Construction method and application of heat-resistant yeast engineering strain producing xylitol under high-temperature aerobic condition

A technology of engineering strains and heat-resistant yeast, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve problems such as ethanol accumulation, low ratio, unfavorable product purification, etc.

Active Publication Date: 2020-09-15
HUAIBEI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this strain is that the conversion ratio of unit glucose to xylose is relatively low, 1g of xylitol can be synthesized for every 2g of glucose consumed, and there is accumulation of ethanol at the end of fermentation, which is not conducive to the purification of the product[16]
The heat-resistant yeast engineering strain YHY013 that the applicant participated in constructing in the early stage can synthesize about 60g / L xylitol by using 20g / L glucose and 80g / L xylose in total. Since xylose is used up, xylose is subsequently consumed as a source of energy and reducing power, so the conversion rate of xylose is low, about 0.75g / g[7]
[0007] In summary, xylitol, as an important compound, is widely used in food, medicine, chemical industry and other fields, but its application is limited by the safety and high cost of industrial production

Method used

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  • Construction method and application of heat-resistant yeast engineering strain producing xylitol under high-temperature aerobic condition
  • Construction method and application of heat-resistant yeast engineering strain producing xylitol under high-temperature aerobic condition
  • Construction method and application of heat-resistant yeast engineering strain producing xylitol under high-temperature aerobic condition

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the preparation of bacterial strain

[0037] 1. Introducing exogenous DNA into heat-resistant yeast

[0038] (1) yeast chemical transformation step

[0039] ①All kinds of modified strains were streaked on the YPD plate and cultured at 37°C for 24 hours.

[0040] ②Take 5mL of liquid YPD, and pick single clones on the YPD plate, culture at 37°C, 250rpm for 18h.

[0041] ③Take 1 mL of the culture and transfer it to a 50 mL Erlenmeyer flask filled with 9 mL of liquid YPD, culture at 37°C, 250 rpm, on a shaker for 5 hours.

[0042] ④ Take out the culture, centrifuge at 5000rpm at room temperature for 3min, discard the supernatant, and keep the bacteria.

[0043] ⑤ Prepare 1mL conversion buffer: 800μL 50% PEG4000; 50μL 4M lithium acetate; 50μL ddH 2 O; 100 μL 1M DTT (dissolved in 10 mM sodium acetate, pH 5.2).

[0044] ⑥Use 200 μL of transformation buffer to resuspend the bacteria, centrifuge at 5000 rpm for 3 minutes, and remove the supernatant.

[0045] ...

Embodiment 2

[0076] Embodiment 2: the engineering strain fermentation situation of construction

[0077] This embodiment is used to test the effect of engineering strains co-utilizing glucose and xylose to ferment xylitol. The results showed that the engineered strain obtained by transforming heat-resistant yeast could efficiently produce xylitol by aerobic fermentation at high temperature (42°C), and almost no by-products were contained in the fermented liquid after fermentation.

[0078] 1. Resuscitate strain YZB194 on a YPE medium plate and culture at 37°C for 1 day.

[0079] 2. Pick a single clone, inoculate in 5mL liquid YPE medium, 37°C, 250rpm, overnight.

[0080] 3. Seed cultivation. Transfer 5mL of liquid YPE medium to 250mLYPE, culture at 37°C, 250rpm, for 48h.

[0081] 4. Preparation of 2.5 LYPDX (60g / L glucose and 100g / L xylose) cultured in a 5L fermenter, sterilized for use.

[0082] 5. Put 250mL seed culture medium into 2.5L fermentation medium, ferment at 42°C, rotation ...

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Abstract

The invention discloses a heat-resistant yeast engineering strain producing xylitol under a high-temperature aerobic condition and a construction method and application thereof. The heat-resistant yeast engineering strain is a heat-resistant yeast engineering strain YZB194, and the classification name of the strain is Kluyveromyces marxianus, the strain is collected in the China General Microbiological Culture Collection Center (CGMCC) in No.3 Yard 1, Beichen West Road, Chaoyang District, Beijing on November 25, 2019 with the collection number of CGMCCC NO.19004. YZB194 synthesizes 203g/L xylitol by using 121 g/L glucose and 205 g/L xylose by batched supplement fermenyation with a fermentation tank under the aerobic condition of 42 DEG C, the synthesis rate of xylitol is 2.99g/L/h, and theconversion rate of xylitol reaches 99% of the maximum theoretical value.

Description

technical field [0001] The invention belongs to the field of microbial metabolism engineering and microbial fermentation engineering, and specifically relates to a heat-resistant yeast engineering strain producing xylitol under high-temperature aerobic conditions, a construction method and application thereof. Background technique [0002] Xylitol is a five-carbon polyol that is a normal intermediate in xylose metabolism [11]. When it is used by the human body, it does not need the promotion of insulin, and it can promote the secretion of a small amount of insulin in the body; when it is used for intravenous infusion, the content of pyruvic acid, lactic acid and glucose in the blood will decrease, and the liver glycogen will increase. ; Non-cytotoxic, can pass through the cell membrane and become the nutrition of the organization. These characteristics make xylitol have important application value. In addition, as a sweetener, xylitol has a cool taste and can prevent denta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/81C12P7/18C12R1/645
CPCC12N9/92C12N9/0006C12Y503/01009C12Y101/01009C07K14/39C12N15/815C12P7/18Y02E50/10
Inventor 张标任丽丽李峰徐大勇曾昕
Owner HUAIBEI NORMAL UNIVERSITY
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