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Recombinant escherichia coli for producing 1, 5-pentamethylene diamine-succinate as well as construction method and application of recombinant escherichia coli

A technology for recombining Escherichia coli and succinate, applied in the biological field, can solve problems such as increased fermentation cost, disadvantage, and inability of cells to grow normally

Active Publication Date: 2021-05-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the method of catalyzing the decarboxylation of lysine is the commonly used method for producing 1,5-pentanediamine. Specifically, the cadA gene is constructed on an inducible plasmid for overexpression, and a large amount of CadA greatly increases the production of 1,5-pentanediamine. production efficiency, but this common method for producing 1,5-pentanediamine has the following defects in the process of fermentatively preparing 1,5-pentanediamine-succinate: First, the overexpression of cadA is harmful to cell growth, because Lysine is an essential substance for cell growth. Overexpression of this enzyme will cause lysine in the cell to be competitively catalyzed into 1,5-pentanediamine, which will lead to insufficient lysine for growth and make cells unable to grow normally
In order to avoid this problem, the cadA gene is usually induced after the cells have grown well. ZL201810195975.2 (CN108531494A) was first fermented for 30 hours aerobically, and fermented for another 6 hours after adding the inducer IPTG. It started after a total of 36 hours of preparation Fermentative production of 1,5-pentanediamine-succinate, a waste of time and carbon source
Secondly, the use of plasmids to overexpress cadA must use antibiotics. ZL201810195975.2 (CN108531494A) uses three antibiotics, kanamycin, chloramphenicol, and streptomycin, which not only increases the cost of fermentation, but also increases the treatment of sewage containing antibiotics cost
Moreover, plasmid loss is an inevitable problem during the overexpression process, resulting in the inability of some cells to express the cadA gene, which is not conducive to the production of 1,5-pentanediamine-succinate

Method used

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  • Recombinant escherichia coli for producing 1, 5-pentamethylene diamine-succinate as well as construction method and application of recombinant escherichia coli
  • Recombinant escherichia coli for producing 1, 5-pentamethylene diamine-succinate as well as construction method and application of recombinant escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0065] Example 1: Construction of recombinant Escherichia coli producing 1,5-pentanediamine-succinate

[0066] 1. Selection of chassis bacteria

[0067] The performance of the chassis bacteria in the present invention is very important, and it must be a succinic acid producing bacteria that hardly produces miscellaneous acids to complete the function of producing succinic acid and pentamethylenediamine at a ratio of 1:1. Otherwise, monocarboxylic acids such as acetic acid and lactic acid produced during bacterial fermentation will also neutralize a certain amount of 1,5-pentanediamine, resulting in a higher molar concentration of 1,5-pentanediamine than succinic acid, and the monocarboxylic acid It is difficult to remove, and because there is only one carboxyl group, it will cause the termination of the polymer chain during the polymerization of nylon, reducing the degree of polymerization and strength of the product. HX028 is a recombinant Escherichia coli that produces succ...

Embodiment 2

[0089] Example 2: Production of 1,5-pentanediamine-succinate by recombinant Escherichia coli HX028-54

[0090] 1. Production of 1,5-pentanediamine-succinate by fermentation

[0091] Both the seed medium and the fermentation medium consisted of:

[0092] Macroelements: Glucose, (NH 4 ) 2 HPO 4 , NH 4 h 2 PO 4 , MgSO 4 ·7H 2 O;

[0093] Trace elements: FeCl 3 ·6H 2 O, CoCl 2 ·6H 2O, CuCl 2 2H 2 O, ZnCl 2 、Na 2 MoO 4 2H 2 O, H 3 BO 3 and MnCl 2 4H 2 o 2 ;

[0094] water.

[0095] The concentration of the above ingredients in the fermentation medium can be respectively:

[0096] Macroelements: Glucose 50g / L-150g / L or 50g / L or 100g / L or 150g / L, NH 4 h 2 PO 4 0.5g / L-5g / L or 0.5g / L or 1g / L or 5g / L, (NH 4 ) 2 HPO 4 1g / L-10g / L or 1g / L or 3g / L or 10g / L, MgSO 4 ·7H 2 O0.1g / L-5g / L or 0.1g / L or 1g / L or 5g / L;

[0097] Trace elements: FeCl 3 ·6H 2 O 0.2μg / L-5μg / or 0.2μg / L or 1.5μg / L or 5μg / L, CoCl 2 ·6H 2 O0.05μg / L-5μg / L or 0.05μg / L or 0.1μg / L or 5μg...

Embodiment 3

[0108] Example 3: Construction of recombinant Escherichia coli producing 1,5-pentanediamine-succinate

[0109] The decarboxylation efficiency of L-lysine is directly related to the production rate of 1,5-pentanediamine. Therefore, in order to further increase the yield of nylon-54 salt, it is necessary to further precisely regulate the expression intensity of the two genes cadB and cadA, so as to further improve the production efficiency of nylon-54 salt. Since the cadB and cadA genes are under the same operon, they are also on the same mRNA after being transcribed into mRNA. Therefore, by precisely regulating the transcriptional intensity of cadB and cadA, the expression intensity of these two genes can be effectively regulated. In order to find the optimal expression intensity of the cadB and cadA genes on the strain HX028-54, the mRNA stable region sequences of the cadB and cadA genes on HX028 were further regulated. The regulation method of the mRNA stable region can be ...

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Abstract

The invention discloses recombinant Escherichia coli for producing 1, 5-pentanediamine-succinate, and compared with recombinant Escherichia coli serving as a starting strain for producing succinic acid, the recombinant Escherichia coli for producing 1, 5-pentanediamine has the advantage that the synthesis capability of 1, 5-pentanediamine is improved. The invention also provides a construction method of the recombinant Escherichia coli for producing the 1, 5-pentanediamine-succinate, and an application of the recombinant Escherichia coli in preparation of the 1, 5-pentanediamine-succinate. The recombinant escherichia coli HX028-54 for producing 1, 5-pentanediamine-succinate, which is constructed by the invention, is fermented for 96 hours to produce 0.94 mol and 207 g / L of 1, 5-pentanediamine-succinate, and glucose and lysine are free from residues, so that later separation and purification are facilitated, and the recombinant escherichia coli has relatively high economic value. On the basis, the production efficiency of the recombinant escherichia coli HX028-54-D which is further optimized is further improved.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli producing 1,5-pentanediamine-succinate and a construction method and application thereof, belonging to the field of biotechnology. Background technique [0002] At present, the global nylon production is about 6 million tons, of which nylon 66 is the earliest industrialized production and the most widely used variety. However, adiponitrile, the key intermediate in the production of nylon 66, has a high price due to high technical barriers and investment thresholds. The current price is about 23,000 yuan / ton. Therefore, if a cheaper nylon product than nylon 66 can be developed, it will greatly promote the development of the nylon industry. Nylon with industrial significance usually has the following three characteristics: 1. Polycondensation of dicarboxylic acid and diamine, 2. Dicarboxylic acid and diamine need to contain more than four CH 2 , 3. The polymer of cyclic lactam needs to contain mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/00C12R1/19
CPCC12N9/88C12Y401/01018C12N15/70C12P13/001
Inventor 张学礼于勇徐洪涛
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI