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Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate

A ketoglutarate and co-expression technology, applied in the fields of fermentation engineering and enzyme engineering, can solve the problems of multiple separations and purifications, troubled costs, etc., and achieve the effect of high-efficiency expression

Active Publication Date: 2016-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, catalase needs to be added during the enzymatic conversion process, and the industrial catalase contains more impurities, which will cause troubles for the separation and purification of α-KG products in the later stage, and the cost of adding catalase from external sources will be higher

Method used

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  • Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate
  • Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate
  • Construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1L-glutamic acid oxidase property research and determination of catalase demand

[0051] The L-glutamate oxidase (LGOX) gene derived from StreptomycesghanaensisATCC14672 (the amino acid sequence is shown in SEQ ID NO.1, the nucleotide sequence is shown in SEQ ID NO.2), and the gene derived from E.coli K12 Catalase gene KatG (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4), through primer 1 (upstream primer: 5'-CATGCCATGGCAATGCTGCCCGCACCGGCCGCCT-3', sequence as in SEQ ID NO.4 ID NO.8; downstream primer: 5'-CCCAAGCTTGTCACGCTGTGTGGATCTCCAAG-3', sequence such as SEQ ID NO.9) and primer 2 (upstream primer: 5'-CATGCCATGGCAATGAGCACGTCAGACGAT-3', sequence such as SEQ ID NO.10; downstream primer: 5 '-CCC AAGCTTG CAGCAGGTCGAAACGGTC-3', sequence such as SEQ ID NO.11) was cloned and expressed in plasmid pET28a, the recombinant plasmid was transferred into E.coli BL21(DE3), and the positive strains were screened and named FXC001 an...

Embodiment 2

[0054] Example 2 Dual-enzyme co-expression strain transcription level regulation

[0055] Using pET28a as the expression vector, three different strategies were set up to construct co-expression strains. Strategy 1 used a single promoter mode to express LGOX and KatG in tandem, and connected KatG through the same RBS sequence as before LGOX, and constructed a successful co-expression strain named F001 ;Strategy 2 adopts the double promoter mode and adds the same promoter and related sequences in front of LGOX and KatG to construct a co-expression strain named F002; Strategy 3 adopts the single promoter mode to directly connect LGOX and KatG through Hind III to construct The successful co-expression strain was named F003. Among them, strategy 1 will transcribe and produce one mRNA, which contains two nucleotide binding sites, and translate two proteins; strategy 2 will transcribe two mRNAs, one large and one small, both containing nucleotide binding sites, and translate two pro...

Embodiment 3

[0059] The influence of embodiment 3SD sequence (Shine-Dalgarno sequence) and ATG interval

[0060] according to Figure 6 Plasmid construction methods to construct recombinant plasmids (wherein RBS* represents the sequence of different SD and ATG intervals), the set intervals are 3bp, 6bp, 9bp and 12bp respectively, and the upstream primers are KatG-rbs1 (sequence such as SEQ ID NO.12), KatG-rbs2 (sequence such as SEQ ID NO.13), KatG-rbs3 (sequence such as SEQ ID NO.14) and KatG-rbs4 (sequence such as SEQ ID NO.15), the downstream primer is KatG-A (sequence such as SEQ ID NO.16) (Table 2), the recombinant strains were constructed and verified by traditional construction methods, and the successfully constructed and verified strains were named FXC003, FXC004, and FXC005 (that is, L-glutamic acid oxidase and catalase passed through The connection sequence of SEQ ID NO.5 was connected), FXC006.

[0061] Table 2 The primers used in the present invention

[0062]

[0063] Fo...

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Abstract

The invention discloses a construction method of dual-enzyme co-expression strains for producing [alpha]-ketoglutarate, and belongs to the technical fields of fermentation engineering and enzyme engineering. According to the construction method, on the basis of analyzing the production of the [alpha]-ketoglutarate from L-glutamic acid, a KatG dosage required by a transformation system is analyzed; and then, the co-enzyme co-expression strains, which are different in expression level, are constructed at a transcription level and a translation level, and the performances of the co-expression strains are evaluated by the production of the [alpha]-ketoglutarate through whole-cell transformation of L-glutamic acid at a shaker level, wherein transformation conditions are as follows: 110g / L of the L-glutamic acid, 2-2.5g / L of bacteria, a phosphate buffer system with pH value at 6.5, 30 DEG C, and 18-24h at 200rpm. The yield of the [alpha]-ketoglutarate from the optimum strain F006 can reach 107.2g / L or above, with a transformation rate of 98.4%, completely replacing exogenous catalase; bacterium concentration is increased, the yield of the [alpha]-ketoglutarate under the condition of 132g / L of a substrate can reach 127.1g / L and a transformation rate is 96.9%.

Description

technical field [0001] The invention relates to a method for constructing a dual-enzyme co-expression strain producing α-ketoglutarate, belonging to the technical fields of fermentation engineering and enzyme engineering. Background technique [0002] α-ketoglutarate (α-KG for short), as an important high-value fine chemical (200,000-250,000 yuan / ton), is widely used in industrial fields such as food, medicine, chemical industry and cosmetics. . In the field of medicine, α-KG can reduce the burden on the kidneys of patients with kidney disease, reduce complications, and promote rapid recovery after surgery; compounded with amino acids such as arginine, it can quickly help athletes replenish energy, and is widely used in functional nutritional enhancement In addition, due to the special chemical properties of α-KG, it is widely used in the chemical synthesis industry. With the continuous expansion of the application field of α-KG, the demand for α-KG in the domestic and int...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C12N15/53C12N1/21C12P7/50
CPCC12N9/0022C12N9/0065C12P7/50C12Y104/03007C12Y111/01006
Inventor 刘立明樊祥臣刘佳林小宝
Owner JIANGNAN UNIV
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