Method for improving absorption and utilization capacities of brewer's yeast xylose

[0018] Example 1: Obtaining DNA fragments for RGT1 knockout

[0019] The DNA fragment used for RGT1 knockout is obtained by fusion PCR technology, the annealing temperature in the amplification conditions is all 60℃, and the process is as follows figure 1 As shown, the specific steps are as follows:

[0020] 1) Using the chromosomal DNA of Saccharomyces cerevisiae BSPC085 (Shandong University, Peng Bingyin master's degree thesis, 2011) as a template, primer rgt1-1 and primer rgt1-2 were used to amplify the DNA fragment Fragment1 (384bp), which is the upstream sequence of RGT1 gene Homologous; amplified with primer rgt1-5 and primer rgt1-6 to obtain a DNA fragment Fragment3 (398 bp), which is homologous to the downstream sequence of RGT1 gene.

[0021] 2) Using plasmid pUG6 (Güldener, U., Heck, S., Fielder, T., Beinhauer, J., and Hegemann, JHA newefficient gene disruption cassette for repeated use inbuddingyeast. Nucleic acids research, 1996, 24, 2519-2524.) as a template, using prime...

[0023] Example 2: Obtaining RGT1 knockout strain

[0024] The DNA fragment Deletioncassette (2139bp) knocked out of RGT1 in Example 1 was used to transform Saccharomyces cerevisiae BSPC085, and the transformation method used the common lithium acetate transformation method (Shandong University, Peng Bingyin master's degree thesis, 2011). The transformants were selected in SC-URA solid medium supplemented with 400 mg / LG418 as the selection pressure and 20 g / L glucose as the carbon source.

[0025] Among them, SC-URA medium contains 1.7g / L yeast basal nitrogen source (YNB, Sangon, China), 5g / L ammonium sulfate (Sangon, China), 0.77g / L mixed amino acid CSM-URA (MPBiomedicals, Solon, OH) , PH4.5. Add 20g / L agarose to the solid medium.

[0026] After PCR verification, the strain whose RGT1 has been knocked out was named BSPC085-RT. The verification method is to use the chromosomal DNA of the transformant as a template and PCR amplification with primers rgt1-1 and primers rgt1-6, and th...

[0027] Example 3: Determination of the expression level of hexose transporter gene in RGT1 knockout strains under different carbon source conditions

[0028] The RGT1 deletion strain BSPC085-RT and the starting strain BSPC085 were cultured in a 50 mL Erlenmeyer flask containing 20 mL SC-URA liquid medium, and cultured at 30°C for 12 hours. Then transfer to two 20mL fresh SC-URA mediums, one uses 20g / L glucose as carbon source, the other uses 20g / L xylose as carbon source, and the inoculum is the initial OD 600 = 0.2. Incubate with shaking at 30°C to OD 600 For 0.8-1.0, collect the bacteria by centrifugation.

[0029] Use UNIQ-10 column Trizol total RNA extraction kit to extract total RNA. Use TaKaRaPrimeScript TM RTReagentKitwithgDNAEraser kit performs genomic DNA removal and reverse transcription to obtain cDNA. The operation of the two kits was carried out in accordance with their instructions.

[0030] Take the cDNA of BSPC085-RT and BSPC085 as templates (the measured concentr...