Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose

A technology of cellobiose and hydrolase, which is applied in the direction of introducing foreign genetic material, hydrolase, biofuel, etc. through the use of vectors, and can solve the problems of lack of efficient utilization of pentose and undisclosed ethanol commercially relevant potency, etc.

Inactive Publication Date: 2013-05-29
LALLEMAND HUNGARY LIQUIDITY MANAGEMENT LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

U.S. Patent No. 5,422,267 discloses the use of glucoamylase in yeast for the production of alcoholic beverages; however, the commercially relevant potency of ethanol is not disclosed
[0026] Furthermore, although yeast cells are known to naturally utilize sugars such as glucose and mannose, they lack the ability to efficiently utilize pentoses such as xylose and arabinose

Method used

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  • Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose
  • Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose
  • Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose

Examples

Experimental program
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Effect test

Embodiment 1

[0371] Embodiment 1: the expression of fungal lignocellulosic enzyme system component in yeast

[0372] In order to generate strains expressing these different enzymes and expected to co-express them, several promoter and terminator pairs were generated for use as expression vectors. The promoter-terminator pairs and the enzyme types tested under their control are listed in Table 3. Genes encoding the various enzymatic activities were cloned into vector pMU1531 using standard molecular biology methods (see, eg, Maniatis, "Molecular Cloning" Cold Spring Harbor Press). figure 2 A schematic diagram of pMU1531 as the backbone cloning vector used is given. This vector contains the ENO1 promoter and terminator from S. cerevisiae and the URA3 and zeocin markers for use in yeast. It was subsequently modified to have various promoter / terminator combinations listed in Table 3.

[0373] Table 3. Promoters and terminators used to express fungal and bacterial genes

[0374]

[0375...

Embodiment 2

[0383] Example 2: Characterization of expression and activity of accessory cellulases

[0384] After strain construction, strains expressing fungal EG1 candidates were grown in 50 mL shake flask cultures with 100 ug / mL zeocin and tested for activity against CMC and Avicel. Figure 3 shows that several active EG1s were found and that several EG1s outperformed a previously used comparable enzyme (T. reesei EG1, M1276) in activity. Based on these data, the best top 6 candidates were selected based on activity against Avicel for further testing on PHW ( Figure 4 and 5 ).

[0385] PHW assays were performed using a pretreated wood substrate (MS149) in the presence and absence of purified yeast-produced CBH1 and CBH2 (2 mg / g each) and Novozyme 188BGL. 2 mL of supernatant from each EG1 expressing strain was used in the assay. A strain expressing TrEG2 from the same plasmid was again used as a control. Results from such assays can be found in Figure 4 and 5 middle. Several EG1...

Embodiment 3

[0398] Embodiment 3: cloning and expression of 5 kinds of synthetic xylanases and 5 kinds of synthetic xylosidases in Saccharomyces cerevisiae

[0399] Expression of xylanases and xylosidases in yeast was examined to broaden the spectrum of enzymatic activity of yeast-produced lignocellulosic systems. Xylanases were selected from public databases and tested for functional expression in yeast on substituted xylans. Xylosidases were selected based on homology to Aspergillus niger xlnD (GH family 3 enzymes), which includes xylosidases from GH family 43. Table 5 (condensed form of Table 4) shows the genes selected for synthesis and the nomenclature of the expression vectors. All genes were cloned under the control of the ENO1 promoter / terminator using the pMU1531 expression plasmid. All the plasmids were transformed into Saccharomyces cerevisiae M0509, and the transformants were confirmed by PCR.

[0400] Table 5. Genes encoding xylanases and xylosidases expressed in Saccharomy...

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Abstract

The present invention is directed to a yeast strain, or strains, secreting a full suite, or any subset of that, full suite, of enzymes to hydrolyze com starch, corn fiber, lignocellulose, (including enzymes that hydrolyze linkages in cellulose, hemicelhiiose, and between lignin and carbohydrates) and to utilize pentose sugars (xylose and arabinose). The invention is also directed to the set of proteins that are well expressed in yeast for each category of enzymatic activity. The resulting strain, or strains can be used to hydrolyze starch and cellulose simultaneously. The resulting strain, or strains can be also metabolically engineered to produce less glycerol and uptake acetate. The resulting strain, or strains can also be used to produce ethanol from granular starch without liquefaction.; The resulting strain, or strains, can be further used to reduce the amount of external enzyme needed to hydrolyze a biomass feedstock during an Simultaneous Saccharification and Fermentation (SSF) process, or to increase the yield of ethanol during SSF at current saccharolytic enzyme loadings. In addition, multiple enzymes of the present invention can be co-expressed in cells of the invention to provide synergistic digestive action on biomass feedstock. In some aspects, host cells expressing different heterologous saccharolytic enzymes can also be co-cultured togetherand used to produce ethanol from biomass feedstock.

Description

Background of the Invention [0001] Biomass is biological material from living or recently living organisms, such as wood, waste, (hydrogen) gas and alcohol fuels. Biomass is based on carbon, hydrogen and oxygen. Nitrogen and small amounts of other atoms including alkali metals, alkaline earth metals and heavy metals can also be found. Metals are commonly found in functional molecules such as porphyrins, including chlorophyll which contain magnesium. Plants specifically combine water and carbon dioxide into sugar building blocks. The required energy is generated from light by chlorophyll-based photosynthesis. On average, 0.1 to 1% of the available light is stored in plants as chemical energy. Sugar building blocks are the starting point for all major fractions, lignin, hemicellulose and cellulose found in land plants. Biomass is widely recognized as a promising source of feedstock for the production of renewable fuels and chemicals. A major obstacle to more widespread ene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N1/19
CPCC12N9/24C12N15/52C12N9/2482C12N15/81C12Y302/01008C12N9/2437C12N9/2405C12Y302/01004C12P7/06C12N9/248C12N9/2445C12Y302/01021C12Y302/01091Y02E50/10
Inventor E·布雷弗诺瓦J·E·麦克布赖德E·威斯沃尔K·S·温格尔N·凯阿扎H·H·豪A·阿盖罗斯F·阿格博格伯C·F·里斯T·巴莱特J·S·巴德斯雷A·S·福斯特A·K·沃纳M·梅隆R·斯金纳I·希克海尔R·邓哈恩C·V·甘地A·贝尔彻V·B·拉吉加西亚A·C·弗罗利赫K·M·德莱奥尔特E·斯通豪斯S·A·特里帕斯J·戈斯林Y-Y·丘H·许
Owner LALLEMAND HUNGARY LIQUIDITY MANAGEMENT LLC
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