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Recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase and applications thereof

A technology of Saccharomyces cerevisiae strain and glucosidase, which is applied in the direction of recombinant DNA technology, fermentation, fungi, etc., to achieve the effects of improving enzymatic hydrolysis efficiency, increasing ethanol production, and simplifying the production process

Inactive Publication Date: 2013-11-27
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The search showed that related recombinant Saccharomyces cerevisiae strains that can continuously and efficiently secrete β-glucosidase on non-selective medium and have high cellobiose metabolism ability and their simultaneous saccharification and fermentation process (SSF) using lignocellulose as raw material The literature and patents used in the application have not been reported

Method used

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  • Recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase and applications thereof
  • Recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase and applications thereof
  • Recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Extract the chromosomal DNA of the yeast Saccharomyces spp. (purchased from ATCC), use the chromosomal DNA as a template, SF-F and SF-R as primers, perform PCR with Fast Pfu polymerase produced by Quanshijin Company, and the amplified product is about 2700bp Bands( figure 1 ).

[0032] Wherein, the above-mentioned SF-F and SF-R primer sequences are:

[0033] SF-F:5’-TATAACTACAAAAAACACATACATAAACTAAAAGGTACCATGTTGATGATAGTACAGC-3’

[0034] SF-R: 5’-TTTTATAATAATTATATTAATCTTAGTTTCTAGACTCGAGTCAAATAGTAAACAGGACAG-3’

[0035] The PCR reaction system is as follows: (primer concentration is 10 μM)

[0036]

[0037] PCR reaction conditions: pre-denaturation at 95°C for 2 minutes, 30 cycles: denaturation at 95°C for 20 seconds, annealing at 54°C for 20 seconds, extension at 72°C for 1min15s. Extend at 72°C for 5 minutes and store at 4°C. Gel recovery and purification of concentrated PCR products.

[0038] Example 2: Acquisition of the loxP-KanMX4-loxP knockout cassette with ...

Embodiment 2

[0039] (1) Culture Saccharomyces cerevisiae CEN.PK530-1D (Hou et al. 2012), and extract chromosomal DNA. Using chromosomal DNA as a template, TPI-F and TPI-R as primers, the Fast Pfu polymerase produced by Quanshijin Company is used for PCR, with recombination arms at both ends and G418 resistance screening marker gene fragments with loxP sites.

[0040] Wherein, the above-mentioned TPI-F and TPI-R primer sequences are:

[0041] TPI-F: 5’-ACCCATCAGGTTGGTGGAAG-3’

[0042] TPI-R: 5’-CAACGCGAAAATGACGCCTC-3

[0043] The PCR reaction system is as follows: (primer concentration is 10 μM)

[0044]

[0045] PCR reaction conditions: pre-denaturation at 95°C for 2 minutes, 30 cycles: denaturation at 95°C for 20 seconds, annealing at 54°C for 20 seconds, extension at 72°C for 1min15s. Extend at 72°C for 5 minutes and store at 4°C. Amplified gene fragments with a size of about 2300 bp were gel recovered, purified and concentrated PCR products.

[0046] Homologous recombination fra...

Embodiment 3

[0054] Carrier CPOT is single-cut with XhoI, and the 8000bp fragment obtained is the same as that of Example 1 figure 1 The primers shown with the homology arms at both ends of the plasmid amplify the expression cassette of β-glucosidase, connect it by DNA one-step isothermal ligation method, and transform E. coli Tran5α competent cells to obtain the recombinant expression vector ( Figure 4 ).

[0055] Embodiment 4: Construction of recombinant Saccharomyces cerevisiae expression strain

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Abstract

The invention provides a recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase, which is named Saccharomyces cerevisiae 102SB, and preserved with a Preservation No. of CGMCC No. 7450 in the China General Microbiological Culture Collection Center on April 11, 2013. The recombinant saccharomyces cerevisiae disclosed by the invention can continuously and efficiently secrete beta-glucosidase in a complex non-selective medium, and the extracellular enzyme activity can reach 1005.3 U / g (dry weight). By using a characteristic that the maximum specific growth rate of cellobiose is consistent with that of glucose, the strain reaches 0.29 h<-1> under the condition of limited oxygen. In SSF taking a cellulose material as a substrate, the yield of ethanol taking microcrystalline cellulose as a substrate is increased by 110%, and the yield of ethanol taking acid-hydrolyzed corncobs as a substrate is increased by 89%. The recombinant saccharomyces cerevisiae strain disclosed by the invention is of great importance in reducing the production cost of simultaneous saccharification and fermentation in the process of cellulosic ethanol production, and simplifying the production process.

Description

technical field [0001] The invention relates to a recombinant Saccharomyces cerevisiae strain continuously and efficiently secreting β-glucosidase BGL1 derived from Saccharomyces fornicosa sp. Background technique [0002] Lignocellulose is one of the most abundant renewable resources on earth, and the production of biofuels such as bioethanol from cellulosic raw materials has broad application prospects (Lynd LR et al., 2002). The production of cellulosic ethanol starts with the hydrolysis of cellulosic raw materials into reducing sugars, which are then converted into ethanol through fermentation. Due to the low degradation efficiency and high cost of enzymatic hydrolysis of cellulase, the conversion of lignocellulose to reducing sugars has been considered as the limiting step for the industrial production of ethanol (van Rooyen et al., 2005). Simultaneous saccharification and fermentation (SSF) conversion of lignocellulose into ethanol can promote the hydrolysis efficienc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P7/10C12R1/865
CPCY02E50/16Y02E50/10
Inventor 侯进沈煜鲍晓明汤红婷
Owner SHANDONG UNIV
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