Astaxanthin synthase of Sphingomonas and its encoding gene and method for genetic manipulation of Sphingomonas

A sphingomonas, coding gene technology, applied in the field of genetic manipulation, astaxanthin biosynthetic enzyme and its coding gene, can solve the problem of no genetic manipulation, etc., to achieve the effect of enriching gene diversity

Active Publication Date: 2017-07-11
WUHAN INST OF BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no gene information reported in Sphingomonas ATCC55669, and there is no literature related to genetic manipulation, even the genus of the strain is rarely reported

Method used

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  • Astaxanthin synthase of Sphingomonas and its encoding gene and method for genetic manipulation of Sphingomonas
  • Astaxanthin synthase of Sphingomonas and its encoding gene and method for genetic manipulation of Sphingomonas
  • Astaxanthin synthase of Sphingomonas and its encoding gene and method for genetic manipulation of Sphingomonas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Detection of Astaxanthin Products in Sphingomonas

[0045] Extract Sphingomonas Sphingomonas ATCC55669 (purchase from ATCC) metabolites, the method is as follows:

[0046] The strains were activated on a #272 medium plate (nutrient broth 8g / L, glucose 5g / L, agar 1.6%) and cultured at 26°C. Pick the colony and culture it in 5mL #272 medium (nutrient broth 8g / L, glucose 5g / L) at 26°C and 220rpm. After 24 hours, transfer to 100mL#272 medium for culture at 26°C and 220rpm, OD 600 At 0.8, transfer to 300mL #272 medium for culture at 26°C and 220rpm, and harvest the bacteria after 60h.

[0047] Centrifuge the bacterial solution at 8000rpm for 10min, collect the bacterial cells, add 10mL extractant (V 丙酮 :V 甲醇 =4:1), shake to break up the cells, extract for 10min, centrifuge at 8000rpm, 4°C for 5min, remove the supernatant, and extract 3 times according to the above steps, collect the supernatant, spin dry, add 3mL acetone to dissolve, and centrifuge at 13000rpm f...

Embodiment 2

[0048] Example 2 Determination of related astaxanthin biosynthesis genes in Sphingomonas

[0049] As can be seen from Example 1, Sphingomonas Sphingomonas ATCC55669 can produce astaxanthin, and this strain contains a biosynthetic pathway capable of producing astaxanthin. Comparing the genome information of Sphingomonas in NCBI (http: / / www.ncbi.nlm.nih.gov / ) Blastp, it was found that there is a MEP pathway in Sphingomonas, which starts from pyruvate IPP and DMAPP were synthesized via dxs, dxr, ispE, ispDF, ispG, ispH. IPP and DMAPP generate astaxanthin through the carotenoid synthesis pathway through crtE, crtB, crtI, crtY, crtZ, and crtW. In this linear pathway of astaxanthin synthesis by Sphingomonas, all the other genes except crtE and crtZ are unique.

Embodiment 3

[0050] Example 3 Amplification of Sphingomonas crtE gene and crtZ gene

[0051] Genomic DNA was extracted from Sphingomonas Sphingomonas ATCC55669, and the extraction method was as follows:

[0052] (1) Take 50mL of fresh bacterial solution to a conical centrifuge tube, centrifuge at 7000rpm×5min, and discard the supernatant.

[0053] (2) Add 10mL of ddH 2 O, break up on the shaker, centrifuge at 7000rpm×5min, and discard the supernatant.

[0054] (3) Add 10 mL of SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris-Cl), disperse on a shaker, centrifuge at 7000 rpm for 5 min, and discard the supernatant.

[0055] (4) Add 5 mL of SET buffer, disperse it on a shaker, add 150 μL of lysozyme (lysozyme, 100 mg / mL, -20°C), bathe in 37°C water for 30-60 minutes, shake slowly every 5-10 minutes, Until the cell wall is completely lysed (to identify the cell wall is completely lysed: take a small amount of bacterial liquid, add 1 drop of 10% SDS, the bacterial liquid is clear and silky). ...

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Abstract

The invention discloses astaxanthin synthetase of sphingomonas, an encoding gene of astaxanthin synthetase and a method for genetic manipulation of sphingomonas. The invention verifies that the sphingomonas can be used for producing astaxanthin, a biological synthesis way capable of producing the astaxanthin is contained, an MEP way and a carotenoid synthesis way are used for producing the astaxanthin in the sphingomonas, and the fact that in the sphingomonas, crtE and crtZ having relatively low homology with the known genes have the function of synthesizing the astaxanthin, can be further proven. Both the enzyme and the encoding gene thereof required by the astaxanthin biosynthetic way of the sphingomonas are not reported in the prior art, so that more resources are provided to the biosynthetic metabolism and transformation of the carotenoid. Genetic manipulation can be carried out on the sphingomonas by taking pBBR1MCS2 as a carrier and taking EGFP as a reporter gene, so that a foundation is laid for the genetic modification of the environmental microorganism of the type and the finding of the degradation mechanisms.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to the astaxanthin biosynthetic enzyme of sphingomonas and its encoding gene, and a method for genetic manipulation in sphingomonas. Background technique [0002] Astaxanthin (also known as astaxanthin or astaxanthin), which belongs to ketone carotenoids, is a strong natural antioxidant. Its unique molecular structure not only makes it have super antioxidant activity, but also has anti-aging, anti-radiation, anti-tumor and prevention of cardiovascular and cerebrovascular diseases. At present, astaxanthin has been widely used in food, feed, and health care products markets. However, the source of natural astaxanthin is very limited. At present, astaxanthin is mostly produced by Pfaffia strains and microalgae produced by traditional mutation technology. However, Pfaffia fermentation has the disadvantage of long fermentation period, and the production technology for obtaining prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N9/90C12N9/12C12N9/10C12N9/02C12N15/52C12N15/61C12N15/54C12N15/53C12N15/70C12N15/74C12P23/00C12R1/01
CPCC12N9/0006C12N9/001C12N9/0071C12N9/0073C12N9/0093C12N9/1022C12N9/1085C12N9/1205C12N9/1241C12N9/88C12N9/90C12N15/66C12N15/70C12P23/00C12Y101/01267C12Y114/13129C12Y117/01002C12Y202/01007C12Y205/01029C12Y205/01032C12Y207/01148C12Y207/0706C12Y406/01012C12Y505/01019
Inventor 刘天罡马田周袁杰朱发银
Owner WUHAN INST OF BIOTECH
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