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Pyruvate high-temperature and high-yield engineered strain and application thereof

A technology of engineering strains and pyruvate, applied in the field of microbial metabolism engineering, can solve the problems of safety and cost in industrial production with limited application, and achieve the effect of reducing cooling energy consumption and reducing bacterial pollution

Active Publication Date: 2017-01-11
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In summary, pyruvic acid is an important chemical product widely used in industrial and scientific research such as pharmaceuticals, daily chemicals, and agricultural chemicals, but its application is limited by the safety and cost of industrial production

Method used

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  • Pyruvate high-temperature and high-yield engineered strain and application thereof
  • Pyruvate high-temperature and high-yield engineered strain and application thereof
  • Pyruvate high-temperature and high-yield engineered strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the preparation of bacterial strain

[0037] 1. The specific operation steps for extracting the yeast genome are as follows:

[0038] ①. Pick a single clone, insert it into 5ml liquid YPD, culture at 37°C, 250rpm for 24h.

[0039] ②. Collect the bacteria by centrifugation at 12000rpm at room temperature for 5sec, and discard the supernatant.

[0040] ③. Resuspend the bacteria in 500μl distilled water, centrifuge at 12000rpm for 5sec to collect the bacteria, and discard the supernatant.

[0041] ④. Take 200μl laboratory self-prepared 1x breaking buffer (TritonX-100 (2% (w / v)), SDS (1% (w / v)), NaCl (100mM), Tris-Cl (10mM, pH8. 0), EDTA (1mM)) to resuspend the bacteria, and transfer the bacteria solution into an EP tube containing 0.3g glass beads (425-600um, sigma, USA).

[0042] ⑤. After adding 200 μl phenol-chloroform solution, shake at high speed for 3 minutes, then add 200 μl 1x TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). Slight shock.

[0043] ⑥. Centri...

Embodiment 2

[0152] The engineering strain fermentation situation of embodiment 2, construction

[0153] This example is used to test the effect of engineering strains to produce pyruvate by co-fermentation of xylose and glucose. The results showed that by engineering Kluyveromyces marxe, the obtained engineered strain could co-ferment glucose and xylose to produce pyruvate at high temperature (42°C). In addition, almost no by-product glycerol was produced during the fermentation process due to knockout of KmGPD1.

[0154] 1. Recover strain YZB058 on a YPD medium plate and culture at 37°C for 1 day.

[0155] 2. Pick a single clone and connect it to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0156] 3. Prepare 30ml xylose glucose culture based on 250ml Erlenmeyer flask. Formula: 20g / l xylose, 40g / l glucose sugar, 10g / l yeast extract, 20g / L bacteriological peptone. Sterilized and ready to use.

[0157] 4. Take an appropriate amount of overnight culture and insert it into 30ml xyl...

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Abstract

The invention uses Kluyveromyces marxianus yeast as a platform to construct a strain of knockout pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase by means of genetic engineering, metabolic engineering, molecular biology and synthetic biology. The strain's growth is restored by metabolic regulation in order to improve the yield and production rate of pyruvate, and then the strain's xylose metabolism is improved, so that it has the ability to produce pyruvate with xylose. Eventually the glucose effect of the engineering strain is removed so that the engineered strain is capable of simultaneously saccharifying lignocellulose, using glucose and xylose and fermenting at high temperature to produce pyruvate. The Kluyveromyces marxianus strain YZB058 obtained by the present invention can produce pyruvate at the same time using glucose and xylose at 42 DEG C. At 42 DEG C, YZB058 is able to produce 29.21 g / l of pyruvate with 40.97 g / l of glucose and 20.37 g / l of xylose for 36 h, the production rate is at a rate of 0.81 g / l / h, and the total yield is at 0.48 g / g.

Description

technical field [0001] The invention relates to the fields of microbial metabolic engineering, sugar metabolism and co-utilization, and microbial fermentation to produce pyruvate. Specifically, the present invention provides a heat-resistant engineered yeast capable of co-fermenting glucose and xylose to produce pyruvate at a higher temperature (42° C.). Background technique [0002] The synthesis methods of pyruvate mainly include chemical synthesis, enzymatic conversion and fermentation. The chemical synthesis method oxidizes lactate (or tartaric acid) into pyruvate in liquid or gas phase, and then hydrolyzes it into pyruvate. It is the main method of pyruvate production at present, and industrial production has been realized. However, the chemical synthesis method has obvious disadvantages such as serious pollution and high cost (Causey et al., 2004). [0003] Enzymatic synthesis of pyruvate has become another hot spot in the research of biotechnology production of pyru...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P7/40C12R1/645
CPCC07K14/39C12N9/0006C12N9/1205C12N9/88C12N9/93C12P7/40C12Y101/01008C12Y101/01009C12Y101/01307C12Y207/01017C12Y401/01001C12Y603/01002
Inventor 洪泂张标王冬梅朱夜琳
Owner UNIV OF SCI & TECH OF CHINA
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