The Experts below are selected from a list of 84 Experts worldwide ranked by ideXlab platform
Chun-zhao Liu - One of the best experts on this subject based on the ideXlab platform.
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Co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum for enhancing hydrogen production via thermophilic fermentation of cornstalk waste
International Journal of Hydrogen Energy, 2012Co-Authors: Li Qian, Chun-zhao LiuAbstract:Abstract A strategic method utilizing the co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum has been developed to improve hydrogen production via the thermophilic fermentation of cornstalk waste. The hydrogen yield in the co-culture fermentation process reached 68.2 mL/g-cornstalk which was 94.1% higher than that in the mono-culture. The hydrogen fermentation process was successfully scaled-up from 125 mL anaerobic bottles to an 8 L continuous stirred tank reactor, and the hydrogen production from cornstalk waste was significantly improved in the bioreactor system due to efficient mixing and mass transfer. The hydrogen yield in the bioreactor reached 74.9 mL/g-cornstalk which was 9.8% higher than that in the 125 mL anaerobic bottle. The present work indicates that the Direct Microbial Conversion of lignocellulosic waste by co-culturing C. thermocellum and C. thermosaccharolyticum is a promising avenue for enhancing hydrogen production.
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enhanced coproduction of hydrogen and methane from cornstalks by a three stage anaerobic fermentation process integrated with alkaline hydrolysis
Bioresource Technology, 2012Co-Authors: Xi-yu Cheng, Chun-zhao LiuAbstract:Abstract A three-stage anaerobic fermentation process including H2 fermentation I, H2 fermentation II, methane fermentation was developed for the coproduction of hydrogen and methane from cornstalks. Hydrogen production from cornstalks using Direct Microbial Conversion by Clostridium thermocellum 7072 was markedly enhanced in the two-stage thermophilic hydrogen fermentation process integrated with alkaline treatment. The highest total hydrogen yield from cornstalks in the two-stage fermentation process reached 74.4 mL/g-cornstalk. The hydrogen fermentation effluents and alkaline hydrolyzate were further used for methane fermentation by anaerobic granular sludge, and the total methane yield reached 205.8 mL/g-cornstalk. The total energy recovery in the three-stage anaerobic fermentation process integrated with alkaline hydrolysis reached 70.0%.
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Hydrogen Production via Thermophilic Fermentation of Cornstalk by Clostridium thermocellum
Energy & Fuels, 2011Co-Authors: Xi-yu Cheng, Chun-zhao LiuAbstract:An efficient hydrogen production process via thermophilic fermentation of cornstalk was developed by Clostridium thermocellum 7072. The hydrogen fermentation process was successfully scaled from a 125 mL anaerobic bottle to a 100 L continuous stirred-tank reactor, and the hydrogen production from cornstalk was significantly improved in the bioreactor system because of good mixing and mass transfer. The hydrogen yield in the 100 L continuous stirred-tank reactor reached 61.4 mL/g of cornstalk, which was higher than that in the 125 mL anaerobic bottle. The present work indicated that Direct Microbial Conversion of lignocellulosic waste via C. thermocellum was a promising avenue for biohydrogen production.
Michael E. Himmel - One of the best experts on this subject based on the ideXlab platform.
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prospect for developing a consolidated bioprocessing cbp strain using xylan as the substrate the case study of yarrowia lipolytica
37th Symposium on Biotechnology for Fuels and Chemicals, 2016Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Min Zhang, Michael E. HimmelAbstract:To achieve the goal of developing a Direct Microbial sugar Conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnDmore » worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan Conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a Direct Microbial Conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica.« less
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Influence of Particle Size on Direct Microbial Conversion of Hot Water-Pretreated Poplar by Clostridium thermocellum
Direct Microbial Conversion of Biomass to Advanced Biofuels, 2015Co-Authors: John M. Yarbrough, Ashutosh Mittal, Yannick J. Bomble, Jessica L. Olstad, Edward J. Wolfrum, Sarah E. Hobdey, Michael E. Himmel, Todd B. VinzantAbstract:Abstract Multiple factors play a role in the Direct Microbial Conversion of biomass. Particle size has been shown to play a significant role in achieving efficient enzymatic hydrolysis of biomass with free cellulase systems. In this study, the Direct Microbial Conversion performance of Clostridium thermocellum is evaluated utilizing hot water extracted poplar sized by sieving to particles ranging between 63 μm to 6 mm, using conditions which maintain the chemical composition. Culture carbon:nitrogen ratios and dry weights were used to differentiate the contribution of Microbial mass from residual poplar mass. This work shows that for poplar, the substrate particle size influences the overall biomass Conversion by C. thermocellum , with particles sized between 63 μm and 250 μm displaying the greatest Conversion (50%). Moreover, the complex nature of biomass (i.e., chemical composition, structure, porosity, etc.) appears to play a greater role than particle size in influencing the overall potential for Microbial Conversion.
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Chapter 1: Feedstock Engineering and Biomass Pretreatments: New Views for a Greener Biofuels Process
Direct Microbial Conversion of Biomass to Advanced Biofuels, 2015Co-Authors: Hui Wei, Michael E. Himmel, Wei Wang, Melvin P. Tucker, Roman BruneckyAbstract:In the future, three crucial factors will determine the likelihood of success for the Direct Microbial Conversion of biomass to advanced biofuels: the properties of the lignocellulosic biomass feedstock, the pretreatment process, and the specific Microbial processing strategy selected. Each step accounts for a substantial portion of total process cost. Here, we extend the technical umbrella for advanced biofuels to technologies including and beyond the scope of the book, such as (1) upstream—new concepts for feedstocks, (2) midstream—specialized pretreatments and cotreatments, and (3) downstream—processing of biomass sugars. The aim of this chapter is not to provide a review of specialized fields, but to highlight new concepts and approaches related to biomass processing. We emphasize the important new trends in “green production” of feedstocks, technologies for “green pretreatment” of biomass, and propose new concepts for tailored chemoprocessing—databases/libraries customized for specific microorganisms applied to specific processes.
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Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.
PloS one, 2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.
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engineering towards a complete heterologous cellulase secretome in yarrowia lipolytica reveals its potential for consolidated bioprocessing
Biotechnology for Biofuels, 2014Co-Authors: Hui Wei, Michael E. Himmel, Stephen R Decker, Wei Wang, Markus Alahuhta, Todd Vander Wall, John O Baker, Larry E Taylor, Min ZhangAbstract:Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass Directly as a carbon source. Toward the development of a Direct Microbial Conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates. Conclusions: Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to Directly convert lignocellulosic substrates to biofuels.
Min Zhang - One of the best experts on this subject based on the ideXlab platform.
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prospect for developing a consolidated bioprocessing cbp strain using xylan as the substrate the case study of yarrowia lipolytica
37th Symposium on Biotechnology for Fuels and Chemicals, 2016Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Min Zhang, Michael E. HimmelAbstract:To achieve the goal of developing a Direct Microbial sugar Conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnDmore » worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan Conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a Direct Microbial Conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica.« less
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Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.
PloS one, 2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.
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engineering towards a complete heterologous cellulase secretome in yarrowia lipolytica reveals its potential for consolidated bioprocessing
Biotechnology for Biofuels, 2014Co-Authors: Hui Wei, Michael E. Himmel, Stephen R Decker, Wei Wang, Markus Alahuhta, Todd Vander Wall, John O Baker, Larry E Taylor, Min ZhangAbstract:Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass Directly as a carbon source. Toward the development of a Direct Microbial Conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates. Conclusions: Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to Directly convert lignocellulosic substrates to biofuels.
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Heterologous Expression of Xylanase Enzymes in Lipogenic Yeast Yarrowia
2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.
Wei Wang - One of the best experts on this subject based on the ideXlab platform.
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prospect for developing a consolidated bioprocessing cbp strain using xylan as the substrate the case study of yarrowia lipolytica
37th Symposium on Biotechnology for Fuels and Chemicals, 2016Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Min Zhang, Michael E. HimmelAbstract:To achieve the goal of developing a Direct Microbial sugar Conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnDmore » worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan Conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a Direct Microbial Conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica.« less
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Chapter 1: Feedstock Engineering and Biomass Pretreatments: New Views for a Greener Biofuels Process
Direct Microbial Conversion of Biomass to Advanced Biofuels, 2015Co-Authors: Hui Wei, Michael E. Himmel, Wei Wang, Melvin P. Tucker, Roman BruneckyAbstract:In the future, three crucial factors will determine the likelihood of success for the Direct Microbial Conversion of biomass to advanced biofuels: the properties of the lignocellulosic biomass feedstock, the pretreatment process, and the specific Microbial processing strategy selected. Each step accounts for a substantial portion of total process cost. Here, we extend the technical umbrella for advanced biofuels to technologies including and beyond the scope of the book, such as (1) upstream—new concepts for feedstocks, (2) midstream—specialized pretreatments and cotreatments, and (3) downstream—processing of biomass sugars. The aim of this chapter is not to provide a review of specialized fields, but to highlight new concepts and approaches related to biomass processing. We emphasize the important new trends in “green production” of feedstocks, technologies for “green pretreatment” of biomass, and propose new concepts for tailored chemoprocessing—databases/libraries customized for specific microorganisms applied to specific processes.
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Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.
PloS one, 2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.
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engineering towards a complete heterologous cellulase secretome in yarrowia lipolytica reveals its potential for consolidated bioprocessing
Biotechnology for Biofuels, 2014Co-Authors: Hui Wei, Michael E. Himmel, Stephen R Decker, Wei Wang, Markus Alahuhta, Todd Vander Wall, John O Baker, Larry E Taylor, Min ZhangAbstract:Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass Directly as a carbon source. Toward the development of a Direct Microbial Conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates. Conclusions: Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to Directly convert lignocellulosic substrates to biofuels.
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Heterologous Expression of Xylanase Enzymes in Lipogenic Yeast Yarrowia
2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.
Hui Wei - One of the best experts on this subject based on the ideXlab platform.
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prospect for developing a consolidated bioprocessing cbp strain using xylan as the substrate the case study of yarrowia lipolytica
37th Symposium on Biotechnology for Fuels and Chemicals, 2016Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Min Zhang, Michael E. HimmelAbstract:To achieve the goal of developing a Direct Microbial sugar Conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnDmore » worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan Conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a Direct Microbial Conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot Directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica.« less
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Chapter 1: Feedstock Engineering and Biomass Pretreatments: New Views for a Greener Biofuels Process
Direct Microbial Conversion of Biomass to Advanced Biofuels, 2015Co-Authors: Hui Wei, Michael E. Himmel, Wei Wang, Melvin P. Tucker, Roman BruneckyAbstract:In the future, three crucial factors will determine the likelihood of success for the Direct Microbial Conversion of biomass to advanced biofuels: the properties of the lignocellulosic biomass feedstock, the pretreatment process, and the specific Microbial processing strategy selected. Each step accounts for a substantial portion of total process cost. Here, we extend the technical umbrella for advanced biofuels to technologies including and beyond the scope of the book, such as (1) upstream—new concepts for feedstocks, (2) midstream—specialized pretreatments and cotreatments, and (3) downstream—processing of biomass sugars. The aim of this chapter is not to provide a review of specialized fields, but to highlight new concepts and approaches related to biomass processing. We emphasize the important new trends in “green production” of feedstocks, technologies for “green pretreatment” of biomass, and propose new concepts for tailored chemoprocessing—databases/libraries customized for specific microorganisms applied to specific processes.
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Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.
PloS one, 2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.
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engineering towards a complete heterologous cellulase secretome in yarrowia lipolytica reveals its potential for consolidated bioprocessing
Biotechnology for Biofuels, 2014Co-Authors: Hui Wei, Michael E. Himmel, Stephen R Decker, Wei Wang, Markus Alahuhta, Todd Vander Wall, John O Baker, Larry E Taylor, Min ZhangAbstract:Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass Directly as a carbon source. Toward the development of a Direct Microbial Conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient degradation of cellulosic substrates. Conclusions: Taken together, this work demonstrates the first case of successful expression of a chimeric CBHI with essentially full native activity in Y. lipolytica, and supports the notion that Y. lipolytica strains can be genetically engineered, ultimately by heterologous expression of fungal cellulases and other enzymes, to Directly convert lignocellulosic substrates to biofuels.
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Heterologous Expression of Xylanase Enzymes in Lipogenic Yeast Yarrowia
2014Co-Authors: Wei Wang, Hui Wei, Markus Alahuhta, Xiaowen Chen, Deborah Hyman, David K. Johnson, Min Zhang, Michael E. HimmelAbstract:To develop a Direct Microbial sugar Conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass Directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a Direct Microbial Conversion process using this organism.