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      Quantitative fermentation of unpretreated transgenic poplar by Caldicellulosiruptor bescii

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          Abstract

          Microbial fermentation of lignocellulosic biomass to produce industrial chemicals is exacerbated by the recalcitrant network of lignin, cellulose and hemicelluloses comprising the plant secondary cell wall. In this study, we show that transgenic poplar ( Populus trichocarpa) lines can be solubilized without any pretreatment by the extreme thermophile Caldicellulosiruptor bescii that has been metabolically engineered to shift its fermentation products away from inhibitory organic acids to ethanol. Carbohydrate solubilization and conversion of unpretreated milled biomass is nearly 90% for two transgenic lines, compared to only 25% for wild-type poplar. Unexpectedly, unpretreated intact poplar stems achieved nearly 70% of the fermentation production observed with milled poplar as the substrate. The nearly quantitative microbial conversion of the carbohydrate content of unpretreated transgenic lignocellulosic biomass bodes well for full utilization of renewable biomass feedstocks.

          Abstract

          Metabolizing lignocellulosic feedstocks to industrial chemicals by microorganisms requires surmounting  the recalcitrance caused by lignin. Here, the authors pair transgenic lignin modified poplar lines with engineered Caldicellusiruptor bescii to achieve biomass solubilization and ethanol conversion without pretreatment.

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          Most cited references12

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          Woody biomass pretreatment for cellulosic ethanol production: Technology and energy consumption evaluation.

          This review presents a comprehensive discussion of the key technical issues in woody biomass pretreatment: barriers to efficient cellulose saccharification, pretreatment energy consumption, in particular energy consumed for wood-size reduction, and criteria to evaluate the performance of a pretreatment. A post-chemical pretreatment size-reduction approach is proposed to significantly reduce mechanical energy consumption. Because the ultimate goal of biofuel production is net energy output, a concept of pretreatment energy efficiency (kg/MJ) based on the total sugar recovery (kg/kg wood) divided by the energy consumption in pretreatment (MJ/kg wood) is defined. It is then used to evaluate the performances of three of the most promising pretreatment technologies: steam explosion, organosolv, and sulfite pretreatment to overcome lignocelluloses recalcitrance (SPORL) for softwood pretreatment. The present study found that SPORL is the most efficient process and produced highest sugar yield. Other important issues, such as the effects of lignin on substrate saccharification and the effects of pretreatment on high-value lignin utilization in woody biomass pretreatment, are also discussed. Published by Elsevier Ltd.
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            Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis

            A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.
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              Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii.

              Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.
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                Author and article information

                Contributors
                ctstraub@ncsu.edu
                rmkelly@ncsu.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                7 August 2019
                7 August 2019
                2019
                : 10
                : 3548
                Affiliations
                [1 ]ISNI 0000 0001 2173 6074, GRID grid.40803.3f, Department of Chemical and Biomolecular Engineering, , North Carolina State University, ; Raleigh, NC 27695 USA
                [2 ]ISNI 0000 0001 2173 6074, GRID grid.40803.3f, Department of Forestry and Environmental Resources, , North Carolina State University, ; Raleigh, NC 27695 USA
                [3 ]ISNI 0000 0004 1936 738X, GRID grid.213876.9, Department of Biochemistry and Molecular Biology, , University of Georgia, ; Athens, GA 30602 USA
                [4 ]ISNI 0000 0001 2173 6074, GRID grid.40803.3f, Department of Forest Biomaterials, , North Carolina State University, ; Raleigh, NC 27695 USA
                [5 ]Present Address: Novozymes Biologicals, Inc., Durham, NC 27709 USA
                Author information
                http://orcid.org/0000-0001-9628-0198
                http://orcid.org/0000-0002-2715-2149
                http://orcid.org/0000-0002-5786-688X
                http://orcid.org/0000-0002-2515-073X
                http://orcid.org/0000-0002-0639-3592
                Article
                11376
                10.1038/s41467-019-11376-6
                6685990
                31391460
                8ac067ed-0b2f-4cde-8ee9-bf78e51911ba
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 12 June 2018
                : 3 July 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/100007917, United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service);
                Award ID: NIFA 2018-67021-27716
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s) 2019

                Uncategorized
                metabolic engineering,applied microbiology,bioalcohols
                Uncategorized
                metabolic engineering, applied microbiology, bioalcohols

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