We would like to express our sincere appreciation to Drs. Kenneth Vogel, Robert Mitchell and Gautam Sarath, Grain, Forage, and Bioenergy Research Unit, Agricultural Research Service, Lincoln, NE for their kind supply of switchgrass for this project. We also thank U.S. Department of Energy for funding to VB through the DOE Great Lakes Bioenergy Research Center (GLBRC) Grant DE-FC02-07ER64494.

<ol>
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The authors have nothing to disclose.

有几个步骤是对演​​化过程的成功至关重要。首先，它的关键是选择合适的选择压力，带动人口发展走向所需要的成功应用所需的表型。下面的选择性压力被选为S.树干毕赤酵母的发展，并在适当的时间施加到引导富集所需表型:12％葡聚糖AFEX CSH（这迫使生长和多样的糖发酵在乙酸和呋喃醛和其它抑制剂的低含量的存在下）增加强度;木糖喂连续培养随着乙醇浓度（这迫使木糖酶诱导减少di...

预计每年1.3十亿干吨木质纤维素生物质可以支持乙醇生产和允许美国30％，以减少其石油消耗。1虽然植物生物质的水解产量糖混合物富含葡萄糖和木糖，通过必要的化学预处理产生发酵抑制剂打破半纤维素和酶的攻击揭露纤维素。乙酸，糠醛和羟甲基糠醛（HMF）被认为是在预处理过程中形成的许多抑制剂中的关键部件。为了移动至木质纤维素乙醇工业向前，研究和程序，以允许能够存活和有效率地运作，以同时使用的己糖和戊糖在这样的抑制化合物的存在是需要的酵母菌株的演变。传统的工业的酵母菌株，例如酿酒酵母的显著附加弱点，是不能有效地fermenT IN植物生物质的水解产物中可用的木糖。 树干毕赤酵母型菌株 NRRL Y-7124（CBS 5773），最近更名Scheffersomyces赤酵母，是土生土长的戊糖发酵酵母是众所周知的木糖发酵成乙醇。2,3株NRRL Y-7124的演变在这里追求的，因为它已经被记录在案，以具有天然酵母菌株的最大潜力来积累的经济可采乙醇量超过40克/升，很少木糖醇副产品。4,5,6在最佳媒体，S.树干毕赤酵母菌株NRRL Y-7124在0.41±0.06克高细胞密度培养物（6克/升的细胞）/克7,8-抗性的产率产生为70g / L的乙醇在40小时（1.75克/升/小时）到发酵抑制乙醇，糠醛和HMF也有报道，9和S.赤酵母已被用于商业规模的乙醇生产中最有前途的天然戊糖发酵酵母位列其中n在木素纤维素。10我们的目标是应用多样化undetoxified木质纤维素水解和乙醇选择压力以迫使进化朝向的适用于工业应用应变NRRL Y-7124的更健壮的衍生物。寻求更强大的功能键都集中在水解糖较快吸收速率，减少了对更高效的混合糖利用率diauxy，以及乙醇和抑制剂更高的公差。 S的应用树干毕赤酵母到undetoxified水解物是研究的主要焦点，以消除与水解产物解毒过程，如加灰过量相关的附加操作费用。 两个工业有前途的水解产物用于强制进化:酶糖化氨纤维膨胀预处理的玉米秸秆水解物（AFEX CSH）和稀酸预处理柳枝水解产物液体（PSGHL）11,12 AFEX预处理技术正在开发最小化生产发酵抑制，而稀酸预处理表示当前最低成本的技术最普遍实行以暴露纤维素生物质进行酶糖化。 PSGHL是从预处理后剩余的纤维素可分离和的特点是丰富的，从水解的半纤维素木糖，但低于葡萄糖。 AFEX CSH和PSGHL组合物，其中被利用来管理演进过程中的关键环节彼此不同。 AFEX CSH是在氨基酸和氨氮源在呋喃醛和乙酸抑制剂较低，但高于PSGHL（ 表1）相比较。 PSGHL呈现木糖是可用的主要糖额外的挑战。因此PSGHL是合适的水解产物为改善木糖利用专门充实，一个弱点防止商业用途使用的酵母。即使是本地戊糖发酵的酵母，对次优糖低聚木糖的依赖本身支持细胞生长和修复变得更因各种原因水解产物更具挑战性:营养不足，抑制造成大面积破坏细胞结构的完整性，并破坏新陈代谢由于氧化还原不平衡9氮的补充，尤其是在形式氨基酸，能代表一个发酵经营显著成本。氮补充对菌株的筛选和排名的影响与柳枝水解探讨。 改进个人进行了使用多个选择压力依赖的S.天然的遗传多样性在不断变化的人口丰富树干毕赤酵母的人口和突变诱导暴露两个不同的水解物，乙醇或UV辐射。选择压力在并联和串联分别适用于探索S的演化进度对能够成长和水解发酵效率所需的树干毕赤酵母衍生物（ 图1）。官能人口日益具有挑战性的水解物的重复培养物的微孔板采用系列稀释为12％的葡聚糖AFEX CSH的要不然PGSHL在20％的固体加载制备完成英寸对木糖乙醇挑战的增长，连续培养应用程序通过丰富的表型示范敏感性较低乙醇木糖利用的镇压进一步提高AFEX CSH适应人群。后者的功能是下面的葡萄糖发酵最近表现出有问题的戊糖利用的菌株NRRL Y-7124 8富集的PSGHL是下探索扩大水解物的功能。 S的推定提高衍生品赤酵母 NRRL Y-7124是使用压力条件和稀释电镀下有针对性的富集，从最普遍的人群挑选的菌落演变过程的每个阶段中分离。相对量纲性能指标（RPI）提供用于基于整体性能，其中动力学行为施加不同的水解物种类和营养素补充剂评估排名的压力。虽然各种改编程序的成功，以提高S的功能树干毕赤酵母中的木质纤维素水解产物先前已被记录，菌株显示出上undetoxified水解经济乙醇生产尚未报道。13-17使用进化过程中这里更详细地进行可视化，Slininger 等人 18开发出了显著改善了菌株亲本菌株NRRL Y-7124，并能产生&gt;为40g / L的乙醇在AFEX CSH和酶糖化柳枝水解产物（SGH）适当补充有氮源。这些新菌株是将来的兴趣到显影木质纤维素到乙醇工业的和作为附加基因组学的研究大厦的受试者19与以前测序株NRRL Y-11545的。在图1中可图示将阐明在开发过程中发生的前奏，进一步菌种改良研究基因变化的历史沿革的不同阶段产生的顶部菌株的基因组学研究。

<table><tbody><tr><td>Cellic Ctec, Contains Xylanase (endo-1,4-)</td><td>Novozymes</td><td>No product number</td><td>www.novozymes.com, 1-919-494-3000</td></tr><tr><td>Cellic Htec, Contains Cellulase and Xyalanase</td><td>Novozymes</td><td>No product number</td><td>www.novozymes.com, 1-919-494-3000</td></tr><tr><td>Toasted Nutrisoy Flour</td><td>Archer Daniels Midland Co. (ADM)</td><td>63160</td><td>ADM, 4666 Faries Parkway, Decatur, IL&amp;nbsp; 1800-37-5843</td></tr><tr><td>Pluronic F-68 (Surfactant)</td><td>Sigma-Aldrich</td><td>P1300</td><td>Sigma-Aldrich</td></tr><tr><td>Difco Vitamin Assay Casamino Acids</td><td>Becton Dickinson and Company</td><td>228830</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, VWR, Daigger</td></tr><tr><td>D,L-tryptophan&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T3300</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, VWR, Daigger</td></tr><tr><td>L-cysteine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C7352</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, Sigma-Aldrich</td></tr><tr><td>Bacto Agar</td><td>Becton Dickinson and Company</td><td>214010</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, VWR, Daigger</td></tr><tr><td>Bacto Malt Extract</td><td>Becton Dickinson and Company</td><td>218630</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, VWR, Daigger</td></tr><tr><td>Bacto Yeast Extract</td><td>Becton Dickinson and Company</td><td>212750</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, VWR, Daigger</td></tr><tr><td>Peptone Type IV from soybean</td><td>Fluka</td><td>P0521-500g</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Fisher Scientific, VWR, Daigger</td></tr><tr><td>Adenine, &gt;99% powder</td><td>Sigma-Aldrich</td><td>A8626</td><td>CAS 73-24-5. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td></tr><tr><td>Cytosine, &gt;99%</td><td>Sigma-Aldrich</td><td>C3506</td><td>CAS 71-30-7. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td></tr><tr><td>Guanine, SigmaUltra</td><td>Sigma-Aldrich</td><td>G6779</td><td>CAS 73-40-5. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td></tr><tr><td>Thymine, 99%</td><td>Sigma-Aldrich</td><td>T0376</td><td>CAS 65-71-4. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td></tr><tr><td>Uracil, 99%</td><td>Sigma-Aldrich</td><td>U0750</td><td>CAS 66-22-8. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td></tr><tr><td>Dextrose (D-Glucose), Anhydrous, Certified ACS</td><td>Fisher Chemical</td><td>D16-500</td><td>CAS 50-99-7. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich</td></tr><tr><td>D-Xylose, assay &gt;99%</td><td>Sigma-Aldrich</td><td>X1500</td><td>CAS 58-86-6. Could use other brands. Multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich</td></tr><tr><td>96-well, flat bottom plates</td><td>Becton Dickinson Falcon</td><td>351172</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Thermo-Fisher, VWR, Daigger</td></tr><tr><td>Wypall L40 Wiper</td><td>Kimberly-Clark</td><td></td><td>towel in microplate boxes to absorb water for humidification; multiple suppliers &lt;em&gt;e.g.&lt;/em&gt;, Thermo-Fisher, uline, Daigger</td></tr><tr><td>Corning graduated pyrex flask, 125 ml, narrow opening (stopper #5)</td><td>Corning Life Science Glass</td><td>4980-125</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Thermo-Fisher, VWR, Daigger</td></tr><tr><td>Innova 42R shaker/incubator, 2.5 cm (1&quot;) rotation</td><td>New Brunswick Scientific (1-800-631-5417)</td><td>M1335-0016</td><td>multiple suppliers: &lt;em&gt;e.g.&lt;/em&gt;, Eppendorf, Thermo-Fisher. Other shaker/incubators with a 2.5 cm (1&quot;) throw could be used.</td></tr><tr><td>Duetz Cover clamp for 4 deep well MTP plates</td><td>Applikon Biotechnology</td><td>Z365001700</td><td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td></tr><tr><td>Duetz System sandwich cover for 96 deep well plates</td><td>Applikon Biotechnology</td><td>Z365001296</td><td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td></tr><tr><td>Duetz System silicone seal (0.8 mm black low evap) for 96 deep well plate cover</td><td>Applikon Biotechnology</td><td>V0W1040027</td><td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td></tr><tr><td>Blue microfiber layer for Duetz system sandwich cover</td><td>Applikon Biotechnology</td><td>V0W1040001</td><td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td></tr><tr><td>96 well, 2 ml square well pyramid bottom plates, natural popypropylene</td><td>Applikon Biotechnology</td><td>ZC3DXP0240</td><td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td></tr><tr><td>Bellco 32 mm silicon sponge plug closures, pk of 25 for 125 ml flasks</td><td>Bellco</td><td>1924-00032</td><td>Thomas Scientific, their Catalog number is 1203K27</td></tr><tr><td>Bellco Spinner Flask, 1968-Glass Dome, Sealable Flange Type, 100 ml working volume. This design no longer manufactured.</td><td>Bellco</td><td>1968-00100 (original Cat. No.)</td><td>Jacketed vessels have lower inlet &amp;amp; upper outlet ports for temp. control with circulating water bath. Vessels are 75 mm in outer diam and 200 mm in height. There are four side ports at ~45&amp;#176; angles and one top port. Port openings appropriate size for size 0 neoprene stoppers (21-22 mm inner diameters on ports).</td></tr><tr><td>Mathis Labomat IR Dryer Oven</td><td>MathisAg</td><td>Typ-Nbr BFA12 215307</td><td>Werner Mathis U.S.A. Inc. usa@mathisag.com, 704-786-6157</td></tr><tr><td>Dual Channel Biochemistry Analyzer</td><td>YSI Life Sciences</td><td>2900D-UP</td><td>www.ysi.com, robotic system for rapid sugars assay in 96-well microplate format</td></tr><tr><td>PowerWave XS Microplate Spectrophotometer</td><td>Bio-Tek Instruments, Inc</td><td>MQX200R</td><td>&lt;a href=&quot;http://www.biotek.com/&quot;&gt;www.biotek.com&lt;/a&gt;</td></tr></tbody></table>

techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

S.树干毕赤酵母 ，使用三个选择培养，其中包括AFEX CSH，PSGHL和乙醇挑战的木糖馈连续培养的组合演变而来的。 图1示出了具有分离株一起执行进化实验的示意图发现无论是最有效地执行整体，或最有效地对测试的水解物中的一个， 表3示出了这些优越菌株NRRL保藏号并总结在实现富集的群体，从每个菌株中分离的过程中施加的适应应力。有些菌株，被视为有一个或?...

Watch this Scientific Journal Video about Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol at JoVE.com

用于将木质纤维素生物转化为乙醇的稳健戊糖发酵酵母的进化技术生物工程|视频

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

适应进化和隔离技术进行了描述和展示，得到了能够迅速消耗己糖和酶戊混合糖的糖化undetoxified水解和超过40克/升乙醇积聚Scheffersomyces树干毕赤酵母株的衍生物NRRL Y-7124。

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

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Universidad de Cartagena UDEC

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Natural Selection

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11.1K 次观看.  National Center for Agricultural Utilization Research. 适应进化和隔离技术进行了描述和展示，得到了能够迅速消耗己糖和酶戊混合糖的糖化undetoxified水解和超过40克/升乙醇积聚Scheffersomyces树干毕赤酵母株的衍生物NRRL Y-7124。 

视频: Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

可再生生物燃料及生化生产的非常规酵母基因工程

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

科研

遗传学

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

环境科学

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

朝仿生学木：纳米纤维素，木质素的装配式自由站立电影和合成聚阳离子

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

化学

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

Pretreatment of Lignocellulosic Biomass with Low-cost Ionic Liquids

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of a variety of lignocellulosic feedstocks. Here we demonstrate the use of these low-cost protic ILs in the deconstruction of lignocellulosic biomass (Ionosolv pretreatment), yielding cellulose and a purified lignin. In the most generic process, the protic ionic liquid is synthesized by accurate combination of aqueous acid and amine base. The water content is adjusted subsequently. For the delignification, the biomass is placed into a vessel with IL solution at elevated temperatures to dissolve the lignin and hemicellulose, leaving a cellulose-rich pulp ready for saccharification (hydrolysis to fermentable sugars). The lignin is later precipitated from the IL by the addition of water and recovered as a solid. The removal of the added water regenerates the ionic liquid, which can be reused multiple times. This protocol is useful to investigate the significant potential of protic ILs for use in commercial biomass pretreatment/lignin fractionation for producing biofuels or renewable chemicals and materials.

The pretreatment of lignocellulosic biomass with protic low-cost ionic liquids is shown, resulting in a delignified cellulose-rich pulp and a purified lignin. The pulp gives rise to high glucose yields after enzymatic saccharification.

木质纤维素生物质预处理低成本离子液体

A number of ionic liquids (ILs) with economically attractive production costs have recently received growing interest as media for the delignification of ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and pectin) embedded within cell walls are highly recalcitrant towards conversion into useful products. Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that increases accessibility of polysaccharides to enzymes for hydrolysis into fermentable sugars. These released sugars can be converted into fuels and chemicals in a biorefinery. Here, we describe a laboratory-scale batch AFEX process to produce pretreated biomass on the gram-scale without any ammonia recycling. The laboratory-scale process can be used to identify optimal pretreatment conditions (e.g., ammonia loading, water loading, biomass loading, temperature, pressure, residence time, etc.) and generates sufficient quantities of pretreated samples for detailed physicochemical characterization and enzymatic/microbial analysis. The yield of fermentable sugars from enzymatic hydrolysis of corn stover pretreated using the laboratory-scale AFEX process is comparable to pilot-scale AFEX process under similar pretreatment conditions. This paper is intended to provide a detailed standard operating procedure for the safe and consistent operation of laboratory-scale reactors for performing AFEX pretreatment of lignocellulosic biomass.

Ammonia Fiber Expansion AFEX Pretreatment of Lignocellulosic Biomass

Ammonia fiber expansion (AFEX) is a thermochemical pretreatment technology that can convert lignocellulosic biomass (e.g., corn stover, rice straw, and sugarcane bagasse) into a highly digestible feedstock for both biofuels and animal feed applications. Here, we describe a laboratory-scale method for conducting AFEX pretreatment on lignocellulosic biomass.

氨纤维膨胀（AFEX）预处理利诺纤维素生物量

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

体内研究 使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

生物学

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

摘要

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此视频中的章节

可再生生物燃料及生化生产的非常规酵母基因工程

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

朝仿生学木：纳米纤维素，木质素的装配式自由站立电影和合成聚阳离子

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

木质纤维素生物质预处理低成本离子液体

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

氨纤维膨胀（AFEX）预处理利诺纤维素生物量

体内研究使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

木质纤维素材料的高通量检测糖化

用于微生物化学和蛋白质生产的光控发酵

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

摘要

探索更多视频

此视频中的章节

可再生生物燃料及生化生产的非常规酵母基因工程

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

朝仿生学木：纳米纤维素，木质素的装配式自由站立电影和合成聚阳离子

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

木质纤维素生物质预处理低成本离子液体

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

氨纤维膨胀（AFEX）预处理利诺纤维素生物量

体内研究 使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性

木质纤维素材料的高通量检测糖化

用于微生物化学和蛋白质生产的光控发酵

体内研究使用酵母中的生物发光报告基因监测代谢转变过程中的转录活性