Lysine decarboxylase mutant and application thereof

A technology of lysine decarboxylase and mutants, applied in the field of one-step production of 1,5-pentanediamine from glucose, which can solve problems such as leakage expression, bacterial toxicity, and inhibition of bacterial growth

Active Publication Date: 2018-11-13
CATHAY R&D CENT CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in addition to the additional addition of IPTG / lactose, the system also has serious leakage expression. Due to the limited concentration of 1,5-pentanediamine that the bacteria itself can tolerate, if the fermentation system expresses lysine decarboxylase in the early stage Too much 1,5-pentanediamine generated will cause toxicity to the bacteria, thereby inhibiting the growth of the bacteria and the process of using glucose to produce L-lysine (Qian, et al., Biotechnol.Bioeng.2011; 108 :93–103)
In the process of one-step production of 1,5-pentanediamine from glucose, if a constitutive promoter is used, the cost of the inducer added in the fermentation process can be effectively saved, but the expression of lysine decarboxylase must be controlled, otherwise The 1,5-pentanediamine produced in the early stage of fermentation will seriously inhibit the growth of bacteria and interrupt the fermentation process

Method used

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  • Lysine decarboxylase mutant and application thereof
  • Lysine decarboxylase mutant and application thereof
  • Lysine decarboxylase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Cloning of lysine decarboxylase gene cadA

[0070] Using primers cadA-SacI-F and cadA-XbaI-R (primers shown in Table 1), the lysine decarboxylase (SEQ ID No: 1) encoding gene cadA (SEQ ID No: 2) was extracted from E. coli MG1655K12 (E. coli MG1655K12, purchased from Beijing Tianenze Biotechnology Co., Ltd.) was amplified from the genome, after double digestion with SacI and XbaI, connected to the same double digested pUC18 plasmid (purchased from Bao Bioengineering (Dalian) Co., Ltd.) in. Use CaCl 2 The competent cells were prepared by the method, and the ligation product was transformed into E.coli BL21 (purchased from Bao Bioengineering (Dalian) Co., Ltd.) cells by the heat shock method. The LB medium was added with ampicillin antibiotics for screening, clone PCR and sequencing After the verification is correct (verification of the CadA sequence is correct), the plasmid is extracted to obtain the pCIB60 plasmid.

[0071] Using the plasmid pCIB60 as a template, the 5'sequ...

Embodiment 2

[0077] Construction and screening of error-prone PCR mutation library of lysine decarboxylase gene CadA

[0078] Using the plasmid pCIB71 constructed in Example 1 as a template, primers cadA-SacI-F and cadA-Xbal-R and GeneMorph II Random Mutagenesis error-prone PCR kit (purchased from Agilent Technologies (China) Co., Ltd.), according to the Instructions for use of the kit: Randomly mutate the cadA gene and introduce restriction sites at both ends of the gene. Using this kit can ensure that the four bases A / T / C / G are mutated at equal frequency, and only need By changing the initial amount of target DNA in the reaction or the number of PCR cycles, the average number of point mutations in each gene in the obtained PCR product is controlled. In this example, by using a higher amount of initial target DNA (450ng) and fewer PCR cycles (20 times), it is possible to control 1-2 nucleotide mutations in each gene, thereby controlling the frequency of amino acid mutations. In order to hav...

Embodiment 3

[0092] Screening results of error-prone PCR mutant library of lysine decarboxylase CadA and identification of mutation sites by sequencing

[0093] The constructed CadA error-prone PCR mutant library was initially screened. Of the 2000 clones, 31 out of the 96-well deep-well plates were judged to be false positive clones; the remaining 1969 clones were screened as 19 clones with color changes. Significantly weaker than the control strain. The 19 clones were re-screened twice (the re-screening method is the same as the initial screening). After re-screening, it was determined that 4 clones had significantly lower lysine decarboxylase activity than the control pCIB71.

[0094] These 4 clones were further inoculated into 5ml LB liquid medium (containing 100μg / ml ampicillin) for cultivation, 37 degrees Celsius, 250rpm (the instrument was purchased from Shanghai Shiping Experimental Equipment Co., Ltd., an extraordinary small-capacity full-temperature constant-temperature culture oscil...

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Abstract

The invention relates to a lysine decarboxylase mutant and application thereof, in particular to lysine decarboxylase modification aiming to allow lysine decarboxylase to be suitable for L-lysine production using microbial fermentation and coupling catalyzing L-lysine decarboxylic reaction to generate 1,5-pentamethylene diamine so as to use glucose to produce the 1,5-pentamethylene diamine in onestep. The lysine decarboxylase mutant has the advantages that the activity-lowered lysine decarboxylase mutant is acquired by using random mutation and high-throughput screening; when recombinant L-lysine production strains containing the lysine decarboxylase mutants are used to produce the 1,5-pentamethylene diamine in one step, the toxic effect of the 1,5-pentamethylene diamine in the system inthe early fermentation stage on thallus when the thallus uses glucose fermentation can be reduced, the balance between L-lysine and the 1,5-pentamethylene diamine can be maintained, the yield of the 1,5-pentamethylene diamine produced by the L-lysine production strains can be increased, and the quantity of the residual L-lysine is less than 2.2g / L.

Description

Technical field [0001] The present invention relates to a mutant of lysine decarboxylase and its application. Specifically, the present invention relates to the modification of lysine decarboxylase to make it suitable for microbial fermentation to produce L-lysine and coupled to catalyze L- The lysine decarboxylation reaction produces 1,5-pentanediamine, which can be produced by one-step process of glucose. Background technique [0002] L-lysine decarboxylase (LDC for short, EC 4.1.1.18) is widely present in microorganisms, insects, animals and higher plants. It can remove a carboxyl group from L-lysine to generate 1,5-pentane Diamine (cadaverine) and CO 2 . However, 1,5-pentanediamine has a wide range of uses. For example, it can be polymerized with dibasic acid to synthesize new nylon, which has high application value in industrial production. At present, microbial production of 1,5-pentanediamine mainly uses the following two methods: microbial fermentation production and mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/74C12N1/21C12P13/08C12P13/00
CPCC12N9/88C12N15/70C12N15/74C12P13/001C12P13/08C12Y401/01018
Inventor 陈玲周豪宏刘修才
Owner CATHAY R&D CENT CO LTD
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