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Building and application of engineered strains capable of producing xylitol and ethanol at high temperature simultaneously with high yield
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A technology of xylitol and xylose, applied in the biological field, can solve problems such as low efficiency, difficulty in removing the inhibitory effect of glucose, and insufficient efficiency
Active Publication Date: 2015-11-18
UNIV OF SCI & TECH OF CHINA
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Glucose and xylose are the two main monosaccharide components of its hydrolyzate. To establish an economical industrial utilization process, it is necessary to efficiently utilize and ferment the two sugars (Haetal., 2011), but the current methods are not efficient enough. Especially when glucose and xylose exist at the same time, due to the glucose inhibition effect, the ability of microorganisms to utilize xylose is often inhibited by glucose, resulting in low efficiency
The construction of various engineering strains has continuously improved the ability to use xylose to ferment xylitol, but the inhibitory effect of glucose is often difficult to remove
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[0043] 1. The specific operation steps for extracting the yeastgenome are as follows:
[0044] 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.
[0046] ③. Resuspend the bacteria in 500μl distilled water, centrifuge at 12000rpm for 5sec to collect the bacteria, and discard the supernatant.
[0047] ④. Take 200μl laboratory self-made 1xbreaking buffer (TritonX-100 (2% (w / v)), SDS (1% (w / v)), NaCl (100mM), Tris-Cl (10mM, pH8.0 ), EDTA (1mM)) to resuspend the bacteria, and transfer the bacteria solution into an EP tube containing 0.3g glass beads (425-600um, sigma, USA).
[0048] ⑤. After adding 200 μl of phenol-chloroform solution, shake at high speed for 3 minutes, and then add 200 μl o...
Embodiment 2
[0189] Fermentation situation of various process bacterial strains constructed in embodiment 2
[0190] Comparison of fermentation results of various process strains constructed
[0191] This example is used to compare the effects of co-fermentation of xylose and glucose to produce xylitol and ethanol by various process strains constructed. The results showed that the co-fermentation ability of the strain constructed by overexpressing the ScGAL2-N376F gene was gradually improved, and the final strain could convert almost all xylose into xylitol and all glucose into ethanol. In addition, knockdown of KmGPD1 almost completely disappeared the by-product glycerol.
[0192] 1. Recover strains on YPD medium plates. Control strain: YZJ015. Experimental strains: YZJ103, YZJ109, YZJ115, YZJ118, YZJ119. Cultured at 37°C for 1 day.
[0193] 2. Pick out single clones respectively, and connect them to 5ml liquid YPD medium. 37°C, 250rpm, overnight.
[0194] 3. Prepare 18 bottles of...
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Abstract
The invention relates to building and application of engineered strains capable of producing xylitol and ethanol at a high temperature simultaneously with a high yield. Concretely, N376F mutant genes of saccharomyces cerevisiae galactose permease (hexosetransporterprotein of hexose for transporting galactose and glucose) (ScGAL2) are effectively expressed in heat-resistant yeast strains YZJ015 by a geneengineering transformation method; meanwhile, K.marxianus glycerol-3-phosphatedehydrogenase (KmGPD1) genes are removed to cut off a glycerol production branch, so that the obtained strains YZJ119 obtain good capability of efficiently co-utilizing and fermenting glucose and xylose to produce the ethanol and the xylitol at a high temperature (higher than 42 DEG C). Under the condition of 42 DEG C, the YZJ119 can simultaneously use 43.95g / l of xylose and 83.11g / l of glucose to produce 40.43g / l of xylitol and 34.66g / l of ethanol in 28 hours; the production speed of the xylitol is 1.44g / l / h; the production speed of the ethanol is 1.24g / l / h; the yield of the xylitol is 0.92g / g; and the yield of the ethanol is 0.42g / g.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular, constructs heat-resistant engineeringyeast that can simultaneously use glucose and xylose to co-ferment to produce ethanol and xylitol at a relatively high temperature, and the production rate is greatly improved. It relates to the field of improving co-fermentation of glucose and xylose to produce ethanol and xylitol through engineeringbacteria transformation. Background technique [0002] Xylitol is a five-carbon polyol and a normal intermediate product of xylose metabolism (Vandeska et al., 1996). Its properties determine that it has important application value in food and medicine. First of all, in the food industry, the sweetness of xylitol is comparable to that of sucrose, and it can be used as a sweetener. Utilized by bacteria, it can prevent dental caries and can be used as an additive in children's anti-cavity food (Domingueze et al., 1997). In addition, since xylitol is a...
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