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Construction and application of heat-resistant yeast engineering bacteria capable of producing glycerine under high temperature aerobiotic condition

A technology of heat-resistant yeast and engineering bacteria, which is applied in the field of construction of heat-resistant yeast engineering bacteria, and can solve problems such as safety and cost of industrial production with limited application.

Active Publication Date: 2020-05-01
HUAIBEI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] To sum up, glycerin is an important chemical product widely used in medicine, daily chemical industry, food and other industries and scientific research, but its application is limited by the safety and cost of industrial production

Method used

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  • Construction and application of heat-resistant yeast engineering bacteria capable of producing glycerine under high temperature aerobiotic condition
  • Construction and application of heat-resistant yeast engineering bacteria capable of producing glycerine under high temperature aerobiotic condition
  • Construction and application of heat-resistant yeast engineering bacteria capable of producing glycerine under high temperature aerobiotic condition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the preparation of bacterial strain

[0045] 1. The specific steps for extracting the yeast genome are as follows:

[0046] ①Pick a single clone, insert it into 5mL liquid YPD, culture at 37°C, 250rpm, for 24h.

[0047] ② Centrifuge at 12000rpm at room temperature for 5sec to collect the bacteria, and discard the supernatant.

[0048] ③ Resuspend the bacteria in 500 μL of distilled water, centrifuge at 12,000 rpm for 5 sec to collect the bacteria, and discard the supernatant.

[0049] ④Take 200 μL of 1x breaking buffer prepared by the laboratory (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).

[0050] ⑤ After adding 200 μL of phenol-chloroform solution, shake at high speed for 3 minutes, add 200 μL of 1x TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), and shake slightly.

[0051] ⑥Centrifu...

Embodiment 2

[0165] Embodiment 2: the engineering strain fermentation situation of construction

[0166] This example is used to test the effects of engineering strains on producing glycerol by fermentation of glucose, fructose and xylose. The results showed that the engineered strain obtained by transforming heat-resistant yeast could efficiently produce glycerol by aerobic fermentation at high temperature (42°C), and almost no by-products were contained in the fermentation broth after fermentation.

[0167] 1. Resuscitate strain YZB115 on a YPD medium plate and culture at 37°C for 1 day.

[0168] 2. Pick a single clone, inoculate in 5mL liquid YPE medium, 37°C, 250rpm, overnight.

[0169] 3. Seed cultivation. Transfer 5mL of liquid YPE medium to 250mLYPE, culture at 37°C, 250rpm, for 48h.

[0170] 4. Preparation 2.5 LYPD, YPF and YPX are cultured in a 5L fermenter and sterilized for use.

[0171] 5. Put 250mL seed culture medium into 2.5L fermentation medium, ferment at 42°C, rotatio...

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Abstract

The invention discloses construction and application of heat-resistant yeast engineering bacteria capable of producing glycerine under high temperature aerobiotic condition. A heat-resistant kluyveromyces marxianus is used as a platform, methods of genetic engineering, metabolic engineering and the like are utilized, a strain having triose phosphate isomerase (TPI1) deletion is constructed, and the constructed strain has the capacity of efficiently producing the glycerine through efficient utilization of glucose, levulose and xylose under the high temperature aerobiotic condition. The obtainedkluyveromyces marxianus strain YZB115 produces 40.32 g / L glycerine through 80g / L glucose, produces 41.84 g / L glycerine through 80g / L levulose and 18.64g / L glycerine through 80g / L xylose under the aerobiotic condition of 42 DEG C, the production rates are respectively 0.84g / L / h, 0.50g / L / h and 0.22g / L / h, the yields are respectively 0.50g / g, 0.50g / g and 0.23g / g, in the fermentation process of the constructed engineering strain, a by-product namely ethanol is not produced, and xylitol is not accumulated when the operation of producing the glycerine through fermentation of xylose is ended.

Description

technical field [0001] The invention belongs to the field of microbial metabolism engineering and microbial fermentation engineering, and in particular relates to the construction and application of heat-resistant yeast engineering bacteria producing glycerol under high-temperature aerobic conditions. The heat-resistant yeast engineering strain of the invention can produce glycerin by fermenting three kinds of monosaccharides (glucose, fructose and xylose) at relatively high temperature (42 DEG C). Background technique [0002] Glycerin (scientific name glycerol) is an important light chemical raw material with a wide range of uses. Used in the paint industry to produce alkyd resins and phenolic resins; in the pharmaceutical industry as solvents and lubricants; in the food industry as sweeteners, humectants and monoglycerides; in the tobacco industry as solvents and lubricants Moisturizing agent; used as raw material of explosive nitroglycerin in the defense industry, antif...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/90C12P7/20C12R1/645
CPCC12N9/90C12N15/905C12P7/20C12Y503/01001Y02E50/10
Inventor 张标任丽丽李峰徐大勇王一晴
Owner HUAIBEI NORMAL UNIVERSITY
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