the source of Gluconobacter oxydans, and then pass the target gene whose sequence is shown in SEQ ID NO: 1 through KpnI and HindIII was connected to the plasmid pBBR1MCS2, and then transformed into E. coli for cloning and amplification to obtain E. col...

Embodiment 2

[0089] Embodiment 2 Single bacteria shake flask fermentation test

[0090] The ketogalactobacillus (pMCS2-Thr.g, pMCS2-Thr.s, pMCS2-Pro.g, pMCS2-Pro.s, pMCS2-His.b) containing the encoding amino acid carrier obtained in Example 1 is carried out seed culture , and then inoculated into the fermentation medium for fermentation, the specific operations are as follows:

[0091] 1) Bacteria ketoglobulina seed cultivation method:

[0092] Take 200 μL of the preserved glycerol bacteria on the resistant solid plate, the resistance contained in the plate is kanamycin, the concentration is 50 μg / mL. Slightly tilted and shaken to make the bacterial solution evenly and completely cover the solid plate, and cultivated at 30°C for 24h. The original strain was cultured on solid plates without resistance for 24 hours.

[0093] 2) single bacteria shake flask fermentation

[0094] Add 200 μL of the original Bacillus ketogalonicum and different recombinant bacteria introduced with different a...

Embodiment 3

[0099] Embodiment 3 mixed bacteria (different amino acid modified bacteria and endophytic bacillus) fermentation test

[0100] Add 200 μL of the original Bacillus ketogalonicum and the different recombinant bacteria introduced into 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 with the endophytic Bacillus glycerol 450 μL was placed in the seed medium, and cultured at 30°C for 12 hours. Different amino acid modified bacteria were inserted into the fermentation medium with the same initial OD. ℃, 250r / min for shake flask fermentation, 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and other different time points for sampling and analysis to detect the output and yield of ketogulonic acid (2-keto-L-gulonic acid) . The results are shown in Table 2, Table 3, Figure 5 , 6 .

[0101] The output (g / l) of the ketogulonic acid (2-KGA) after table 2 different bacterial strain fermentation cult...

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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 engineering strain of Bacillus ketoclonicus and its preparation method and application. Background technique [0002] Vitamin C (English: Vitamin C, also known as L-ascorbic acid) is an acidic hexose derivative, which is a dilute alcohol type hexonolactone. It is mainly derived from fresh fruits and vegetables and is a water-soluble substance necessary for maintaining 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, and both the reduced and oxidized forms have physiological activities. In organisms, vitamin C is for the formation of antibodies and collagen, tissue repair (including some redox effects), metabolism of phenylalanine, tyrosine, and folic acid, utilization of iron and carbohydrates, synthesis of fat and protein, and maintenance Necessary for immune function, hydr...

Claims

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Application Information

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