Acetic acid and xylose co-used Kluyveromyces marxianus strain and screening method

A technology of yeast strain and acetic acid is applied in the field of genetic breeding of industrial microorganisms, which can solve problems such as shortage and limit the application of cellulosic ethanol, and achieve the effects of good growth, reduction of production cost and improvement of yield.

Active Publication Date: 2019-04-09
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on its inhibitor resistance is still relatively

Method used

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  • Acetic acid and xylose co-used Kluyveromyces marxianus strain and screening method
  • Acetic acid and xylose co-used Kluyveromyces marxianus strain and screening method
  • Acetic acid and xylose co-used Kluyveromyces marxianus strain and screening method

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Screening of hydrolyzate-resistant yeast Kluyveromycesmarxianus DX-5

[0038] Straw steam explosion pretreatment liquid is obtained by using pulverized straw, material-water ratio 1:10, 1% sulfuric acid, 130°C, 2h. The hydrolyzate contains 30g / L xylose, 3g / L acetic acid and 0.9g / L formic acid. Add 2% glucose, 0.2% yeast extract, and 1% peptone to 10% extract hydrolyzate to make a medium for initial directed evolution. Inoculate the starting strain of Kluyveromycesmarxianus, the optical density value at 620nm (OD620) ≈ 0.3; culture at 40°C, 150rmp / min for 48 hours, when the OD620≥2.0, collect the cells; add 2% glucose to the 15% extraction hydrolyzate according to OD620 ≈ 0.3 , yeast extract 0.2%, and peptone 1% to continue directional domestication. Repeat this step to increase the hydrolyzate content in the acclimatization medium to 100%. But when OD620≥2.0, collect the cells, smear on the 100% hydrolyzate medium plate containing 1.5% agar, and pick a sin...

Embodiment 2

[0039] Embodiment 2: The growth status of Kluyveromycesmarxianus DX-5 in different concentrations of acetic acid

[0040] Kluyveromycesmarxianus DX-5 was cultured in YPD medium at 40°C for 24 hours, and then inoculated into different concentrations and different types of medium according to OD620≈0.3. Kluyveromyces marxianus DX-5 can grow with different concentrations of acetic acid as the sole carbon source, and adding organic nitrogen sources can promote the utilization of acetic acid compared with inorganic nitrogen sources. With the increase of acetic acid concentration, the growth rate of yeast cells decreased and the depletion time of acetic acid prolonged. In the organic nitrogen source, 13g / L acetic acid can be completely consumed in 130h, and the OD620 reaches about 2. See attached for specific results figure 2 , image 3 .

Embodiment 3

[0041] Example 3: KluyveromycesmarxianusDX-5 co-utilization of xylose and acetic acid status

[0042] Kluyveromycesmarxianus DX-5 was cultured in YPD medium at 40°C for 24 hours, and then inoculated into medium containing 10g / L acetic acid and 20g / L xylose according to OD620≈0.3.

[0043] Kluyveromycesmarxianus DX-5 can utilize both acetic acid and xylose. Compared with xylose growth without acetic acid, addition of high concentration of acetic acid decreased the rate of xylose utilization but increased xylitol production. See attached for specific results Figure 4 .

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Abstract

The invention discloses an acetic acid and xylose co-used Kluyveromyces marxianus strain with a biological preservation number of CGMCC No.16757. The strain is obtained by long-term orient domestication of Kluyveromyces marxianus through cellulosic hydrolysate and is prepared by the following steps: picking single yeast colonies from a YPD flat plate and carrying out 40 DEG C overnight culture; reinoculating collected cells to a fresh culture medium containing the cellulosic hydrolysate with certain concentration, enabling an optical density value of the culture medium at 620nm to be approximate to 0.3; culturing for 24 to 48 hours at 40 DEG C and 150rmp/min; when OD620 of the acetic acid and xylose co-used Kluyveromyces marxianus strain is greater than or equal to 2.0 (5 to 7 generations), collecting the cells; repeating the steps until the growth speed of the cells is obviously improved under the stress condition of an inhibitor with the concentration; improving the concentration ofthe cellulosic hydrolysate and starting new repeated culture; after continuous domestication, coating the YPD flat plate containing the cellulosic hydrolysate with the collected cells and culturing at40 DEG C for 24 to 48 hours; picking the single colonies with good growth situation and putting into a YPD liquid medium, and culturing at 40 DEG C and 150rmp/min for 24 to 48 hours; preserving dominant resistance clones in 30 percent glycerinum.

Description

technical field [0001] The invention belongs to the field of genetic breeding of industrial microorganisms, and relates to a Kluyveromyces marx strain and a screening method for co-utilization of acetic acid and xylose. Background technique [0002] Lignocellulose is the most abundant renewable biomass resource, and using it as a substrate for fuel ethanol production can meet my country's demand for energy and is conducive to environmental protection. The production of fuel ethanol from lignocellulose is of great economic and social significance. Inhibitors in cellulose hydrolyzate, especially the toxic effect of acetic acid on microorganisms, is one of the main bottlenecks facing the development of cellulosic ethanol (Palmqvist, E. & Hahn-Hagerdal, B. Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresour. Technol. 2000, 74, 17–24). Judging from the current literature reports, the acetic acid resistance of the cellulose hydrolyzate ferme...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N1/02C12R1/645
CPCC12N1/02C12N1/16C12N1/145C12R2001/645
Inventor 袁文杰杜聪相瑞娟李益民
Owner DALIAN UNIV OF TECH
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