Yeast engineering bacterium of high productive glycerin

A yeast engineering, yeast technology, applied in the fields of fermentation and biology, can solve the problem of unsatisfactory glycerol yield

Inactive Publication Date: 2007-06-06
SI CHUAN HEBEN BIOTIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the glycerol yield of powdered Pichia pastoris is still unsatisfactory

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Cloning of 3-phosphate glycerol dehydrogenase gene from Dunaliella salina

[0056] 1. Collection of Dunaliella salina

[0057] Dunaliella salina (Dunaliella salina) was purchased from the algae bank of Wuhan Institute of Hydrobiology.

[0058] 2. Poly A + RNA isolation (Poly A + RNA isolation)

[0059] Total RNA of Dunaliella salina was extracted with Trizol reagent (Gibco, NY, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis. Dunaliella salina mRNA was extracted using the kit instructions provided by Oligotex mRNA Kits (Qiagen).

[0060] 3. Construction of Dunaliella salina cDNA library (Cloning of Full-length cDNA)

[0061] The cDNA phage library of Dunaliella salina was constructed using the kit instructions provided by the Smart cDNA Library Construction Kit (Clon Tech) with λTriplEX2 as the vector.

[0062] 4. Cloning of Full-length cDNA

[0063] By comparing the 3-phosphate glycerol dehydrogenases of a large numb...

Embodiment 2

[0129] Isolation of Pichia powder pastoris strains

[0130]From the molasses of Leshan Qianwei Sugar Factory, the hypertonic medium was used as the selective medium (60% glucose YPD medium), and the hypertonic yeast was isolated, which was confirmed as powdery Pichia pastoris through microbial identification, and could be produced in 70% It grows in the above concentrations of glucose medium and has the ability to produce glycerol.

Embodiment 3

[0132] Construction of Powdery Pichia Genetic Engineering Strain Kaiya-0917

[0133] (1) Competent preparation of powdered Pichia pastoris

[0134] a. Use a sterile toothpick to pick a single colony of Pichia pastoris powder (Example 1) from the YEPD plate and place it in 2 mL of liquid YEPD medium, culture at 28° C. with shaking at 200 rpm overnight.

[0135] b. The next day, transfer to 200mLYEPD medium at 28°C and shake at 200rpm until OD 600 = Between 1.0 and 1.3.

[0136] c. Transfer the cells to a 50 mL centrifuge tube at 4000 rpm, centrifuge at 4°C for 10 min, and discard the supernatant.

[0137] d. Add an equal volume of deionized water into the centrifuge tube, resuspend the cells, and centrifuge at 4000rpm at 4°C for 10min. Discard the supernatant.

[0138] e. Add half the volume of deionized water into the centrifuge tube, resuspend the cells, and centrifuge at 4000rpm at 4°C for 10min. Discard the supernatant.

[0139] f. Add half volume of 1M sorbitol into ...

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Abstract

The invention has supplied a yeast germ for producing glycerine with high productivity and its preparing method and application. In the yeast germ, the Dunaliella salina glycerol phosphate dehydrogenize is introduced, so that the glycerine with good quality can be produced with high productivity and low cost.

Description

technical field [0001] The invention relates to the fields of fermentation and biotechnology, and more particularly relates to a high-yield glycerol-yielding yeast engineered bacterium and its preparation method and application. Background technique [0002] As a basic chemical raw material, glycerin is widely used in thousands of products in food, military industry, daily chemical medicine and other industries. In China, it has always been extracted from the by-products of oil saponification. Its output has long been unable to meet the needs of social development. In 2001, the domestic glycerin output was 40,000 tons, and the import was 40,000 tons; with the improvement of living standards, the gap of glycerin will further expand. [0003] The production of glycerin mainly includes synthesis method, oil saponification product extraction method and fermentation method. With the continuous development of industrial production and the improvement of people's living standards...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N9/04C12N15/53C12N15/81C12P7/20C12P1/02
Inventor 王小行李钢杨滔蒋彦杨会强曾昌耀
Owner SI CHUAN HEBEN BIOTIC ENG
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