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Recombinant yarrowia lipolytica T30pED with high yield of campesterol as well as construction method and application of recombinant yarrowia lipolytica T30pED

A Yarrowia lipolytica and campesterol technology, applied in the field of recombinant Yarrowia lipolytica, can solve the problems of limited campesterol production, low DHCR7 activity, and failure to achieve high yield, etc.

Active Publication Date: 2021-11-05
SHAANXI HEALTHFUL BIOLOGICAL ENG
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AI Technical Summary

Problems solved by technology

The prior art found that in the process of using yeast cells to produce campesterol, only by expressing the DHCR7 gene and knocking out the ERG5 gene can not achieve the purpose of high yield. In addition, due to the key enzyme 7-dehydrocholesterol reductase DHCR7 in its synthetic pathway The activity is not high, resulting in the production of campesterol is limited

Method used

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  • Recombinant yarrowia lipolytica T30pED with high yield of campesterol as well as construction method and application of recombinant yarrowia lipolytica T30pED
  • Recombinant yarrowia lipolytica T30pED with high yield of campesterol as well as construction method and application of recombinant yarrowia lipolytica T30pED
  • Recombinant yarrowia lipolytica T30pED with high yield of campesterol as well as construction method and application of recombinant yarrowia lipolytica T30pED

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Experimental program
Comparison scheme
Effect test

Embodiment 1T

[0045] Construction and screening of embodiment 1TEF promoter mutation library

[0046] 1. Construction of the first reporter gene EGFP recombinant strain and detection of relative fluorescence intensity

[0047] 1.1 Construction of the first reporter gene EGFP expression vector

[0048] Download the EGFP sequence (GenBank: MN623123.1) from NCBI, send it to Synthesis, connect it to the universal plasmid pUC57, design primers EGFP-F-HindⅢ / EGFP-R-SalⅠ with restriction sites, and use the synthetic plasmid pUC57 as The template was amplified to obtain the EGFP fragment, and the pJN44 vector and the EGFP fragment were double-digested respectively with HindⅢ / SalⅠ as restriction sites and ligated to obtain the recombinant plasmid pJN44-EGFP. The recombinant plasmid was identified with primers EGFP-F-HindⅢ / EGFP-R-SalⅠ. The size of the target fragment EGFP was 732bp, which was consistent with the size of the synthetic fragment, indicating that the recombinant plasmid was successfully ...

Embodiment 2

[0094] Example 2: Construction of a Yarrowia lipolytica recombinant strain that increases campesterol production: construction of a recombinant strain expressing the DHCR7 gene

[0095] The target gene DHCR7 used for expression comes from zebrafish, and the obtained sequence is codon-optimized by Nanjing Jinsirui, as shown in SEQ No.3. The rare codon with low utilization rate is replaced by the preferred codon in the host to simplify the gene For the secondary structure of post-transcriptional mRNA, remove the motifs that are not conducive to high-efficiency expression, add motifs that are conducive to expression, and adjust the GC content so that the target gene can be efficiently expressed in the host. The DHCR7 fragment was connected with the pJN44 and T30pJN44 fragments to obtain the pJN44-lacZ and T30pJN44-lacZ recombinant plasmids, which were identified by PCR and then transferred to the Yarrowia lipolytica engineering strain pE by the method of the yeast transformation k...

Embodiment 3

[0096] Embodiment 3: the fermentation experiment of recombinant bacterial strain

[0097] 1. Shake flask fermentation of recombinant strains

[0098] After activating the recombinant strains pED and T30pED expressing the DHCR7 gene, pick a single colony and insert it into a 10mL / 50mL vial of liquid YPD seed medium, and culture it at 30°C and 200r / min for 24h. Then, 2% inoculum was transferred to 50mL / 250mL liquid YPD medium, fermented at 30°C and 200r / min for 48h, and then the cells were collected and transferred to 50mL / 250mL liquid YPD medium for further fermentation for 96h. Cells were collected for extraction and detection of campesterol.

[0099] Extraction method of campesterol:

[0100] (1) Put 1mL of fermentation broth in an anaerobic tube, add 100uL of cholestanol standard (4mg / mL stock solution is diluted 100 times);

[0101] (2) Add 2.5 mL of 3 mol / L potassium hydroxide methanol solution, saponify at 90°C, take out the anaerobic tube for shaking for a few seconds...

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Abstract

The invention relates to recombinant yarrowia lipolytica T30pED with high yield of campesterol as well as a construction method and application of the recombinant yarrowia lipolytica T30pED. The strong promoter TEF30 provided by the invention can be used for constructing a recombinant strain of the yarrowia lipolytica, and verification shows that the highest yield of the campesterol in shake flask fermentation reaches 664.1 mg / L (33.21 mg / g DCW) and the highest yield of the campesterol in a 5L fermentation tank reaches 1305.2 mg / L (68.73 mg / g DCW), which are obviously higher than the yield of a wild type promoter strain, so that the strong promoter has outstanding industrial application significance.

Description

【Technical field】 [0001] The present invention relates to recombinant Yarrowia lipolytica, especially the strong promoter TEF30 used to improve the expression of DHCR7 gene in Yarrowialipolytica (Yarrowialipolytica), and also relates to the recombinant solution of high-yielding campesterol containing the strong promoter. A construction method of Yarrowia lipolytica T30pED, and the application of the recombinant Yarrowia lipolytica. 【Background technique】 [0002] Steroid drugs are a special class of polycyclic terpenoids that widely exist in animals and plants in nature. They have a variety of physiological activities and uses, especially in the medical field. They are an important class of clinical drugs. Anti-inflammatory, anti-allergic, antibacterial and antiviral effects. At present, the production methods of steroid drugs mainly include chemical synthesis, plant extraction and biosynthesis. The production process of the chemical synthesis method is complex, the produc...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/81C12N15/67C12N15/65C12N15/56C12N15/53C12P33/00C12R1/645
CPCC07K14/395C12N15/815C12N15/67C12N15/52C12N15/65C12N9/001C12P33/00C12Y103/01021
Inventor 孟永宏苟元元郭建琦牛永洁杨璐
Owner SHAANXI HEALTHFUL BIOLOGICAL ENG
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