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Construction and application of engineering strains that co-utilize glucose and xylose to produce xylitol

A glucose and xylitol technology, applied to the biological field, can solve the problem of not fully utilizing the glucose and xylose co-utilization ability of heat-resistant yeast

Active Publication Date: 2021-08-13
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventor's previous research realized the co-utilization of glucose and xylose by expressing exogenous xylose-specific transporters in Kluyveromyces marxe, and the resulting final strain YZJ119 could utilize corncobs (including corncob residues (CCR) and hydrolyzate) simultaneously produce 44.58g / L ethanol and 32.03g / L xylitol (Zhang et al., 2016), but the glucose and xylose co-utilization of this strain is only realized at the level of sugar transport, Glucose and xylose co-utilization capacity of thermotolerant yeast has not been fully exploited

Method used

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  • Construction and application of engineering strains that co-utilize glucose and xylose to produce xylitol
  • Construction and application of engineering strains that co-utilize glucose and xylose to produce xylitol
  • Construction and application of engineering strains that co-utilize glucose and xylose to produce xylitol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Preparation of strains:

[0063] 1. Construction of a heat-resistant yeast strain produced by glucose and xylose to produce xylitol:

[0064] Reconstruction of genetically modified strains is performed by heat-resistant yeast YHJ010 (see HONG ET Al., 2007).

[0065] 1) Yeast Chemical Transformation Steps:

[0066] 1. Waiting to be transformed into the YPD board, cultured overnight at 37 ° C.

[0067] 2. Pick a monoclonal from the YPD plates of overnight culture, inoculated into 5 ml of fresh YPD liquid medium, 37 ° C, 250 rpm, and cultured overnight.

[0068] 3. Take 500 μl of culture to transfer to a 5 ml fresh YPD liquid medium test tube, 37 ° C, 250 rpm, and shaker for 5 h.

[0069] 4. Take out the culture, centrifuge at normal temperature, 3 minutes, discard the supernatant, and retain the bacteria.

[0070] 5. Formulated 500 μL of converted buffer: 400 μL 50% PEG4000; 50 μl of 2M acetate; 50 μl of 1 M DTT (dissolved in 10 mM sodium acetate, pH 5.2).

[0071]...

Embodiment 2

[0160] Example 2. Water-resistant yeast strain glucose inhibitory release

[0161] This example is used to verify that the engineered strain in the present invention is compared to whether the glucose inhibitory effect is released in the wild-type strain. The results show that the glucose inhibitory effect of YLM001 has been greatly released, which can grow in a variety of non-glucose carbon sources of 2-deoxyl glucose (2-DG), and KMGLK knockout is not in the glucose inhibitory effect. Any effect is produced; KmmiG1 is only a slight growth advantage compared to the control YWD016 compared to the control YWD016 compared to the control YWD016 compared to the control YWD016 compared to the control YWD016, which contains 2-DG. The growth state of the glycerol culture is basically consistent with the control, indicating that Kmmig1 knockout inhibition in glucose is not released inhibition of xylose and glycerol utilization; at the same time verifying the YLM005 obtained in YLM001, the ...

Embodiment 3

[0167] Example 3. Fermentation conditions of transforming strains under conditions of 42 ° C

[0168] This example is used in comparison of different engineered strains for glucose and xylose sharing effect. The results indicate that the strain YHY013 of all engineering genes is used to use glucose and xylose to produce xylitol.

[0169] 1. In the YPD medium plate, the strain was recovered: control strain: yzj119, experiment strain: YLM005, YHY006, YHY008 and YHY013, 37 ° C for 1 day.

[0170] 2. Pick a monoclonal, followed by 5 ml of liquid YPD medium. 37 ° C, 250 rpm, overnight.

[0171] 3. Formulated 30 ml of fermentation medium is dispensed into 250 ml tapered flask. Formulation: 20g / L glucose, 80 g / L xylose, 10g / L yeast extract, 20 g / L bacteriology protein, sterilization.

[0172] 4. Take an appropriate amount of overnight culture to access 30 ml of xylose fermentation medium to make them initial OD 600 It was cultured in 1,42 ° C, 250 rpm.

[0173] 5. Sampling at 0H,...

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Abstract

The present invention finds that the glucose inhibitory effect in the heat-resistant yeast with hexokinase KmHXK knockout is eliminated to a large extent, realizing the co-utilization of various sugars and glucose, and overexpressing glucokinase KmGLK can restore the glucose metabolism after KmHXK knockout In the present invention, it is found that the bacterium still maintains the characteristics of releasing the glucose inhibitory effect, and can be used as a platform strain; the present invention successfully obtains a heat-resistant yeast strain YHY013 that can efficiently co-utilize glucose and xylose to produce xylitol with this platform strain. The deposit number is CGMCC No.15347. The strain can effectively utilize glucose and xylose in natural fermentation substrates such as xylose mother liquor or corncob hydrolyzate to ferment xylitol under the condition of 42°C, and the yield of xylitol produced by using corncob hydrolyzate exceeds Other strains have been reported.

Description

Technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention has found that the glucose inhibitory effect in heat-resistant yeast in hexin kmhxk knockout is greatly eliminated, achieving a variety of sugars and glucose, and can restore KmHxk knockout after the glucose kinase Kmglk can restore the glucose knockout The influence of metabolism, in the present invention, the bacterium is found to maintain the characteristics of the glucose inhibitory effect, which can be used as a platform strain; the present invention is based on the platform strain, and the formation of the xylose metabolic path of the bacteria is modified, and it can be efficient. The heat-resistant yeast strain YHY013 with glucose and xylose was produced by glucose and xylose, which was deposited as CGMCC 15347. The strain can effectively produce xylitol and xylose fermentation in a natural fermentation substrate such as xylose, or corn hydrolyzate, and pr...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P7/18C12R1/645
CPCC12N9/1205C12P7/18C12Y207/01001
Inventor 洪泂华艳王冬梅
Owner UNIV OF SCI & TECH OF CHINA
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