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Red yeast engineering bacterium for high-yield production of astaxanthin and construction method thereof

A construction method, red yeast technology, applied in the biological field, can solve problems such as high price, limited output, and failure to meet market demand, and achieve the effect of improving metabolic flux

Active Publication Date: 2017-01-11
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natural astaxanthin is available for human use, but the output is very limited, far from meeting the market demand, and the price is expensive
Extracting astaxanthin from crustacean aquatic product waste such as shrimp and crab faces difficulties in raw material sources and collection, and the output is limited

Method used

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  • Red yeast engineering bacterium for high-yield production of astaxanthin and construction method thereof
  • Red yeast engineering bacterium for high-yield production of astaxanthin and construction method thereof
  • Red yeast engineering bacterium for high-yield production of astaxanthin and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1, plasmid construction

[0025] 1.1 Cloning of genetic elements

[0026] 1) Acquisition of genes ADH2, ALD6, CrtE, CrtYB, CrtS and CrtR:

[0027] The genomic DNA of Phaffia rhodozyma was extracted by the glass bead breaking method, and the genomic DNA of Phaffia rhodozyma was used as a template, and the primer sequences of each gene recorded in Table 1 were respectively amplified by KAPAHiFi high-fidelity DNA polymerase Genes were amplified by PCR, and the amplification system was 25ul (2×KAPA Mix, 12.5ul; 0.5ul each of 10uM primers; template 1ul; add water to make up to 25ul), and each gene sequence was obtained.

[0028] The amplification conditions are: pre-denaturation at 95°C for 3 minutes; denaturation at 98°C for 20 seconds; annealing at 60-72°C for 15 seconds; minute.

[0029] 2) Synthesis of genes ACS, PHK and PTA: Escherichia coli acetyl-CoA synthetase gene ACS, Aspergillus niger phosphoketolase gene PHK, Bacillus subtilis phosphate acetyltransf...

Embodiment 2

[0076] Embodiment 2, the construction of Phaffia rhodozyme engineering bacteria

[0077] 2.1 Obtaining the expression cassette

[0078] The plasmid pCrtS described in Table 6 was used as a template, the CYPUP-NF and CYPDN-NR described in Table 7 were used as primers, and the CrtS-Hgr-CYP61 expression cassette was amplified by PCR using KAPA HiFi high-fidelity DNA polymerase.

[0079] The plasmid pCrtE described in Table 6 was used as a template, rDNA1-F and rDNA2-R described in Table 5 were used as primers, and the CrtE-G418-rDNA expression cassette was amplified by PCR using KAPA HiFi high-fidelity DNA polymerase.

[0080] The plasmids pStH and pSR described in Table 6 were respectively digested with SmaI to obtain the expression cassette CrtS-tHMGR-Hgr-CYP61 or CrtS-CrtR-Hgr-CYP61.

[0081] Use AflII to digest the plasmids pCrtE, pEYB, pKA, pA3 and pPDH recorded in Table 6, respectively, to obtain the expression cassettes CrtE-G418-rDNA, CrtE-CrtYB-G418-rDNA or PHK-PTA-BLM-...

Embodiment 3

[0092] Embodiment 3, engineering strain culture produces astaxanthin

[0093] 3.1 Strain shake flask culture

[0094] The strains CBS 6938, XD4 and SXD were respectively activated on the YM solid plate and cultured at 22°C for 4 days. Single colonies were picked and inoculated into 250ml shake flasks filled with 20ml CNM medium, cultured at 22°C for 48h, and used as seed culture solution. Then the various seed liquids obtained were inoculated into 250ml shake flasks containing 30ml CNM medium according to the inoculum amount of 8%, respectively, cultivated at 22°C for 3 days, then cultivated at 18°C ​​for 3 days, and set aside.

[0095]Among them, the composition of CNM medium is glucose 40g / L, peptone 3g / L, yeast extract 1.5g / L, corn steep liquor 1g / L, (NH 4 ) 2 SO 4 0.8g / L, MgSO 4 ·7H 2 O 0.5g / L, KH 2 PO 4 1.5g / L, K 2 HPO 4 0.3g / L, CaCl 2 2H 2 O 0.1g / L, biotin 1mg / L, soybean oil 2ml / L.

[0096] 3.2 Strain fermenter culture (taking strain SXD as an example)

[0...

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Abstract

The invention relates to a red yeast engineering bacterium for high-yield production of astaxanthin and a construction method thereof, and belongs to the technical field of biology. The red yeast engineering bacterium is an xanthophyllomyces dendrorhous strain SXD, and is preserved in China General Microbiological Culture Collection Center, the preservation number is CGMCC No.10519, and the preservation date is February 4th, 2015. The xanthophyllomyces dendrorhous engineering bacterial strain SXD is obtained by a metabolic engineering method, and metabolic flux of astaxanthin synthesizing route of xanthophyllomyces dendrorhous is substantially improved. The highest astaxanthin content of the xanthophyllomyces dendrorhous engineering bacterial strain SXD is 4.4mg / g dry cell weight, after optimal cultivation, the astaxanthin content of the bacterial strain reaches 7.1mg / g dry cell weight, the maximum biomass is 83.8g / L, and an industrial exploitation prospect is provided. The SXD bacterial strain is not treated by any mutagenesis, the astaxanthin yield can be increased continuously and significantly by means of a mutation breeding, and production cost of yeast astaxanthin is hopeful to reduce continuously.

Description

technical field [0001] The invention relates to a rhodotorula engineering bacterium with high astaxanthin production and a construction method thereof, belonging to the field of biotechnology. Background technique [0002] Astaxanthin is a terpenoid natural pigment, which belongs to the category of carotenoids. The astaxanthin molecule is connected by two terminal ring structures through the middle polyene, and the molecular formula is C 40 h 52 o 4 . Due to the chirality of the third hydroxyl carbon atom of the two terminal β-ionone structures, there are three stereoisomers of astaxanthin, namely (3S,3S'), (3R,3R'), (3R,3S' ). Astaxanthin molecules contain conjugated double bonds, hydroxyl groups, and ketone groups, which are both lipophilic and hydrophilic. These molecular characteristics endow astaxanthin with unique biological activities and functions. [0003] Astaxanthin has a strong antioxidant effect. The conjugated double bond in the astaxanthin molecule can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12R1/645
Inventor 王士安李福利
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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