Yarrowia lipolytica with high yield of beta-carotene and application thereof

A technology of Yarrowia lipolytica and strains, applied in the field of bioengineering, can solve problems such as unfavorable industrial production of strains, hinder normal growth of cells, etc., and achieve the effects of efficient synthesis and improvement of cell growth

Pending Publication Date: 2022-07-29
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the existing research is mainly on the overexpression of related genes in metabolic pathways, which hinders the normal growth of cells and is not conducive to the industrial production of strains

Method used

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  • Yarrowia lipolytica with high yield of beta-carotene and application thereof
  • Yarrowia lipolytica with high yield of beta-carotene and application thereof
  • Yarrowia lipolytica with high yield of beta-carotene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Amplification of Gene Elements and Preparation of Target Plasmids

[0055] (1) Preparation of target gene

[0056] According to the nucleotide sequence of the geranylgeranyl diphosphate synthase encoding gene crtE from Xanthophyllomyces denrorhous, the nucleotide sequence of the phytoene synthase encoding gene crtYB and the phytoene de The nucleotide sequence of the saturase encoding gene crtI was codon-optimized and entrusted to General Biosystems (Anhui) Co., Ltd. for codon optimization and synthesis, and then each gene fragment was inserted into the plasmid 113-TEF-GPD (experimental Room preservation), obtain the plasmid 113-TEF-GPD-crtIEYB, the plasmid structure is as follows figure 1 shown. The gene sequences of crtE, crtYB and crtI are shown in SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No: 3.

[0057] Using the PAN7-1 plasmid (preserved in the laboratory) as a template, the hph was amplified by PCR, and the obtained fragment was inserted into the plasmi...

Embodiment 2

[0105] Embodiment 2, the construction of recombinant bacteria

[0106] The original strain Yli-CAH is based on the conventional Yarrowia lipolytica po1f strain, and geranylgeranyl diphosphate synthase (CrtE), phytoene synthase ( CrtB), phytoene desaturase (CrtI), 3-hydroxy-3-methylglutaryl CoA reductase (tHMGR) and acetyl CoA carboxylase (ACC) expression cassettes; construction methods see Patent: 202210480414.3.

[0107] (1) Construction of recombinant bacteria Yli-II

[0108] The plasmid 113-TEF-GPD-crtIEYB containing the crtI-crtYB-crtE gene expression cassette was introduced into Yarrowia lipolytica Yli-CAH, and the crtI-crtYB-crtE expression cassette was integrated into the genome Not I site to obtain the recombinant strain Yli -II. The specific method is as follows:

[0109] ①The original Yarrowia lipolytica Yli-CAH strain was cultured overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose) to prepare competent cells.

[0110] ② 113-T...

Embodiment 3

[0122] Embodiment 3, the application of recombinant bacteria in the production of β-carotene

[0123] 1. Cultivation of engineered bacteria

[0124] The starting strains of Yarrowia lipolytica Yli-CAH, recombinant strain Yli-II, recombinant strain Yli-III, and recombinant strain Yli-IV in Example 2 were used to produce β-carotene. The specific method is as follows: take out the strain from the seed preservation tube, insert it into the YPD test tube with 1% inoculum, and cultivate it at 30°C for 24 hours to obtain seed liquid; inoculate the seed liquid with 1% inoculum in 50ml of fermentation medium, at 25°C, Shaking at 220 rpm for 7 days. 2mM H was added exogenously during 24h fermentation 2 O 2 , every 24h timing sampling detection.

[0125] The fermentation medium contained 20 g / L glucose, 10 g / L yeast extract and 20 g / L tryptone.

[0126] 2. Extraction of beta-carotene

[0127] (1) Take 1 ml of the mixed fermentation broth and centrifuge at 12,000 rpm for 3 min (wash w...

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Abstract

The invention discloses yarrowia lipolytica with high yield of beta-carotene and application of the yarrowia lipolytica. The recombinant strain is prepared by the following steps: inserting geranyl geranyl diphosphate synthase, phytoene dehydrogenase, phytoene synthetase / phytoene cyclase, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl coenzyme A carboxylase into a genome of an initial strain po1f to construct a strain yarrowia lipolytica Yli-CAH; and inserting at least one expression cassette of crtE, crtI, crtYB, citrate lyase (ACL) or tHMGR again to obtain the recombinant expression vector. The construction method of the recombinant yarrowia lipolytica is simple to operate, efficient and capable of removing the speed limiting step of an MVA pathway, more acetyl CoA transfer flux flows to synthesis of beta-carotene, cell growth can be improved by overexpressing a citrate lyase coding gene, and industrial utilization is facilitated.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to the construction and application of recombinant Yarrowia lipolytica expression host bacteria for producing beta-carotene. Background technique [0002] β-Carotene is mainly extracted from natural raw materials such as plants and algae by organic solvent extraction method, ultrasonic-assisted extraction method, etc.; The disadvantage is that it cannot meet the needs of the market. At present, chemical synthesis is one of the methods for large-scale production of β-carotene. Using ionone as raw material, through a series of complex chemical reactions, β-carotene is generated through wittig reaction. However, due to the existence of various isomers and a certain burden on human metabolism, there are many controversies in its use. Microorganisms have a short growth cycle, and can use raw materials such as sugars to achieve mass production in a short period of time, and enzyme catalysi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P23/00C12R1/645
CPCC12N15/815C12N9/001C12N9/1085C12N9/0006C12N9/93C12N15/52C12P23/00C12Y205/01001C12Y103/99029C12Y205/01032C12Y205/01099C12Y101/01088C12Y604/01002
Inventor 章文明王靖楠姜岷信丰学蒋羽佳姜万奎周大伟
Owner NANJING UNIV OF TECH
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