Expression of beta-glucosidase in yeast to improve ethanol production
A technology of glucosidase and ethanol, which is applied in the field of expression of β-glucosidase, can solve the problems of not accurately characterizing biofuels, and requiring accuracy.
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example 1
[0143] Example 1: Addition of beta-glucosidase to stillage
[0144] Fermentation broths from submerged cultures expressing eight different glycosyl hydrolases, including ABG54 beta-glucosidase, were prepared for analysis using 96-well Millipore filter plates to remove cell clumps by filtration. After filtration, use 96 well ZEBA TM Spin desalting plates (ThermoFisher, catalog number: 89807 ), each enzyme in the culture broth was exchanged into 20 mM sodium acetate solution (pH 5.0, 0.005%) )middle.
[0145] Using an Agilent HPLC 1290 INFINITY TM system using Waters ACQUITY C4BEH 300 column (1.7 μm, 1 x 50 mm), quantification of protein obtained from each culture. A six minute program was used, starting with an initial gradient from 5% to 33% acetonitrile (Sigma-Aldrich) in 0.5 minutes, followed by a gradient from 33% to 48% acetonitrile in 4.5 minutes , followed by a step gradient to 90% acetonitrile. Enzyme sample preparations were quantified using a protein standard ...
example 2
[0151] Example 2: Materials and Methods of Examples 2-8
[0152] Liquefaction preparation
[0153] By adding 600ppm urea, 0.124SAPU / g ds FERMGEN TM 2.5X (acid fungal protease), 0.33 GAU / g ds variant Trichoderma glucoamylase (TrGA) and 1.46 SSCU / g ds Aspergillus alpha-amylase (AkAA), adjusted to pH 4.8 to prepare liquefaction (ground corn slurry). To evaluate strains expressing glucoamylase, the dose of glucoamylase was reduced to 0.1 GAU / g ds. In some experiments, 5 g / L cellobiose was also added to the liquefaction.
[0155] Yeast cells were seeded into 2 mL of YPD in a 24-well plate, and the cells were grown overnight. A 5 ml aliquot of the prepared liquefaction was added to serum vials (Chemglass, catalog number: CG-4904-01 ) and yeast was added to each vial to a final OD of about 0.2-0.4. The cap of the vial was twisted and pierced with a needle (BD, catalog number: 305111 ) for ventilation (to release CO 2 ), followed by incubation at 32°C...
example 3
[0166] Example 3: Screening for β-glucosidase expression in Saccharomyces cerevisiae
[0167] The genes encoding four different β-glucosidases (SEQ ID NOs: 1-4) (codon-optimized for S. cerevisiae) were cloned between the SpeI and NotI restriction sites in pJT257 (described in US Patent No. 9,181,566). In the resulting plasmid, the expression of β-glucosidase is under the control of the FBA1 promoter and FBA1 terminator from Saccharomyces cerevisiae (see, eg, WO 2018 / 111792). A second set of plasmids was constructed containing the same four β-glucosidases encoding but with the native signal sequence removed (SEQ ID NO: 5-8) and with mating factor alpha (MFα; SEQ ID NO: 9) from Saccharomyces cerevisiae. Gene with signal sequence replacement. Plasmids were named as shown in Table 2. Note that FAB is derived from beta-glucosidase moieties from Fusarium vesticilloies, Rasamsonia emersonii and Hypocrea jecorina (Trichoderma) The hybrid enzyme of , which is described in WO 201212...
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