Method for producing 1, 4-butanediamine by using recombinant escherichia coli

A technology for recombining Escherichia coli and butanediamine, applied in the field of genetic engineering, can solve the problems of long transformation period, low yield, low yield and the like, and achieve the effect of improving specific enzyme activity

Active Publication Date: 2021-06-08
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, arginine is used as a substrate to synthesize 1,4-butanediamine, which has disadvantages such as low yield. For example, in the method disclosed by Yu Deng et al., the method of overexpressing arginine decarboxylase and arginine Aminase combined with a well-controlled fermentation strategy, but the yield of 1,4-butanediamine is only 26.21g·L -1 , and there are shortcomings such as low yield and long conversion cycle (at least 48 hours) (disclosed in the paper "Highly efficient whole-cell biosynthesis of putrescine by recombinantEscherichia coli")

Method used

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  • Method for producing 1, 4-butanediamine by using recombinant escherichia coli
  • Method for producing 1, 4-butanediamine by using recombinant escherichia coli
  • Method for producing 1, 4-butanediamine by using recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Construction of recombinant bacteria Escherichia coli BL21 / pXMJ19-speA-speB, Escherichia coliBL21 / pXMJ19-A533P-speB, Escherichia coli BL21 / pXMJ19-D531R-speB and Escherichiacoli BL21 / pXMJ19-I534G-speB

[0064] (1) According to the gene sequence of speA (nucleotide sequence shown in SEQ ID NO.2) and speB (nucleotide sequence shown in SEQ ID NO. 4), design the PCR primer F of arginine decarboxylase gene 1 and R 1 , PCR primer F of agmatine enzyme gene 2 and R 2 .

[0065] f 1 : 5'-ggtcgactctagaggatccaaaggaggaaaatcatgtctgacgacatgtctatggg-3'

[0066] R 1 : 5'-ttccacacattatacgagccgatgattaattgtcaagaattcttactcatcttcaagataagtataaccg-3'

[0067] f 2 : 5'-gtataatgtgtggaattgtgagcggataacaaaaaaggaggacaaccatgagcaccttaggtcatcaatac-3'

[0068] R 2 : 5'-gtatcaggctgaaaatcttctctcatccgaattcttactcgccctttttcgccgcctg-3'

[0069] (2) Cloning of arginine decarboxylase gene and agmatine enzyme gene

[0070] Using the total DNA of Escherichia coli str.K-12substr.MG1655 as...

Embodiment 2

[0092] Embodiment 2: the specific enzyme activity determination of arginine decarboxylase mutant

[0093] Specific steps are as follows:

[0094] (1) Obtaining wild-type arginine decarboxylase and mutant A533P, D531R and I534G crude enzyme solutions

[0095] The recombinant E. coli Escherichia coli BL21 / pXMJ19-speA-speB, Escherichia coli BL21 / pXMJ19-A533P-speB, Escherichia coli BL21 / pXMJ19-D531R-speB and Escherichia coli BL21 / pXMJ19-I534G-speB prepared in Example 1 were respectively prepared Added at a concentration of 10 μg·mL -1 Streak culture on the chloramphenicol-resistant LB solid medium plate, pick a single colony and inoculate them to a concentration of 10 μg·mL -1 In chloramphenicol-resistant 10 mL LB liquid medium, shake the flask for 12 hours at a temperature of 37° C. and 200 rpm to obtain a seed solution.

[0096] The seed solution was inoculated with an inoculation volume of 1% (v / v) to a concentration of 10 μg mL -1 In chloramphenicol-resistant 50mLLB liquid...

Embodiment 3

[0106] Example 3: Production of 1,4-butanediamine by recombinant Escherichia coli whole cells

[0107] Specific steps are as follows:

[0108] (1) The recombinant bacteria Escherichia coli BL21 / pXMJ19-speA-speB, Escherichia coli BL21 / pXMJ19-A533P-speB, Escherichia coli BL21 / pXMJ19-D531R-speB and Escherichia coli BL21 / pXMJ19-I534G- After speB was activated by streaking on the LB solid plate, pick a single colony and inoculate it in 10 mL with a concentration of 10 μg·mL -1 In the chloramphenicol-resistant LB liquid medium, shake the flask for 12 hours under the conditions of 37 ° C and 200 rpm to obtain the seed solution;

[0109] (2) Transfer the prepared seed solution to 200mL TB liquid medium with an inoculum size of 1% (v / v), culture it at 37°C and 200rpm for 2 hours, and then add IPTG with a final concentration of 0.5mM. Low-temperature induction at 16°C and 200rpm for 12 hours to obtain a culture medium;

[0110](3) The culture solution was centrifuged at 8000 rpm and ...

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Abstract

The invention discloses a method for producing 1, 4-butanediamine by using recombinant escherichia coli, which belongs to the genetic engineering technology, and is characterized in that an arginine decarboxylase mutant and agmatine enzyme are successfully expressed by using a high-copy plasmid pXMJ19 to construct recombinant bacteria. Whole-cell transformation results show that the original bacteria do not have the capability of excessively accumulating 1, 4-butanediamine, and the recombinant bacteria can realize excessive accumulation of 1, 4-butanediamine; and meanwhile, based on the characteristic that the genetically engineered strain can excessively accumulate 1, 4-butanediamine, enzymatic conversion is successfully adopted to produce 1, 4-butanediamine by taking arginine as a substrate. IPTG (isopropyl-beta-d-thiogalactoside) is added in a cell culture process to induce enzyme expression, and magnesium ions and coenzyme pyridoxal phosphate are added in a transformation system according to enzymatic properties of the arginine decarboxylase. Finally, 72.6 g.L <-1 > 1, 4-butanediamine can be accumulated through 24-hour conversion, and the yield of agmatine serving as an intermediate product is 0.14 g/L.

Description

technical field [0001] The invention relates to a method for producing 1,4-butanediamine by using recombinant Escherichia coli, and belongs to the technical field of genetic engineering. Background technique [0002] 1,4-Butanediamine (also known as 1,4-diaminobutane or putrescine) belongs to amino acid derivatives, as a component of polymers, drugs, agricultural chemicals, surfactants and other additives, it has many industrial application. 1,4-Butanediamine is currently used in the synthesis of nylon-4,6 by polycondensation with adipic acid. Nylon-4,6 has become an excellent engineering plastic due to its high melting point, high crystallinity, high heat resistance, high mechanical strength and excellent solvent resistance. have wide applications. Therefore, 1,4-butanediamine has a very broad market application prospect. [0003] The production of 1,4-butanediamine on an industrial scale mainly relies on the chemical synthesis of hydrogenation of succinonitrile, which ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/55C12P13/00C12R1/19
CPCC12N9/88C12N15/70C12N9/78C12P13/001C12Y401/01019C12Y305/03011
Inventor 徐美娟饶志明杨凤玉杨套伟张显
Owner JIANGNAN UNIV
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