Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Engineered strain for producing ethanol and xylitol by efficient utilization of xylitose fermentation

A technology for engineering strains and yeast strains, applied in the directions of fermentation, genetic engineering, microorganism-based methods, etc., can solve the problems of residual substrates, insufficient utilization, and bottlenecks in the purification of product xylitol, so as to improve yield and production. rate, release of glucose inhibitory effect, the effect of great application prospects

Active Publication Date: 2019-12-20
青岛普罗百世生物科技有限公司
View PDF4 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the ratio of glucose and xylose in the hydrolyzate of agricultural waste corncobs is within a certain range, and the existing engineering strains for the production of ethanol and xylitol have extremely high requirements for the ratio of glucose and xylose in the substrate, therefore Xylose in the hydrolyzate of agricultural waste corncobs cannot be fully utilized, resulting in residues in the substrate, which not only cannot fully utilize the hydrolyzate, but also provides a bottleneck for the purification of the product xylitol

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Engineered strain for producing ethanol and xylitol by efficient utilization of xylitose fermentation
  • Engineered strain for producing ethanol and xylitol by efficient utilization of xylitose fermentation
  • Engineered strain for producing ethanol and xylitol by efficient utilization of xylitose fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 constructs heat-resistant yeast strain

[0027] Knockout of xylose reductase gene XR and xylitol dehydrogenase gene XDH in K. marxianu, while overexpressing multiple copies of Neurospora crassa xylose reductase gene NcXR and glucose / xylose symporter gene CiGXF1 , constructed and obtained a heat-resistant yeast strain;

[0028] The specific steps for constructing a heat-resistant yeast strain are described below.

[0029] 1. Extract the yeast genome

[0030] ① Kluyveromyces marxii NBRC177 strain was streaked on a YPD plate, a single clone was picked, inserted into 5ml liquid YPD, cultured at 37°C, 250rpm for 24h.

[0031] ② Centrifuge at 12000rpm at room temperature for 5sec to collect the bacteria, and discard the supernatant.

[0032] ③ Resuspend the bacteria in 500 μl of distilled water, centrifuge at 12,000 rpm for 5 sec to collect the bacteria, and discard the supernatant.

[0033] ④Take 200μl laboratory self-prepared 1x breaking buffer (TritonX-100...

Embodiment 2

[0196] Example 2 Recombinant expression of multiple copies of the N376F mutant gene of Saccharomyces cerevisiae galactose permease in the above-mentioned heat-resistant yeast strain

[0197] 1. Plasmid construction:

[0198] 1) Construction of pQDJL001:

[0199] The genome of Saccharomyces cerevisiae W303 was extracted, and the extracted genome of Saccharomyces cerevisiae was diluted 100 times as a template, with SCGAL2-ECORI-F and SCGAL2-NOTI-R as primers, and PCR was performed using PrimeSTARHS DNA polymerase (Dalian Bao Biology) , the product obtained is ScGAL2, and the ScGAL2 gene and the YEUGAP vector were digested with EcoR I and Not I, and ligated to obtain the plasmid pQDJL001.

[0200] The specific operation is as follows:

[0201] (1) ScGAL2 PCR system:

[0202] 5x PrimeSTAR Polymerase Buffer 40μl 2.5mM dNTP mix 16μl 10 μM SCGAL2-ECORI-F 4μl 10 μM SCGAL2-NOTI-R 4μl Saccharomyces cerevisiae genomic DNA 2μl PrimeSTAR DNA ...

Embodiment 3

[0313] Co-fermentation effect of the recombinant bacterial strain constructed in embodiment 3 of the present invention

[0314] To compare whether the various process strains constructed can make glucose and xylose fully utilized at the same time by adjusting the addition of glycerol when the ratio of glucose and xylose is consistent. The results showed that in the strain constructed by overexpressing the ScGAL2-N376F gene, xylose could be utilized in the presence of glucose, and the inhibitory effect of glucose was partially released. Moreover, by adding glycerol, all glucose and xylose in the substrate are utilized ( figure 1 ).

[0315]1. Recover strains on YPD medium plates. Control strain: YQD005; experimental strains: YQDJL001, YQDJL002, YQDJL003, YQDJL004. Cultured at 37°C for 1 day.

[0316] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.

[0317] 3. Prepare 12 bottles of 30ml fermentation medium and dist...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an engineered strain for producing ethanol and xylitol by efficient utilization of xylitose fermentation. According to the engineered strain, a galactose permease N376F mutant gene is multi-expressed in a heat-resistant yeast strain; and the heat-resistant yeast strain knocks out a xylose reductase gene NcXR and a xylitol dehydrogenase gene XDH, and meanwhile overexpresses aplurality of copied Neurosporacrassa xylose reductase genes NcXR and Kluyveromyces marxianus of a glucose / xylose symporter gene CiGXS1. According to the obtained K.marxianus engineered strain capableof producing the ethanol and the xylitol by efficient utilization of agricultural waste corncob hydrolysate fermentation, all glucose and xylose in a hydrolysis product can be completely converted into the ethanol and the xylitol, and the utilization rates of the glucose and the xylose are maximized. The prepared recombinant engineered strain can produce the ethanol and the xylitol at the high temperature by simultaneously utilizing a mixed sugar solution of the fermented glucose and the fermented xylose, and the yield and the production rate of the xylitol are greatly increased without affecting the ethanol output.

Description

technical field [0001] The invention belongs to the technical field of bioengineering strain construction, and in particular relates to a K.marxianus engineering strain that efficiently utilizes xylose to ferment ethanol and xylitol, that is, a kind of K.marxianus engineering strain that can efficiently utilize agricultural Engineering strains for the production of ethanol and xylitol by fermentation of waste corncob hydrolyzate. Background technique [0002] Xylitol is a five-carbon polyol, which is a normal intermediate product of xylose metabolism (Vandeska et al., 1996). Its properties determine that it has important application value in food, medicine, etc. Ethanol is an alcohol A kind of alcohol, commonly known as alcohol. Ethanol has a wide range of uses, and in terms of bioenergy, it has received much attention in recent years because of its potential as a substitute for fossil fuels. [0003] Xylose and glucose, which are raw materials for the production of xylito...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/56C12N15/53C12N15/31C12P7/10C12P7/18C12R1/645
CPCC12N9/0006C12N9/2402C07K14/39C12P7/10C12P7/18C12Y101/01009C12Y101/01307Y02E50/10
Inventor 张佳
Owner 青岛普罗百世生物科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com