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5-aminolevulinic acid production strain and its construction method and application

A technology of aminolevulinic acid and Corynebacterium glutamicum, applied in the biological field, can solve the problems of low yield, staying at the level of several grams, low ALA production level, etc.

Active Publication Date: 2020-06-09
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

However, the production level of ALA produced by existing strains is still low, and the ALA production reported in the literature is mostly at the level of a few grams, and the output of a few literature reports reaches more than ten grams, such as the two-stage fermentation process reported by Yang P et al. Produce ALA with a yield of 14.7 g / L (Yang P et al., Applied and Environmental Microbiology 2016,82:2709–2717); the yield of ALA production process reported by Zhu CC et al is 11.5 g / L (Zhu CC et al., Biotechnology and Bioengineering 2019, 116:2018–2028)
However, the yield of existing methods for producing ALA is still low, and there is an urgent need in this field for strains with higher production levels and a wider range of raw material utilization in order to reduce production costs and promote large-scale applications.

Method used

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  • 5-aminolevulinic acid production strain and its construction method and application
  • 5-aminolevulinic acid production strain and its construction method and application
  • 5-aminolevulinic acid production strain and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 hemA Expression optimization vector construction

[0044] According to the NCBI released Rhodopseudomonas palustris ( Rhodopseudomonas palustris ) ATCC 17001 ALA synthetase coding gene sequence (GenBank: JQ048720.1), designed primer RBS2- hemA -F (SEQ ID NO.1) and hemA -R (SEQ ID NO. 2). With pZWA1 vector (WO2014121724A, in this vector hemA The RBS sequence is RBS1) as a template to obtain the RBS2 with RBS2 hemA Gene fragments, the PCR amplification system is as follows: PCR amplification parameters are: 94°C for 10 min, 94°C for 20 s, 55°C for 30 s, 72°C for 40 s, 30 cycles, and 72°C for 5 min. target segment Eco RI and Sma I double digestion treatment, and then under the action of DNA ligase, the obtained fragment was ligated with the plasmid pEC-XK99E that was also digested, and the ligated product was transformed into DH5α competent cells, and the kanamycin-resistant plate was coated and cultured overnight , pick positive clones for colony PCR ...

Embodiment 2

[0046] Example 2 hemA Effect of expression optimization on ALA synthesis

[0047] The above recombinant vector pZRA10 was transformed into Corynebacterium glutamicum ATCC 13032 to obtain recombinant strain ATCC13032 / pZRA10. The above-mentioned recombinant strain ATCC13032 / pZRA10 and the original strain ATCC13032 / pZWA2 were inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and 20 g / L glucose, respectively, and cultured at 30 °C, 200 rpm for 12 h. According to the initial OD of 0.5, a 500 mL Erlenmeyer flask filled with 50 mL of fermentation medium was transferred, cultured at 30°C and 200 rpm for 3 h, and then IPTG with a final concentration of 100 μM was added. concentration. Wherein the shake flask fermentation medium formula is: Na 2 HPO 4 12H 2 O 17.1 g / L, KH 2 PO 4 3.0 g / L, NaCl 0.5 g / L, NH 4 Cl 1.0 g / L, MgSO 4 2mM, CaCl 2 0.1mM, glucose 50 g / L, yeast powder 2 g / L, glycine 6 g / L. The final concentration of kanamycin was 50 µg / mL. ALA de...

Embodiment 3p

[0050] Example 3 ppc Expression optimization vector construction

[0051] Utilize the same technical method of embodiment 1, through primer design and PCR amplification, obtain respectively with RBS2-RBS7 ppc Gene fragment, the primer sequence used RBS2- ppc -F, RBS3- ppc -F, RBS4- ppc -F, RBS5- ppc -F, RBS6- ppc -F, RBS7- ppc -F and ppc -R are respectively shown in Table 2, and the sequences of RBS2-RBS7 are respectively shown in Table 3. The PCR amplification system was as follows: PCR amplification parameters were: 94°C for 10 min, 94°C for 20 s, 55°C for 30 s, 72°C for 2 min, 30 cycles, and 72°C for 5 min. target segment Sma I and XbaI double digestion treatment, and then under the action of DNA ligase, the obtained fragment was connected to the pZRA8 vector of the same digestion treatment, and the ligation product was transformed into DH5α competent cells, and the kanamycin resistance plate was coated for overnight culture, and the challenge The posi...

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Abstract

The invention provides a 5-aminolevulinic acid producing strain, and a construction method and application thereof. According to the invention, a ribosome binding site (RBS) sequence of 5-aminolevulinic acid synthetase and phosphoenolpyruvate carboxylase in a starting strain is optimized, so that capacity of producing 5-aminolevulinic acid by the strain is greatly improved, and the strain is suitable for large-scale production.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a recombinant bacterial strain with high yield of 5-aminolevulinic acid, as well as the construction method and application of the bacterial strain. Background technique [0002] 5-aminolevulinic acid (5-aminolevulinic acid, ALA) is a biosynthetic heme, chlorophyll, VB 12 Precursors of tetrapyrrole compounds, such as tetrapyrrole, widely exist in animals, plants and microorganisms. ALA is widely used in the fields of medicine, agriculture, feed and health food, and is a high value-added bio-based chemical with great development value. In recent years, the use of microorganisms to ferment and produce ALA through the C4 pathway has been industrialized. [0003] With the continuous development of metabolic engineering technology, there are more and more studies on the construction of ALA production strains by means of metabolic engineering, and t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/00C12R1/15
CPCC12N9/1029C12N9/88C12N15/77C12P13/005C12Y203/01037C12Y401/01031
Inventor 孙际宾陈久洲郑平饶德明王钰周文娟郭轩马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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