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Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof

A technology of aminolevulinic acid and engineering bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of long cultivation period, many by-products, cumbersome steps, etc., and achieve low cost and controllability The effect of high reliability and simple process

Inactive Publication Date: 2012-03-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the production of ALA mostly adopts the chemical synthesis method, which has cumbersome steps, many by-products and low yield; while the production of ALA by biological mutagenesis is mainly to optimize the mutant strain of Rhodobacter sphaeroides, and the highest yield of ALA reported is 27mmol / L, but its cultivation period is long and the cost is high

Method used

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  • Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof
  • Engineering bacterium producing 5-glycyl ethylformic acid and construction and application method thereof

Examples

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Effect test

Embodiment 1

[0023] The 5-aminolevulinic acid engineering bacterium that obtains, its construction comprises the following steps:

[0024] 1. Extraction of recombinant plasmid pET28a-A.R-hemA

[0025] 1) Inoculate the engineering bacteria with the preservation number CGMCC No.1332 into the liquid medium, shake and culture at 37°C for 12 hours, and harvest the bacteria in the logarithmic growth phase;

[0026] 2) Take 3ml of bacterial culture solution (2 times, 1.5ml each time) in an Eppendorf tube, centrifuge at 13,000rpm for 30s, discard the supernatant (as complete as possible);

[0027] 3) Add 100ul of ice-precooled solution I (50mmol / L glucose; 25mmol / L TrisCl (pH8.0); 10mmol / LEDTA (pH8.0), can be prepared in batches, steam sterilized for 15min, and stored at 4°C) , shake vigorously to completely resuspend the cells, and let stand at room temperature for 5-10 minutes;

[0028] 4) Add 200ul of newly prepared solution II (0.2mol / L NaOH; 1% SDS), quickly invert 4 times, mix gently to mi...

Embodiment 2

[0039] The method for producing 5-aminolevulinic acid with engineering bacteria comprises the following steps:

[0040] 1. Use an inoculation needle to dip a small amount of bacterial liquid from the glycerol tube of CGMCC No.1938 engineering bacteria Rosetta (DE3)-pET28a-A.R.hemA, and add 30 μg / ml kanamycin and 34 μg / ml chloramphenicol Streak on the LB medium plate and cultivate overnight in a 37°C oven;

[0041] 2. Pick a single clone and inoculate it in a 250ml shake flask containing 50ml LB medium at a rotation speed of 200rmp, and culture at 37°C for 8h to obtain first-grade seeds;

[0042] 3. Take 2ml of primary seeds and inoculate them in a 500ml shake flask containing 100ml of fermentation medium, and culture them at 37°C and 200rpm for 3 hours to obtain secondary seeds. Wherein the initial fermentation medium is composed of 1% peptone, 0.5% yeast powder, 0.3% succinic acid, 0.2% glycine and 0.2% glucose in mass volume ratio, and the pH value is adjusted to 5.9 with s...

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Abstract

The invention discloses a method to prepare engineering bacteria of 5-glycyl propionic acid and methods for using them, which comprises the following steps: incorporating radial agrobacteriocin; setting the preserved number at CGMCC No. 1938; activating engineering bacteria with LB medium flat; getting monoclonal of the engineering bacteria; seeding to shake; culturing; getting first-grade seed; seeding the first-grade in shake; culturing; getting second-grade seed; seeding the second-grade seed in fermenter; culturing; cooling and evoking with isopropyl-beta-D-sulfo-galactose-glycoside; culturing continually; proceeding supplementing material culture. This engineering bacteria possesses higher ALA synthase expression, simple craft, low cost and good industrial prospect.

Description

technical field [0001] The invention relates to an engineering bacterium for producing 5-aminolevulinic acid and its construction and application method. Background technique [0002] 5-Aminolevulinic acid (5-aminolevulinic acid, ALA) is widely present in organisms, and it is the synthesis of heme, chlorophyll, vitamin B 12 common precursor of tetrapyrrole compounds. As a photodynamic agent (photodynamic agent, PDT), ALA has a wide range of uses in the fields of agrochemicals and medicine. In the field of agriculture, ALA has many functions such as weeding, insecticide, increasing plant stress resistance and promoting plant growth, etc., and is easy to degrade without residue, non-toxic to humans and animals, and has become a very promising pollution-free green agrochemical. In the field of medicine, as a new generation of photodynamic agents, it has significant advantages such as low toxicity and high selectivity. It is used to treat various cancers such as skin cancer, b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/52C12P13/00C12R1/19
Inventor 林建平傅维琦岑沛霖
Owner ZHEJIANG UNIV
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