Gene involved in the biosynthesis of lycopene, recombinant vector comprising the gene, and transformed microorganism with the recombinant vector
A recombinant vector, phytoene technology, applied in biochemical equipment and methods, introduction of foreign genetic material using a vector, recombinant DNA technology, etc., can solve the problems of low lycopene content and difficulty in developing a production process, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0048] Example 1: Cloning of novel genes (crtE, crtB and crtI) for lycopene biosynthesis from a metagenomic library in seawater
[0049] To obtain the crtE, crtB and crtI genes required for lycopene biosynthesis, genomic DNA (metagenomics) was directly obtained from seawater to construct a metagenomic library. On the basis of the fact that lycopene is red, clones with red color were selected and sequenced to confirm their identity.
[0050] First, microorganisms were collected from bulk seawater by membrane filtration, and metagenomic DNA was obtained from seawater. Because the size of most microorganisms is 0.2 to 10 μm (microns), first use a peristaltic pump to pass a large amount of seawater through a filter with a pore size of 10 μm (microns), mainly remove various types of suspended solids with a size larger than 10 μm (microns), and then pass through the pore size The 0.2μm (micron) filter selectively recovers only microorganisms with a size above 0.2μm (micron). Chrom...
Embodiment 2
[0063]Example 2: Preparation of a recombinant vector comprising a lycopene biosynthesis gene derived from a seawater metagenomic library
[0064] The crtE, crtB and crtI genes cloned in Example 1 were inserted into the expression vector pTrc99A (Amannm E. et al., (1998) Gene, 69:301-305).
[0065] First, in order to insert the crtE gene into the pTrc99A vector, the following primer pairs were synthesized.
[0066] f5E-f:
[0067] 5′-TGGAATTCTACATCAGGAGGTAATAAATATGATAAGCCCTATATCCAC-3′
[0068] f5E-r:
[0069] 5′-TAGGATCCCTCGAGATGCATTATCATGGGAGCTTCGCTCGGAGC-3′
[0070] Using the vector pBF5-crt prepared in Example 1 as a template, amplification was performed using primers to obtain a DNA fragment of about 0.85 kb including the crtE gene. The obtained DNA fragment was purified using Qiagen PCR Purification Kit (Qiagen), digested with restriction enzymes EcoRI and BamHI, and introduced into pTrc99A vector digested with the same restriction enzymes, named pT-f5crtE. Next, in o...
Embodiment 3
[0084] Example 3: Production of lycopene in recombinant E. coli
[0085] It was confirmed whether lycopene biosynthesis proceeded in the E. coli strain transformed with the vector pT5-LYC-idi prepared in Example 2.
[0086] First, Escherichia coli MG 1655 was transformed with the vector pT5-LYC-idi. Each single colony of transformed Escherichia coli was inoculated into 5 mL (milliliter) of 2YT medium (16 g / L pancreatic peptone, 10g / L yeast extract and 5g / L NaCl), shake culture at 37°C for 8 hours. 600 µl (microliter) of the obtained culture solution was inoculated into 30 ml (milliliter) of 2YT medium supplemented with 1% glycerol and 100 µg / ml (microgram / ml) ampicillin, and cultured at 30°C for 48 hours.
[0087] After the cell culture is completed, take an appropriate amount of culture medium, and calculate the dry weight of the cells (gDCW / L), the output of lycopene (mg lycopene / L, referred to as mg / L hereinafter) and the content (mg Lycopene / gDCW, hereinafter referred t...
PUM
| Property | Measurement | Unit |
|---|---|---|
| size | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More