Yeast with high tolerance to inhibitors derived from lignocellulosic biomass and construction method thereof

A lignocellulosic and biomass technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as increasing production costs, increasing saccharification and fermentation production costs, and hindering the commercialization of cellulosic ethanol

Active Publication Date: 2021-10-19
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But there are the following problems: commonly used pretreatment processes such as acid method and alkali method will produce a large number of inhibitors, including weak acids, furans and phenolic compounds, which seriously affect the subsequent microbial reproduction, cell activity, product yield and production rate , thus increasing production costs for subsequent steps such as saccharification and fermentation
If the first strategy is adopted in biofuel production, the inhibitor removal procedure will greatly increase the production cost and hinder the commercialization of cellulosic ethanol. Therefore, improving the inhibitor tolerance of fermenting microorganisms is considered to be a better solution

Method used

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  • Yeast with high tolerance to inhibitors derived from lignocellulosic biomass and construction method thereof
  • Yeast with high tolerance to inhibitors derived from lignocellulosic biomass and construction method thereof
  • Yeast with high tolerance to inhibitors derived from lignocellulosic biomass and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Changes in the expression level of the nitroreductase KmHBN1 gene in the presence of different inhibitors.

[0048] First, the inventors measured the changes in the expression level of the nitroreductase KmHBN1 gene in the original strain YHJ010 in the presence of different inhibitors by Real-time PCR. Five groups were set up, namely no inhibitor group, inhibitor mixture group (acetic acid, furfural+5-hydroxymethylfurfural, phenolic mixture (catechol, syringaldehyde, vanillin, 4-hydroxybenzaldehyde )), acetic acid inhibitor group, phenolic mixture inhibitor group (catechol, syringaldehyde, vanillin, 4-hydroxybenzaldehyde), furan mixture inhibitor group (furfural+5-hydroxymethylfurfural) .

[0049] The specific operation is as follows.

[0050] 1. Take out the strain YHJ010 from the -80°C refrigerator, inoculate it into YPD solid medium according to the usual method, and culture it overnight at 37°C.

[0051] 2. On the next day, pick a single clone in YPD li...

Embodiment 2

[0087] The construction of embodiment 2 nitroreductase gene KmHBN1 overexpression bacterial strain:

[0088] 1. Construction of nitroreductase expression vector.

[0089] 1) Cloning of nitroreductase gene. According to the sequence design of the nitroreductase gene (GenBank accession No.: MT383676) in Genbank, primers 10054Up-F (SEQ ID No: 5) and 10054Down-R (SEQ ID No: 6). Using the genomic DNA of the YHJ010 strain as a template, PCR was performed using PrimeSTAR HS DNA polymerase (Dalian Baobio) to obtain a fragment with a length of about 1.7 kb. The fragment was treated with A by the usual method and ligated with pGEM-T Easy (Promega, USA) to form plasmid pZN001. The specific construction process is as follows:

[0090] (1) Extraction of Kluyveromyces marx genome

[0091] a) Take out the preserved yeast strain from the refrigerator at -80°C and inoculate it in YPD solid medium, and culture it overnight in a 37°C incubator.

[0092] b) Pick colonies and inoculate them ...

Embodiment 3

[0145] Example 3 Tolerance test of bacterial strain YZN013 to various inhibitors

[0146] In this example, the inventors tested the tolerance of the nitroreductase gene overexpression mutant strain YZN013 to various inhibitors. The experiment was divided into two groups, the control group (strain YZN012, which does not express nitroreductase, but the auxotrophy is the same as YZN013) and the overexpression group (YZN013).

[0147] 1) Streak the above-mentioned strains YZN012 and YZN013 onto YPD solid medium and cultivate them in a 37°C incubator;

[0148] 2) Pick a single clone into 5mL YPD liquid medium and culture overnight (37°C, 250rpm) to the logarithmic phase;

[0149] 3) Configure synthetic medium (yeast nitrogen base, YNB, Shanghai Sangong) 0.67%, 2% glucose, inhibitor mixture) liquid medium containing inhibitor mixture, and distribute 30mL into Erlenmeyer flasks ;

[0150] Here, the inhibitor mixture is 1.9g / L acetic acid+0.95g / L furan derivative (furfural and 5-hy...

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Abstract

The invention provides a Kluyveromyces marxianus with the preservation number of CGMCC No.21814. The Kluyveromyces marxianus has excellent tolerance to various toxic inhibition substances, including weak acid compounds, furan compounds and phenolic compounds, which are generated in the pretreatment process of lignocellulose biomass. The culture delay phase of the strain in an inhibitor mixture is obviously shortened, the yield and production speed of ethanol in anaerobic culture containing inhibitors and glucose are improved, and the production rate of ethanol is improved when pretreated corncobs are used for fermentation. The invention also provides a method for constructing the strain and a method for improving the tolerance of the kluyveromyces marxianus to various toxic inhibitors derived from lignocelluloses.

Description

technical field [0001] The invention relates to the field of microorganisms and biotechnology. In particular, it relates to a heat-resistant Kluyveromyces marx with improved biological characteristics, a method for constructing it and a method for improving the tolerance of bioengineered bacteria to inhibitors when utilizing lignocellulosic biomass. Background technique [0002] Due to the advantages of wide sources, low cost, and less pollution, lignocellulose is favored by researchers as a bioenergy and green manufacturing. In lignocellulose, cellulose, hemicellulose, and lignin are tightly combined through molecular bonds to form a complex structure that is not easy to degrade. [0003] Lignocellulose is mainly composed of cellulose, hemicellulose, lignin and some inorganic compounds, which are tightly combined to form a polymer compound that is not easy to degrade. The compact and complex structure of lignocellulose makes the hydrolysis efficiency of cellulase low. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/10C12N15/81C12R1/645
CPCC12N9/0006C12P7/10C12N15/815C12Y101/01284Y02E50/10
Inventor 洪泂张妮妮王冬梅
Owner UNIV OF SCI & TECH OF CHINA
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