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A kind of Yarrowia esterolytica yw100-1 and its application

A technology of Yarrowia and yeast, which is applied in the field of construction of Yarrowia lipolytica engineering bacteria, can solve the problems that need to be improved, cannot be accumulated in large quantities, and the degradation rate of pyruvate is fast.

Active Publication Date: 2021-07-27
浙江英沃迪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although Yarrowia lipolytica has a good ability to produce pyruvate, the conversion rate and synthesis rate of pyruvate from glycerol still need to be improved, and the degradation rate of pyruvate is too fast to accumulate in large quantities.

Method used

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  • A kind of Yarrowia esterolytica yw100-1 and its application
  • A kind of Yarrowia esterolytica yw100-1 and its application
  • A kind of Yarrowia esterolytica yw100-1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of Yarrowia lipolytica genetically engineered bacteria with high pyruvate production

[0038] Process such as figure 1 As shown, the construction steps of Yarrowia lipolytica YW100-1 are as follows:

[0039] 1. Construction of E14 plasmid

[0040] Amplification of GUT1: By designing primers YW238 and YW239, GUT1 was amplified with the genome of wild-type Yarrowia lipolytica AS2.1405 (purchased from Guangdong Microbial Culture Collection Center, numbered GIM 2.187) as a template, with a size of about 1.5kb, the nucleotide sequence is shown in SEQ ID NO.1.

[0041] Amplification of the promoter pEXP1: by designing primers YW405 and YW240, using the wild-type Yarrowia lipolytica AS2.1405 genome as a template, pEXP1 was amplified, with a size of about 1 kb and a nucleotide sequence of SEQ ID NO.9 Show.

[0042] Amplification of GUT2: By designing primers YW241 and YW242 and using the wild-type Yarrowia lipolytica AS2.1405 genome as a template, GU...

Embodiment 2

[0092] Embodiment 2: the comparison of the shake flask fermentation of wild-type AS2.1405 and recombinant engineered bacteria YW100-1

[0093] The wild-type Yarrowia lipolytica strain AS2.1405 and the recombinant engineered strain YW100-1 were respectively inoculated in YPD liquid medium, and cultured in a 250mL Erlenmeyer flask at 30°C and 200rpm for 24h to obtain seed liquid; Concentration OD 600 =0.05 were inoculated in YNG medium, each strain was analyzed in three parallel groups, fermented at 30°C, 200rpm for 16h to OD 600 When about 4.5, get 10mL fermented liquid and measure the concentration of pyruvic acid and glycerin with high performance liquid chromatography, the result sees figure 2 .

[0094] Determination of pyruvate concentration: take 10 mL of fermentation broth, centrifuge at 6000 rpm for 5 min, collect the supernatant, and measure the content of pyruvate with a C18 column. Mobile phase: 0.1% phosphoric acid; flow rate: 1ml min -1 ; Injection temperature...

Embodiment 3

[0096] Embodiment 3: the comparison of wild-type AS2.1405 and engineering bacteria YW100-1 fermenting and producing pyruvate in a 20L fermenter

[0097] Seed medium: 2g / L glycerol, 0.4g / L tryptone, 0.2g / L yeast extract, 0.24g / L KH 2 PO 4 , 1.7g / L K 2 HPO 4 ·3H 2 O, the solvent is distilled water, and the pH value is natural.

[0098] Fermentation medium composition: 60g / L glycerol, 10g / L (NH 4 ) 2 SO 4 , 1.4g / L MgSO 4 ·7H 2 O, 2g / L KH 2 PO 4 , 0.8g / L CaCl 2 , 0.5g / L NaCl, 1μg / L vitamin B1, the solvent is distilled water, and the pH value is natural.

[0099]Inoculate the wild-type Yarrowia lipolytica strain AS2.1405 and the engineered strain YW100-1 in YPD liquid medium, culture overnight at 30°C, then transfer 200ml to 3L seed medium, and culture in shake flasks at 30°C for 18h, then Inoculate in a 20L fermenter equipped with 12L fermentation medium with a volume concentration of 10% inoculum, and ferment for 1 hour. During the whole fermentation and cultivation...

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Abstract

The invention discloses Yarrowia lipolytica YW100‑1 and its application. The Yarrowia lipolytica YW100‑1 overexpresses glycerol kinase GUT1, glycerol kinase GUT2, and glycerol in Yarrowia lipolytica. Dehydrogenase enzyme GCY1, dihydroxyacetone kinase DAK1, dihydroxyacetone kinase DAK2 and NADH kinase POS5 were obtained by simultaneously knocking out the diacylglycerol acyltransferase gene DGA2 and the 3‑phosphate glycerol dehydrogenase gene GPD2 in Yarrowia lipolytica of. When the bacterial strain of the invention uses glycerol as a carbon source, the output of pyruvate increases by 66.2 percent to 121.2 g / L, and provides an excellent bacterial strain for the industrial production of pyruvate.

Description

[0001] (1) Technical field [0002] The invention relates to a construction method and application of Yarrowia lipolytica engineering bacteria, belonging to the technical field of bioengineering. [0003] (2) Background technology [0004] Pyruvate, as an intermediate in the microbial metabolic pathway, is also one of the important organic acids, and has a wide range of functions in the fields of biochemical, pharmaceutical, food and science. In the pharmaceutical industry, pyruvate is an important pharmaceutical intermediate, which can be used to synthesize levodopa, anti-inflammatory and analgesic cinecofen, anti-tuberculosis drug isoniazid calcium pyruvate, thimidazole drugs, etc. In addition, pyruvate ( Such as calcium pyruvate, potassium pyruvate, creatine pyruvate, etc.) are widely used in weight loss health care products. In the food industry, as a food additive, pyruvic acid has natural antiseptic and fresh-keeping effects. In the chemical industry, ethyl pyruvate is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/40C12R1/645
CPCC12N9/0006C12N9/1029C12N9/1205C12N15/815C12P7/40C12Y101/01006C12Y101/05003C12Y203/0102C12Y207/01029C12Y207/0103C12Y207/01086
Inventor 袁围钟爽孙杰汪钊
Owner 浙江英沃迪生物科技有限公司
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