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Recombinant yarrowia lipolytica bacterial strain as well as construction method and application thereof

A yeast strain, Yarrowia lipolysis technology, applied in the field of genetic engineering, can solve the problems of large by-products and pollution, long plant extraction cycle, etc., and achieve high yield effect

Active Publication Date: 2015-07-29
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a common phytosterol, campesterol is extracted from plants, but the plant extraction period is long, and the by-products and pollution are relatively large
At present, there is no report on the synthesis of campesterol as a product in microorganisms, but there are studies on the final synthesis of progesterone and hydrocortisone using it as an intermediate product

Method used

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  • Recombinant yarrowia lipolytica bacterial strain as well as construction method and application thereof
  • Recombinant yarrowia lipolytica bacterial strain as well as construction method and application thereof
  • Recombinant yarrowia lipolytica bacterial strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Construction of single gene expression cassette

[0051] Shown in SEQ ID NO: 4 (optimized Rattus norvegicus-derived dhcr7 gene), shown in SEQ ID NO: 5 (optimized rice-derived dhcr7 gene) and SEQ ID NO: 6 (optimized toad-derived Add BsaI restriction sites to both ends of the nucleotide sequence of the dhcr7 gene) to obtain the exogenous target gene, and connect it to the pUC57-simple plasmid;

[0052] The endogenous promoter EXP1 (sequence shown in SEQ ID NO: 27) and the terminator XPR2 (sequence shown in SEQ ID NO: 28) of Yarrowia lipolytica were selected, and a pair of reverse BsaI restriction sites were constructed between the two point to form an expression cassette, and add NotI restriction sites at both ends of the expression cassette, and connect it to the pUC57-simple plasmid;

[0053] The pUC57-simple plasmid connected with the exogenous target gene and the pUC57-simple plasmid connected with the expression cassette were digested with BsaI, and th...

Embodiment 2

[0055] Example 2: Obtaining of fragment 1 (gene fragment upstream of erg5 site) and fragment 2 (gene fragment downstream of erg5 site)

[0056] Using the nucleotide sequences shown in SEQ ID NOs: 15-20 as primers, the nucleotide sequences shown in SEQ ID NOs: 7-9 were connected by OE-PCR to obtain fragments containing NotI restriction sites at both ends, Connected into the pEASY-Blunt plasmid, and carried out NotI digestion to obtain Fragment 1, as shown in SEQ ID NO:21.

[0057] Using the nucleotide sequences shown in SEQ ID NOs: 22-25 as primers, the nucleotide sequences shown in SEQ ID NOs: 10-11 were connected by OE-PCR to obtain fragments containing NotI restriction sites at both ends, Connected into the pEASY-Blunt plasmid, carried out NotI digestion, and obtained fragment 2, as shown in SEQ ID NO:26.

[0058] Table 1 OE-PCR primers

[0059]

[0060]

Embodiment 3

[0061] Embodiment 3: Construction of recombinant Yarrowia lipolytica strain

[0062] The single gene expression cassette in Example 1, fragment 1 and fragment 2 in Example 2 were transformed into lipolytic yeast by the lithium acetate method, and the above-mentioned fragments were transformed by homologous recombination between the fragments using the principle of homologous recombination of the yeast itself. The source sequences were joined and integrated into the genome by recombination with homologous sequences at the erg5 locus on the yeast genome. After transformation, the yeast was screened using SC-drop solid medium (synthetic yeast nitrogen source YNB6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L, solid supplemented with 2% agar powder), and the obtained transformants were After separation and purification by streaking, transfer to liquid medium and culture for 24 hours, extract the yeast genome as a template, carry out PCR verification, confirm the correct recomb...

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Abstract

The invention relates to the technical field of genetic engineering and particularly discloses a recombinant yarrowia lipolytica bacterial strain as well as a construction method and an application thereof. A genome of the recombinant yarrowia lipolytica bacterial strain comprises a nucleotide sequence as shown in any one of SEQ ID NO: 1-3. The construction method comprises the following steps of constructing a key gene dhcr7 in a biosynthesis way of campesterol expressed by a single-gene expression cassette through an OE-PCR (overlap extension-polymerase chain reaction) method; cutting down the single-gene expression cassette through restriction enzyme, transferring the single-gene expression cassette into yeast at one time to perform homologous recombination among fragments and integration of a special erg5 lotus of the genome, screening a completely-assembled converter through an auxotrophic culture medium and genome PCR to obtain a novel recombinant yarrowia lipolytica bacterial strain. The novel recombinant yarrowia lipolytica bacterial strain can be applied to biosynthesis of campesterol, and a relatively high yield can be kept.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and more specifically relates to a recombinant Yarrowia lipolytica strain and its construction method and application. Background technique [0002] The major needs in the fields of human health, energy, and the environment have led to the rapid development of synthetic biology. Synthetic biology technology has achieved remarkable results in the synthesis of important products, the production of clean energy, and the maintenance of human health. Using synthetic biology technology to construct campesterol artificial cells can make up for the shortcomings of chemical and enzymatic methods, and the production process is green and clean, which has great advantages. The functional gene module is formed by organically remodeling and linking the gene elements (promoter, transcriptional regulatory region, ribosome binding site, open reading frame, terminator, etc.) according to the needs of ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P33/00C12R1/645
Inventor 肖文海杜昊星王颖元英进
Owner TIANJIN UNIV
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