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Recombinant cellulose diastatic enzyme cocktail, recombinant yeast complex strain, and use thereof

A yeast and transformant technology, applied in the field of expression cassettes containing the expression cassettes, can solve the problems of limited bioethanol production, low ethanol production efficiency, and inability to effectively decompose cellulose substrates

Active Publication Date: 2016-04-13
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a host cell for producing such a recombinant enzyme, Saccharomyces cerevisiae is excellent in ethanol fermentation ability, and is commonly used as an ethanol production strain, and attempts to introduce cellulolytic ability to the strain and use for production of recombinant fiber have been made Many studies on cellulolytic enzymes (Lynd et al., 2002, Microbiol.Mol.Biol.Rev., 66, 506), but because the secretion productivity of cellulolytic enzymes is lower than that of molds, it is less likely to be used as the purpose of mass production of recombinant enzymes. Low
[0008] In this way, although the existing yeast has excellent bioethanol fermentation ability, it does not have the ability to decompose cellulosic biomass at all, so when using non-food resource cellulosic biomass as a raw material to produce bioenergy, there is an unavoidable problem. The problem of using high-priced cellulolytic enzymes, and in order to solve this problem, the development of recombinant strains into which foreign cellulolytic enzyme genes have been introduced has been carried out numerous times, but the produced enzymes cannot be efficiently decomposed due to low enzyme secretion productivity Problems with Cellulose Matrix in Media
Therefore, the production of bioethanol using the produced sugar (sugar) is limited
[0009] In recent years, there have been reports in Saccharomyces cerevisiae expressing cellulose glucoamylase genes excavated from several types of molds and confirming the production of bioethanol using microcrystalline cellulose (Avicel), but the amount of secreted enzymes is still insufficient. And the production efficiency of ethanol is low, and the problem of needing to supply external enzymes (M.Ilmen et al., 2011, Biotechnol Biofuels.4:30, 2011)

Method used

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  • Recombinant cellulose diastatic enzyme cocktail, recombinant yeast complex strain, and use thereof
  • Recombinant cellulose diastatic enzyme cocktail, recombinant yeast complex strain, and use thereof
  • Recombinant cellulose diastatic enzyme cocktail, recombinant yeast complex strain, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1: use bacterial strain and experimental material

[0088] Ensuring yeast-based hemicellulase, endoglucanase, exoglucanase and β-glucanase glucosidase gene

[0089]In order to effectively utilize endo-xylanase (endo-xylanase) TrXynII (sequence numbers 1, 2) and β-xylosidase (beta-xylosidase) TrBxl (sequence numbers 3, 4) derived from Trichoderma reesei ) and endo-glucanase (endo-glucanase) TrEGL2 (sequence numbers 5, 6) genes, using the nucleotide sequence information of the National Center for Biotechnology Information (NCBI). Regarding Trichoderma reesei, the strain (KCTC6968) distributed by the Bioresource Center was cultured in a potato dextrose agar medium (PotatoDextroseAgar, Difco) at 25°C for 5 days, and after only the mold mycelium that had completed the culture was filtered and recovered, The T.H. Al-Samarrai mold genomic DNA extraction method was used (T.H. Al-Sammarrai et al., 2000, Letters in Applied Microbiology, 30, 53-56).

[0090] In ...

Embodiment 2

[0101] Example 2: Hemicellulase by introduction of a translational fusion partner (TFP), Secreted production of endoglucanase and exoglucanase

[0102] In order to express the two hemicellulase, one endoglucanase and seven exoglucanase genes ensured by Example 1 in the Saccharomyces cerevisiae strain, primer LNK39 (SEQ ID NO: 45) was used from each gene After amplification with GT50R (SEQ ID NO. 46), 24 protein secretion fusion partners (Korean Patent No. 10-0975596, TFP1 to TFP24) vectors that assist protein secretion expression were introduced into Y2805 (Matapep4:: HIS3prb1can1his3-200ura3-52) strain. The sequences of TFP1 to TFP24 are shown in Table 3 below.

[0103] [table 3]

[0104]

[0105]

[0106]

[0107] Since the polymerase chain reaction product amplified by LNK39 (SEQ ID NO: 45) and GT50R (SEQ ID NO: 46) primers contains the same sequence as the vector above 40base, if it is introduced into yeast cells together with the linearized vector, the Cro...

Embodiment 3

[0109] Example 3: Hemicellulase, endogenous Activity Confirmation of Exoglucanase and Exoglucan

[0110] In order to confirm the activity of the protein secreted from each transformant, the activity analysis method for each protein as described below was used.

[0111] 3-1. Confirmation of endoxylanase activity of secreted TrXynII protein

[0112]To confirm the activity of the secreted TrXynII protein, the DNS (dinitirosalicylic acid, dinitrosalicylic acid) quantification method (Miller, G.L., Anal. Chem, 55:952-959, 1959) was used. Add 100 μl matrix solution (100 mM potassium phosphate solution containing 1% oat xylan (optspeltxylan), pH 5.0) to 100 μl enzyme solution, react at 60° C. for 30 minutes, add 700 μl DNS (3,5-Dinitrosalicylicacid, 3 , 5-dinitrosalicylic acid) solution, and treated at 100°C for 5 minutes, and then measured at absorbance at 540 nm after cooling with cold water. As a result of analyzing the activity of endoxylanase (Table 4), the activity of T...

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Abstract

The present invention relates to: an expression cassette which has an increased protein secretion in yeast compared to wild-type and which comprises a polynucleotide for coding a translational fusion partner (TFP) and a polynucleotide for coding a protein selected from the group consisting of: TrXynII, TrBx1, TrEGL2, PaCEL1, PaCel2, TeCBH1, NfCBH1, HgCBH1, CtCBH1, ClCBH2, and CfCex1; an expression vector comprising the same; a transformant in which the expression vector is introduced into a host cell; and a complex strain comprising two or more of the transformants. In addition, the present invention relates to a method for producing hemicellulase, endoglucanase, and exoglucanase, or a method for producing bioethanol, comprising a step for culturing the transformants. Furthermore, the present invention relates to a cellulase cocktail comprising endoglucanase and exoglucanase produced by the method and beta-glucosidase, and a method for saccharifying biomass using the cocktail. Moreover, the prevent invention relates to a method for producing bioenergy or useful biochemicals from cellulosic biomass, using a single strain which produces the cellulase, or two or more strains in combination.

Description

technical field [0001] The present invention relates to an expression cassette for expressing a protein having increased secretion ability in yeast compared with a wild-type protein, an expression vector comprising the expression cassette, a transformant formed by introducing the expression vector into a host cell, and two or more The composite strain of the above-mentioned transformant, wherein the above-mentioned expression cassette comprises a polynucleotide encoding a translational fusion partner (Translationalfusion partner, TFP) and a pair selected from TrXynII, TrBxl, TrEGL2, PaCEL1, PaCel2, TeCBH1, NfCBH1, HgCBH1, A polynucleotide encoding the proteins of CtCBH1, ClCBH2 and CfCex1. Furthermore, the present invention relates to a method for producing hemicellulase, endoglucanase or exoglucanase and a method for producing bioethanol comprising a step for culturing the above transformants. Further, the present invention relates to a recombinant cellulase mixture comprisi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/62C12P7/06
CPCC12N9/2437C12N9/2445C12P7/10C07K2319/02Y02E50/10Y02P20/10
Inventor 孙廷薰成奉炫裴贞勋李超龙金美辰金炫辰龙焕雄林光默
Owner KOREA RES INST OF BIOSCI & BIOTECH
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