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Heat-resistant engineered yeast strains for high-temperature high-efficiency xylose fermentation and application of heat-resistant engineered yeast strains

A technology of yeast strains and heat-resistant yeast, which is applied in application, fermentation, genetic engineering, etc., can solve the problem of low fermentation capacity and achieve the effect of increasing ethanol production

Inactive Publication Date: 2013-01-16
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This engineered bacterium solves the problem of low xylose fermentation ability of heat-resistant yeast at high temperature, and the ethanol production is greatly increased. Compared with the previous Saccharomyces cerevisiae system, there is no product accumulation of xylitol and glycerol

Method used

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  • Heat-resistant engineered yeast strains for high-temperature high-efficiency xylose fermentation and application of heat-resistant engineered yeast strains
  • Heat-resistant engineered yeast strains for high-temperature high-efficiency xylose fermentation and application of heat-resistant engineered yeast strains
  • Heat-resistant engineered yeast strains for high-temperature high-efficiency xylose fermentation and application of heat-resistant engineered yeast strains

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The preparation of embodiment 1 bacterial strain

[0049] 1. Optimize the XI codon

[0050] The XI codon derived from the fungus Orpinomyces.sp (rumen fungus) is not necessarily the optimal codon in yeast, and if the original XI gene is used directly, the expression level may be low or even difficult to express. Under the premise of not changing the amino acid sequence, the codons in K. marxianus yeast were used for optimization, and primers for cloning were added at both ends. The obtained codon-optimized xylose isomerase gene sequence has a length of 1334 bp, as shown in SEQ ID NO:1.

[0051] 2. Synthesis of XI gene

[0052] The XI gene is divided into four fragments F1, F2, F3, and F4, whose lengths are F1 (356bp), F2 (354bp), F3 (354bp), and F4 (354bp). The sequences are recorded as SEQ ID NOs: 2-5. Each fragment is passed through the Tmprime tool ( http: / / prime.ibn.a-star.edu.sg / ) was cut into 11 oligonucleotides (including 6 positive strands (A strand) a...

Embodiment 2

[0205] Determination of xylose isomerase activity in the domesticated strain of embodiment 2

[0206] 1. Cultivate the acclimated strains YRL005, YHJ010 and YRL003 in 5ml liquid YPX medium for 48 hours.

[0207] 2. The bacterial cells were recovered by centrifugation at 1500 rpm at 4° C. for 10 min, and washed twice with 500 μl sterile water.

[0208] 3. Suspend the cells in 1ml buffer A (50mM Tris-HCl, 25mM NaCl, pH 8.0).

[0209] 4. The resulting suspension was disrupted by sonication. Vibra-Cell VC505 sonicator (Connecticut, USA) was used at 40% intensity for 10 min.

[0210] 5. Centrifuge at 15,000 rpm for 20 minutes at 4° C., and recover the supernatant as a crude enzyme solution.

[0211] 6. Configure 275 μl of xylose isomerase enzyme activity reaction system, including 100 μl of crude enzyme liquid and final concentration of 50 mM Na 3 PO 4 buffer (pH 8.0) and 40mM MgCl 2 .

[0212] 7. Add xylose with a final concentration of 20mM to start the enzyme activity r...

Embodiment 3

[0218] Embodiment 3 The growth situation of the domesticated bacterial strain under the synthetic medium

[0219] This example is used to understand the growth of the domesticated strain, its comparison with the wild-type strain NBRC1777 and its improvement over the strain YRL003 before domestication.

[0220] 1. Recover strains on YPD medium plates. Wild strain: NBRC1777. Domesticated strain: YRL005. Xylose metabolic pathway knockout strain: YRL002. Strain before acclimatization: YRL003. Cultured at 37°C for 1 day.

[0221] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0222] 3. Prepare 12 bottles of 50ml xylose synthesis medium and pack them in 250-ml Erlenmeyer flasks. Formula: 20g / l xylose, 6.7g / l YNB. Add amino acids respectively: leucine (30mg / ml), uracil (20mg / ml), tyrosine (20mg / ml). Sterilized and ready to use.

[0223] 4. Take an appropriate amount of overnight culture and transfer it to a ste...

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Abstract

The invention relates to heat-resistant engineered yeast strains for xylose fermentation. A heat-resistant yeast strain Kluyveromyces marxianus in which xylose reductase and xylitol dehydrogenase genes are knocked out is taken as a host, and a heat-resistant yeast expression vector containing a xylose isomerase gene of which a codon is optimized is transformed into the host. The invention also relates to domesticated strains of the heat-resistant engineered yeast strains for the xylose fermentation, a preparation method for the domesticated strains and application of the domesticated strains.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the field of improving xylose fermentation through engineering bacteria transformation. Background technique [0002] The increase of greenhouse gas emissions and global warming, the depletion of petroleum resources and the increasing energy demand, as well as national energy security issues make the utilization and development of renewable energy one of the most important issues in the 21st century. Among them, the production of fuel ethanol from biomass is an important direction. Lignocellulose is the most abundant renewable biological resource on earth (about 200X10 9 tons of production), China produces about 1 billion tons of agricultural and forestry wastes each year (Reddy and Yang, 2005; Yinbo et al., 2006), but only 3% of them are used in non-food fields such as paper and pulp (Kamm and Kamm, 2004). [0003] Natural lignocellulose is mainly plant fiber raw material, and it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/61C12P7/06C12R1/645
CPCY02E50/17Y02E50/10
Inventor 洪泂汪荣亮高晓连
Owner UNIV OF SCI & TECH OF CHINA
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