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

An engineering and xylose technology, applied in the biological field, can solve the problems of low rate, low xylitol concentration and efficiency, and a production rate of only 0.24g.

Active Publication Date: 2015-03-11
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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 xylitol high-temperature and high-yield engineered strains
  • Construction and application of xylitol high-temperature and high-yield engineered strains
  • Construction and application of xylitol high-temperature and high-yield engineered 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 genome of Neurospora extracted was diluted 100 times as a template, and PrimeSTAR HS DNA was used polymerase (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 Bao Biology). 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 again...

Embodiment 2

[0263] Example 2. Fermentation of strains containing different promoters and XR genes under synthetic medium

[0264] This example is used to compare the effect 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.

[0265] 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.

[0266] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0267] 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.

[0268] ...

Embodiment 3

[0275] Example 3. Fermentation of strains containing different copies of the NcXR gene at 42°C

[0276] 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.

[0277] 1. Recover strains on YPD medium plates. Control strains: YZJ003, YZJ007. Experimental strains: YZJ012, YZJ014, YZJ015, YZJ017. Cultured at 37°C for 1 day.

[0278] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0279] 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 glycerin 50g / L xylose, 15g / L glycerin, 10g / L yeast extract, 20g / L bacteriological pe...

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Abstract

The invention discloses construction and application of xylitol high-temperature and high-yield engineered strains. Xylose reductase genes from different sources are efficiently expressed in heat-resistant yeast Kluyveromyces marxianus through a gene engineering method, so that the strain can be used for producing a large amount of xylitol by efficiently using and fermenting xylose under higher temperature (which is higher than 42 DEG C) through a xylose reductase metabolism path. Heat-resistant engineered yeast strains YZJ015 and YZJ017 capable of using xylose to carry out fermentation are constructed, and the preservation numbers are respectively CGMCC No.7819 and 7820. The yeast strains can be applied to produce xylitol with higher yield and higher production rate under the temperature of 42-45 DEG C by fermentation through different xylose concentrations. Furthermore, the strain YZJ017 (CGMCC No.7820) can also be used for preparing xylitol from glycerinum and xylose through fermentation.

Description

[0001] This application is a divisional application of the invention patent application with application number 201310399915.X. technical field [0002] 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 [0003] 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/18C12R1/645
CPCC12P7/18C12N1/145C12R2001/645
Inventor 洪泂张佳
Owner UNIV OF SCI & TECH OF CHINA
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