Construction and application of high-temperature high-yield xylitol engineering strains

A xylitol, engineering technology, applied in the direction of fungi, microorganism-based methods, using vectors to introduce foreign genetic material, etc., can solve the problems of low xylitol concentration and efficiency, low rate, and production rate of only 0.24g

Inactive Publication Date: 2015-04-22
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] There have been some studies on the production of xylitol by fermenting xylose with thermostable yeast. Among them, Mueller et al. used K. hours, the production rate is only 0.24g / L / h (Mueller et al., 2011)
Prakash et al. used the isolated heat-resistant yeast Debaryomyces hansenii strain to ferment 100g / L xylose at 40°C to produce 68.6g / L xylitol, and the fermentation efficiency was 0.69g / g, but the rate was low, only 0.44g / L L / h (Prakash et al., 2011)
In addition, there are some studies that use xylose to ferment ethanol at high temperature, and produce xylitol as a by-product, and the concentration and efficiency of xylitol produced in these fermentations are lower (Kumar et al., 2009; Zhang et al., 2013)
The above studies have not fully exploited the potential of thermotolerant yeast to ferment xylose at high temperature to produce xylitol. Our research has greatly improved the ability of thermotolerant yeast to Alcohol industry has great application prospects

Method used

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  • Construction and application of high-temperature high-yield xylitol engineering strains
  • Construction and application of high-temperature high-yield xylitol engineering strains
  • Construction and application of high-temperature high-yield xylitol engineering strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1. Preparation of bacterial strains:

[0072] 1. Acquisition of various XR genes:

[0073] 1). Acquisition of NcXR gene (Woodyer et al., 2005):

[0074] The genome of Neurospora 74-OR23-1VA (purchased from Fungal Genetics Stock Center 2489, School of Biological Sciences, University of Missouri, Kansas City) was extracted, and the extracted genome of Neurospora was diluted 100 times as a template, and PrimeSTAR HS DNA was polymerized Enzyme (Dalian Baobiology) and NCXR-F1 (SEQ ID No.1), NCXR-R2 (SEQ ID No.4) primers for PCR, the product obtained is the NcXR gene (SEQ ID No.21 ), and insert the gene into the pMD18-T vector (Dalian Baobiology). Since the NcXR gene has introns, the T vector inserted into NcXR was used as a template, and PrimeSTAR HS DNA polymerase and NCXR-F1 (SEQ ID No.1), NCXR-R1 (SEQ ID No.2), NCXR- F2 (SEQ ID No.3), NCXR-R2 (SEQ ID No.4) primer PCR exon gene respectively, then fusion, obtain the exon gene fusion fragment of NcXR, clone aga...

Embodiment 2

[0267] Example 2. Fermentation of strains containing different promoters and XR genes under synthetic medium

[0268] This example is used to compare the effects of different XR genes and different promoters on the production of xylitol. The results showed that the effect of NcXR gene on producing xylitol was better than that of PsXR, and the promoter P ScGAPDH and P KmGAPDH Little effect, but with P KmGAPDH The effect is slightly better.

[0269] 1. Recover strains on YPD medium plates. Control strains: YZB001, YLUA005. Experimental strains: YZJ001, YZJ003, YZJ005, YZJ006, YZJ007, YZJ008. Cultured at 37°C for 1 day.

[0270] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0271] 3. Prepare 39 bottles of 50ml xylose synthesis medium and distribute them in 250ml Erlenmeyer flasks. Formula: 50g / L xylose, 10g / L yeast extract, 20g / L bacteriological peptone, 10g / L glycerin. Sterilized and ready to use.

[0272]...

Embodiment 3

[0279] Example 3. Fermentation of strains containing different copies of the NcXR gene at 42°C

[0280] This example is used to understand the comparison of the fermentation of strains containing different copies of the NcXR gene under high temperature (42° C.) conditions with the fermentation of strains containing one copy of the NcXR gene. The results prove that increasing the NcXR copy can make Increased fermentation capacity.

[0281] 1. Recover strains on YPD medium plates. Control strains: YZJ003, YZJ007. Experimental strains: YZJ012, YZJ014, YZJ015, YZJ017. Cultured at 37°C for 1 day.

[0282] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0283] 3. Prepare 27 bottles of 30ml xylose synthesis medium and pack them in 250ml Erlenmeyer flasks. Formula: 50g / L xylose, 10g / L yeast extract, 20g / L bacteriological peptone, 15g / L glycerol 50g / L xylose, 15g / L glycerin, 10g / L yeast extract, 20g / L bacteriological pe...

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Abstract

The invention discloses a construction and application of high-temperature high-yield xylitol engineering strains. Xylose reductase genes from different sources are efficiently expressed in heat-resistant yeast Kluyveromyces marxianus in the method of genetic engineering so that the strains can efficiently use and ferment xylose to produce a large amount of xylitol at a high temperature of larger than 42 DEG C through a metabolic pathway of xylose reductase. A heat-resistant engineering yeast strain YZJ015 and a heat-resistant engineering yeast strain YZJ017 capable of fermenting through xylose are constructed, and the preservation numbers are CGMCC No.7819 and 7820 respectively. The yeast strains can be used at a temperature between 42 DEG C and 45 DEG C. The xylitol can be produced in a high yield and at a high production speed by means of fermentation of xylose of different concentrations. Besides, the YZJ017 strain (CGMCC No.7820) can also be used for fermentation of glycerin and xylose to produce xylitol.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the field of improving the production of xylitol by fermenting xylose at high temperature through the transformation of engineering bacteria. The invention constructs a heat-resistant engineering yeast strain capable of producing xylitol by fermenting xylose at relatively high temperature and greatly increasing the production rate of xylitol. Background technique [0002] Xylitol is a five-carbon polyol that is a normal intermediate in xylose metabolism (Vandeska et al., 1996). When it is used by the human body, it does not need the promotion of insulin, and it can promote the secretion of a small amount of insulin in the body; when it is used for intravenous infusion, the content of pyruvic acid, lactic acid and glucose in the blood will decrease, and the liver glycogen will increase. ; Non-cytotoxic, can pass through the cell membrane and become the nutrition of the organization. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/18C12R1/645
Inventor 洪泂张佳
Owner UNIV OF SCI & TECH OF CHINA
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