A kind of L-carnitine-producing Escherichia coli genetically engineered bacteria and its construction method and application

A technology of genetically engineered bacteria and Escherichia coli, applied in the field of bioengineering, can solve the problems of high production cost, intolerance to croton betaine, and failure to reach the level of industrial production, and achieve the effect of simple control and low production cost

Active Publication Date: 2018-06-12
武汉中科光谷绿色生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN101014709A uses γ-butyl betaine hydroxylase derived from Neurospora crassa, and uses γ-butyl betaine as a substrate to carry out enzyme conversion to produce L-carnitine. The method involves many enzyme purification steps, and the enzyme conversion reaction is assisted There are many factors and the production cost is too high
The conversion method of croton betaine as substrate has its unique advantages, the substrate is cheap, and can be prepared by dehydration by splitting waste D-carnitine. resistant strains
EP0457735A1 uses the screened Proteus mirabilis to transform croton betaine with a substrate concentration of 12% to produce L-carnitine, with a molar conversion rate of 40-43%, and the conversion rate needs to be improved
The conversion of croton betaine by whole-cell enzymes can overcome the high-concentration intolerance of the bacterial strain to the substrate. For example, CN96117166.9 uses an ultraviolet mutagenic strain of Escherichia coli to convert croton betaine, and the L-carnitine output is up to 15-20g. / L, the conversion rate is 40-50%, this value has not yet reached the level of industrial production, and the output and conversion rate need to be improved

Method used

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  • A kind of L-carnitine-producing Escherichia coli genetically engineered bacteria and its construction method and application
  • A kind of L-carnitine-producing Escherichia coli genetically engineered bacteria and its construction method and application
  • A kind of L-carnitine-producing Escherichia coli genetically engineered bacteria and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Construction of Escherichia coli genetic engineering bacteria producing L-carnitine

[0033] (1) According to the caiTABCDE operon in the genome sequence of Escherichia coli, design forward primer F-caiBCD-NdeI:5'-GGGCATATGGATCATCTACCCATGCC-3 (SEQ ID NO.1)' and reverse primer R-caiBCD-KpnI:5 '-ATTGGTACCCAACCGTGAGCTATTACGCC-3' (SEQ ID NO.2) or R-caiBCD-XhoI:5'-ATTCTCGAGCAACCGTGAGCTATTACGCC-3' (SEQ ID NO.9), NdeI and KpnI restriction sites (underlined) were added to both ends of the primer.

[0034] Forward primer F-caiF-NdeI:5'-CACGCCCAT ATGTGTGAAGGATATGTTGAAAAAC-3'(SEQ ID NO.3) and reverse primer R-caiF-xhoI:5'-CAGCTCGAGTTAACGACGCATACTCTT TGACAA-3'(SEQ ID NO.4) of CaiF ). Forward primer F-caiT-EcoRI of caiT: 5'-GGGGAATTCATGAAGAATGAAAAGAGAAAAACGG-3' (SEQ ID NO.5), reverse primer R-caiT-HindIII: 5'-GCCAAGCTTTTAATCTTTCCAGTTCTGTTTCGCG-3' (SEQ ID NO.6).

[0035] (2) Using Escherichia coli genomic DNA as a template, using TaKaRa’s DNA polymerase primestar and designed pr...

Embodiment 2

[0052] 1. Construction of Escherichia coli genetic engineering bacteria producing L-carnitine

[0053] Step 1: Amplify the caiBCD fragment from the genome of Escherichia coli wild strain BW25113, the primer design is forward: 5'-GGGCATATGGATCATCTACCCATGCC-3' (SEQ ID NO.1); the primer design is reverse: 5'-ATTCTCGAGCAACCGTGAGCTATTACGCC-3 '(SEQ ID NO.9); Step 2: select the plasmid pET28a of an IPTG-induced promoter as a carrier, insert the caiBCD fragment at the NdeI / XhoI restriction site of the multi-cloning site, and obtain the overexpression plasmid pET28a-caiBCD; step Three: Transfer the overexpression plasmid pET-caiBCD into the E. coli expression strain BL21(DE3) to obtain the genetically engineered bacteria BL21(DE3) / pET28a-caiBCD; Step four: Extend the genes caiF and caiT, caiF from E. coli W3110 The forward and reverse primers were: 5'-CACGCCCAT ATGTGTGAAGGATATGTTGAAAAAC-3'(SEQ NO.3) and 5'-CAGCTCGAGTTAACGACGCATACTCTTTGACAA-3'(SEQ NO.4). The forward and reverse primers...

Embodiment 3

[0063] 1. Construction of Escherichia coli genetic engineering bacteria producing L-carnitine

[0064] Same as step 1 of Example 2 above.

[0065] 2. Preparation of the whole cell enzyme source of Escherichia coli genetically engineered bacteria BL21(DE3) / (pET28a-caiBCD+pACYCD-caiT-caiF)

[0066] (1) Preparation of enzyme production medium: glycerol 10ml / L, peptone 5g / L, yeast powder 15g / L, KH 2 PO 4 5g / L, K 2 HPO 4 5g / L, sodium fumarate 4g / L and croton betaine 4g / L, pH 6. Sterilize.

[0067] (2) The inoculum size is 1% (V / V). After the Escherichia coli genetically engineered bacteria are gradually activated and cultured, they are placed in a container equipped with an enzyme-producing medium, and 1 mM IPTG is added for aerobic culture at 37° C. for 6 hours.

[0068] (3) Centrifuge at 10° C., 8000 rpm, and collect the bacterial cells. The cells were washed twice with 67 mmol / l phosphate buffer at pH 7.4.

[0069] 3. Whole-cell enzymatic conversion of croton betaine to ...

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Abstract

The invention relates to a construction method of escherichia-coli gene engineering bacterium generating L-carnitine and an application. The gene engineering bacterium is characterized by converting encoded crotonbetaine into three key genes of the L-carnitine, namely caiB, caiC and caiD, transferring the three key genes, a transporter encoding gene caiT and a positive regulator caiF into escherichia coli for overexpression, controlling by an anaerobic promoter or adopting IPTG to induce expression of related genes for synthesis of the L-carnitine, and simultaneously deleting aceK gene for encoding isocitrate dehydrogenase phosphatase / kinase of the escherichia coli. After the gene enginering bacterium is cultured by production enzyme, and thalli are collected as a whole-cell enzyme source; the crotonbetaine is directly converted to generate the L-carnitine, after conversion, the yield of the L-carnitine can reach more than 30g / L, the molar conversion rate is 42% at highest, the yield and the conversion rate are increased by more than 60 times than those of wild plants, and the construction method reaches the leading level of biological-process preparation reported domestically. The gene engineering bacterium has a high conversion rate for substrate, the enzyme-reaction process is simple, the cost of production materials is low, the resource is saved and no pollution is caused.

Description

technical field [0001] The invention relates to an L-carnitine-producing Escherichia coli genetically engineered bacterium, a construction method and application thereof, and belongs to the technical field of bioengineering. Background technique [0002] L-carnitine (L-carnitine, L-Carnitine), also known as vitamin B T , is a vitamin product related to fatty acid metabolism in animals. When animals lack carnitine due to congenital or metabolic diseases, it will cause body fatigue and many cardiovascular diseases. At present, L-carnitine is not only used for infants and the infirm in the form of enhanced nutrition, but also used as a drug for lowering blood fat, losing weight and treating cardiovascular diseases. Due to its remarkable curative effect, it is attracting great interest and attention from people. In recent years, L-carnitine has been widely used in the fields of medicine, health care and food, and has been regulated as a statutory multi-purpose application agen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/00C12R1/19
Inventor 黄娇芳孙立洁陈祥松袁丽霞王纪吴金勇朱薇薇王刚姚建铭史吉平魏东
Owner 武汉中科光谷绿色生物技术有限公司
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