Recombinant strain for producing ALA through fermentation and preparation method thereof

A technology of recombinant strains and strains, which is applied in the fields of metabolic engineering and biomanufacturing, can solve the problems of high price, high production cost, and expensive price, and achieve the effects of optimized culture conditions, high bioaccumulation, and low cost of culture medium

Inactive Publication Date: 2017-07-21
UNIV OF JINAN
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above-mentioned method for producing 5-ALA mainly adopts the model organism Escherichia coli, but for practical production applications, there are many disadvantages in using Escherichia coli as the production strain
First of all, the LB medium used to cultivate Escherichia coli is more expensive, resulting in higher production costs
Second, when culturing recombinant E. coli strains, antibiotics need to be added, which poses some challenges to production costs and subsequent sewage treatment
Finally, using Escherichia coli as a production strain requires a large amount of expensive IPTG (isopropylthiogalactopyranoside) to be added in the pot cultivation process, which also poses a huge challenge to production cost control

Method used

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  • Recombinant strain for producing ALA through fermentation and preparation method thereof
  • Recombinant strain for producing ALA through fermentation and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Construction of recombinant expression ALA synthetase plasmid pGAPZB-ALA (see figure 1 )

[0020] 1. Primer design and PCR reaction

[0021] According to GenBank Saccharomyces cerevisiae The source of 5-aminolevulinic acid synthase gene (accession number is M26329), design two PCR primers:

[0022] ScALAF 5'-ATCTGAATTC ATG CAGCTCCATTTTTG-3'

[0023] ScALAR 5'-TGACTCGAG TTA CTGCTTGATACCACTAGAAAC-3'

[0024] The primer (ScALAF) at the 5' end of the designed gene contains the initiation codon ATG, and an EcoRI restriction site is introduced at the same time. The primer at the 3' end of the gene (ScALAR) contained the stop codon TAA, and introduced the XhoI restriction site.

[0025] Saccharomyces cerevisiae DNA was extracted using the Takara Yeast Genome Extraction Kit.

[0026] Using Saccharomyces cerevisiae chromosomal DNA as a template, add a certain amount of primers and long-chain high-fidelity DNA polymerase PrimeSTAR ® GXL DNA Polymerase and dNTP mixt...

Embodiment 2

[0031] 1. Preparation of Competent Pichia pastoris

[0032] Inoculate Pichia pastoris GS115 in 500mL YPD medium, and cultivate to OD at 28-30°C 600 Centrifuge at 1.3-1.5, 5000rpm / min for 5min, and wash the cell pellet with 100mL ice-bath pre-cooled sterile water, 20mL ice-bath pre-cooled sterile water and 20mL 1 mol / L ice-bath pre-cooled sorbitol, respectively. After each wash, centrifuge at 5000rpm for 5min to collect the cells, and finally add 200 μL of 1M ice-bath pre-cooled sorbitol to suspend.

[0033] 2. Electrotransformation of the recombinant expression vector into Pichia pastoris GS115 cells

[0034] Linearize about 10 μg of the constructed recombinant expression plasmid pGAPZA-ALA with AvrⅡ restriction endonuclease, extract with phenol and chloroform, and recover the linear DNA by ethanol precipitation and dissolve it in 10 μL sterile water. At the same time, linearize the empty pGAPZA plasmid with the same enzyme digestion And recovered as a control. The above l...

Embodiment 3

[0061] Inoculate the screened recombinant strains in a test tube of 3 mL of YPD medium, culture overnight at 30°C 220r / min, and transfer 1% inoculum into a shaker flask containing 100mL of medium (10g / L yeast extract, 10g / L L peptone, 0.05mol / L potassium phosphate buffer pH6.0, 13.4g / L yeast basic nitrogen source containing ammonium sulfate, 4×10 -4 g / L biotin, 2g / L glycerol). Cultivate at 30°C 220r / min. From the 48th hour, 0.2% glycerol was added every 24 hours, precursors (succinic acid 7g / L, glycine 3g / L) were added every 48h, and metabolic inhibitor levulinic acid 5g / L. After 6 days of continuous culture, the ALA production reached the highest. The ALA production of the recombinant bacteria reached 1.2g / L, compared with 0.048g / L of the host bacteria, the yield increased by about 25 times.

[0062] It should be noted that the Pichia pastoris in this example was only fermented in shake flasks, and did not fully exert its advantages of high bioaccumulation (up to 130g / L dr...

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Abstract

The invention provides a recombinant methanol utilization type yeast and especially provides a recombinant bacterium provided with an exogenous ALA(5-aminolevulinic acid) synthetase expression vector, a method for producing ALA by utilizing the recombinant bacterium and a method for establishing the expression vector and obtaining a recombinant engineering strain. The recombinant bacterium having the characteristics is fermented in a culture medium containing glycerin, a proper amount of glycine, succinic acid and a certain carbon source, and high-level ALA accumulation can be obtained. The novel methanol utilization type pichia pastoris can achieve green and pollution-free production of 5-aminolevulinic acid and can be widely applied to the field of agriculture and feeds.

Description

technical field [0001] The invention relates to the fields of metabolic engineering and biomanufacturing, in particular to a method for using DNA recombination technology to construct methanol-utilizing yeast engineering strains and using the strains to produce ALA. Background technique [0002] 5-Aminolevulinic acid (ALA) is an important intermediate metabolite in organisms, it is the synthesis of porphyrin, chlorophyll, heme and vitamin B 12 Precursors of these substances are ubiquitous in animals and plants in nature. In recent years, ALA has been recognized as a pollution-free green agricultural chemical because of its non-toxicity to humans and animals, easy degradation in the environment, and no residue. It can be used as a green herbicide, insecticide, Plant growth promoters, etc. In the medical field, 5-aminolevulinic acid can be used in light-driven therapy to treat a variety of cancers and has therapeutic effects on some other diseases. [0003] At present, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P13/00C12R1/84
CPCC12N9/1029C12N15/815C12N2800/60C12P13/005C12Y203/01037
Inventor 叶春江王凯王元秀
Owner UNIV OF JINAN
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