Name: techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol
Uploaded: 2016-10-24T13:00:00
Description: Watch this Scientific Journal Video about Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol at JoVE.com

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.

1.アッセイのための材料および装置の起動準備<ol><li>進化、単独での使用とランク付け手続きの前処理反応で18〜20％の初期バイオマス乾燥重量を使用して加水分解物を準備します。 Slininger らは詳細な方法のために2015年18進化 、単離またはランク付けに使用される窒素サプリメントN1またはN2とAFEX CSH、PSGHL、およびSGHを準備するために参照してください。各加水分解型の組成?...

<ol>
	<li>Perlack, R. D., Stokes, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. , (2011).</li><li>Prior, B. A., Kilian, S. G., duPreez, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermentation+of+D-xylose+by+the+yeasts+Candida+shehatae+and+Pichia+stipitis.">Fermentation of D-xylose by the yeasts Candida shehatae and Pichia stipitis.</a> Process Biochem. 24 (1), 21-32 (1989).</li><li>Kurtzman, C. P., Suzuki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+ascomycete+yeasts+that+form+coenzyme+Q-9+and+the+proposal+of+the+new+genera+Babjeviella,+Meyerozyma,+Millerozyma,+Priceomyces+and+Scheffersomyces.">Phylogenetic analysis of ascomycete yeasts that form coenzyme Q-9 and the proposal of the new genera Babjeviella, Meyerozyma, Millerozyma, Priceomyces and Scheffersomyces.</a> Mcoscience. 51 (1), 2-14 (2010).</li><li>Slininger, P. J., Bothast, R. J., Okos, M. R., Ladisch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+ethanol+production+by+xylose-fermenting+yeasts+presented+high+xylose+concentrations.">Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations.</a> Biotechnol. Lett. 7 (6), 431-436 (1985).</li><li>Slininger, P. J., Bothast, R. J., Ladisch, M. R., Okos, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimum+pH+and+temperature+conditions+for+xylose+fermentation+by+Pichia+stipitis.">Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis.</a> Biotechnol. Bioeng. 35 (7), 727-731 (1990).</li><li>Slininger, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+and+kinetics+of+xylose+fermentation+by+Pichia+stipitis.">Stoichiometry and kinetics of xylose fermentation by Pichia stipitis.</a> Annals NY Acad. Sci. 589, 25-40 (1990).</li><li>Slininger, P. J., Dien, B. S., Gorsich, S. W., Liu, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+source+and+mineral+optimization+enhance+D-xylose+conversion+to+ethanol+by+the+yeast+Pichia+stipitis+NRRL+Y-7124.">Nitrogen source and mineral optimization enhance D-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124.</a> Appl. Microbiol. Biotechnol. 72 (6), 1285-1296 (2006).</li><li>Slininger, P. J., Thompson, S. R., Weber, S., Liu, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+xylose-specific+enzymes+by+ethanol+in+Scheffersomyces+(Pichia)+stipitis+and+utility+of+repitching+xylose-grown+populations+to+eliminat+diauxic+lag.">Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminat diauxic lag.</a> Biotechnol. Bioeng. 108 (8), 1801-1815 (2011).</li><li>Slininger, P. J., Gorsich, S. W., Liu, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+nutrition+and+physiology+impact+inhibitor+tolerance+of+the+yeast+Pichia+stipitis+NRRL+Y-7124.">Culture nutrition and physiology impact inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124.</a> Biotechnol. Bioeng. 102 (3), 778-790 (2009).</li><li>Agbogbo, F. K., Coward-Kelly, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+ethanol+production+using+the+naturally+occurring+xylose-fermenting+yeast,+Pichia+stipitis.">Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis.</a> Biotechnol. Lett. 30 (9), 1515-1524 (2008).</li><li>Balan, V., Bals, B., Chundawat, S., Marshall, D., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignocellulosic+pretreatment+using+AFEX.">Lignocellulosic pretreatment using AFEX.</a> Biofuels: Methods and protocols, Methods in Molecular Biology. 581, 61-77 (2009).</li><li>Jin, M., Gunawan, C., Uppugundla, N., Balan, V., Dale, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+integrated+biological+process+for+cellulosic+ethanol+production+featuring+high+ethanol+productivity,+enzyme+recycling,+and+yeast+cells+reuse.">A novel integrated biological process for cellulosic ethanol production featuring high ethanol productivity, enzyme recycling, and yeast cells reuse.</a> Energ. Environ. Sci. 5 (5), 7168-7175 (2012).</li><li>Nigam, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+xylose-fermenting+yeast+Pichia+stipitis+for+ethanol+production+through+adaptation+on+hardwood+hemicellulose+acid+prehydrolysate.">Development of xylose-fermenting yeast Pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehydrolysate.</a> J. Appl. Microbiol. 90 (2), 208-215 (2001).</li><li>Nigam, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+production+from+wheat+straw+hemicellulose+hydrolysate+by+Pichia+stipitis.">Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis.</a> J. Biotechnol. 87 (1), 17-27 (2001).</li><li>Hughes, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Random+UV-C+mutagenesis+of+Scheffersomyces+(formerly+Pichia)+stipitis+NRRL+Y-7124+to+improve+anaerobic+growth+on+lignocellulosic+sugars.">Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars.</a> J. Ind. Microbiol. Biotechnol. 39 (1), 163-173 (2012).</li><li>Bajwa, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutants+of+the+pentose-fermenting+yeast+Pichia+stipitis+with+improved+tolerance+to+inhibitors+in+hardwood+spent+sulfite+liquor.">Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor.</a> Biotechnol. Bioeng. 104 (5), 892-900 (2009).</li><li>Bajwa, P. K., Pinel, D., Martin, V. J. J., Trevors, J. T., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+improvement+of+the+pentose-fermenting+yeast+Pichia+stipitis+by+genome+shuffling.">Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling.</a> J. Microbiol. Methods. 81 (2), 179-186 (2010).</li><li>Slininger, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolved+strains+of+Scheffersomyces+stipitis+achieving+high+ethanol+productivity+on+acid-+and+base-pretreated+biomass+hydrolyzate+at+high+solids+loading.">Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid- and base-pretreated biomass hydrolyzate at high solids loading.</a> Biotechnol. Biofuels. 8:60, 1-27 (2015).</li><li>Jeffries, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+lignocellulosic-bioconverting+and+xylose-fermenting+yeast+Pichia+stipitis.">Genome sequence of the lignocellulosic-bioconverting and xylose-fermenting yeast Pichia stipitis.</a> Nature Biotechnol. 25 (3), 319-326 (2007).</li><li>Zabriski, D. W., Armiger, W. B., Phillips, D. H., Albano, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermentation+media+formulation.">Fermentation media formulation.</a> Trader's Guide to Fermentation Media Formulation. , 1-39 (1980).</li><li>Syzbalski, W., Bryson, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+studies+on+microbial+cross+resistance+to+toxic+agents.+I.+Cross+resistance+of+Escherichia+coli+to+fifteen+antibiotics.">Genetic studies on microbial cross resistance to toxic agents. I. Cross resistance of Escherichia coli to fifteen antibiotics.</a> J. Biotechnol. 64 (4), 489-499 (1952).</li><li>Klinke, H. B., Thomsen, A. B., Ahring, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ethanol-producing+yeast+and+bacteria+by+degradation+products+produced+during+pre-treatment+of+biomass.">Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass.</a> Appl. Microbiol. Biotechnol. 66, 10-26 (2004).</li><li>Almeida, J. R. M., Bertilsson, M., Gorwa-Grauslund, M. F., Gorsich, S., Liden, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+effects+of+furaldehydes+and+impacts+on+biotechnological+processes.">Metabolic effects of furaldehydes and impacts on biotechnological processes.</a> Appl. Microbiol. Biotechnol. 82, 625-638 (2009).</li><li>Allen, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Furfural+induces+reactive+oxygen+species+accumulation+and+cellular+damage+in+Saccharomyces+cerevisiae.">Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae.</a> Biotechnol. Biofuels. 3, (2010).</li><li>Weisburger, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenic,+carcinogenic,+and+chemopreventive+effects+of+phenols+and+catechols:+the+underlying+mechanisms.">Mutagenic, carcinogenic, and chemopreventive effects of phenols and catechols: the underlying mechanisms.</a> ACS Symposium Series. 507, 35-47 (2009).</li><li>Slininger, P. J., Dien, B. S., Lomont, J. M., Bothast, R. J., Ladisch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+kinetic+model+for+computer+simulation+of+growth+and+fermentation+by+Scheffersomyces+(Pichia)+stipitis+fed+D-xylose.">Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose.</a> Biotechnol. Bioeng. 111 (8), 1532-1540 (2014).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+metabolic+profiling+revealed+limitations+in+xylose-fermenting+yeast+during+co-fermentation+of+glucose+and+xylose+in+the+presence+of+inhibitors.">Comparative metabolic profiling revealed limitations in xylose-fermenting yeast during co-fermentation of glucose and xylose in the presence of inhibitors.</a> Biotechnol. Bioeng. 111 (1), 152-164 (2014).</li><li>Slininger, P. J., Branstrator, L. E., Bothast, R. J., Okos, M. R., Ladisch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth,+death,+and+oxygen+uptake+kinetics+of+Pichia+stipitis+on+xylose.">Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose.</a> Biotechnol. Bioeng. 37 (10), 973-980 (1991).</li></ol>

いくつかのステップは、進化のプロセスの成功に不可欠でした。まず、成功したアプリケーションのために必要とされる目的の表現型に向かって人口の進化を推進するための適切な選択圧を選択するためのキーです。以下の選択応力はS.のために選択しました開発をstipitisし、所望の表現型について濃縮を導くために適切なタイミングで適用される：12％（酢酸およびフランアル?...

リグノセルロース系バイオマスの推定年間13億ドライトンは、エタノールの生産を支援し、米国は30％、その石油消費量を削減する可能性があります。1グルコースとキシロースの豊富な植物バイオマス加水分解利回り糖混合物は、発酵阻害物質が必要な化学的前処理によって生成されますがヘミセルロースを分解し、酵素の攻撃のためにセルロースを露出させます。酢酸、フルフラール、およびヒドロキシメチルフルフラール（HMF）は、前処理の間に形成する多くの阻害剤の中で重要な構成要素であると考えられています。フォワードリグノセルロースエタノール産業を移動させるために、研究手順は、生存し、効率的に必要とされるそのような阻害化合物の存在下でヘキソース及びペントース糖の両方を使用するように機能し得る酵母株の進化を可能にします。例えばサッカロマイセス・セレビシエなどの伝統的な工業用酵母株、かなりの追加の弱点は、効率的にfermenできないことです植物バイオマスの加水分解物で使用可能なキシロースのt。 ピキアstipitis型株 NRRL Y-7124（5773 CBS）は、最近、名前が変更さScheffersomycesのstipitisは、よくキシロースをエタノールに発酵することが知られているネイティブのペントース発酵酵母である。2,3ここに追求された株NRRL Y-7124の進化それが報告されているので、最適なメディアで4,5,6。少しキシリトール副産物と40グラム/ Lを超えると経済的に回収エタノールを蓄積するネイティブの酵母株の最大の可能性を持っている、S。 stipitis株 NRRL Y-7124は0.41±0.06N・G / G、高細胞密度培養（6グラム/ L細胞）である。7,8抵抗の収率で40時間（4 cm G / L / hr）で70グラム/ Lのエタノールを生産します発酵エタノール、フルフラールの阻害剤、およびHMFも報告されている、 図9およびS. stipitisは、商業規模のエタノールproductioで利用できる最も有望なネイティブペントース発酵酵母の中にランクされていますnはリグノセルロースから。10我々の目的は、産業用途に適した株NRRL Y-7124のより強固な派生物に向かって進化を強制するために、多様なundetoxifiedリグノセルロース加水分解物とエタノールの選択圧を適用することでした。求められる改善された機能のうち、キーが濃縮された加水分解物中の速い糖取り込み率、より効率的な混合糖利用のための減少diauxy、エタノールおよび阻害剤の高い公差ました。 S.の応用undetoxified加水分解物へのstipitisは 、overlimingなどの加水分解物の無害化処理に関連した追加営業費用を排除するための研究の主要な焦点でした。 二つの工業的に有望な加水分解物は、進化を強制的に適用した：糖化アンモニア繊維拡張前処理トウモロコシ茎葉加水分解物（AFEX CSH）を酵素や酸で前処理スイッチグラスの加水分解液（PSGHL）を希薄11,12 AFEX前処理技術がに開発されています。希薄酸前処理が最も一般的に酵素糖化用セルロース系バイオマスを露出するように練習し、現在最も低コストの技術を表しながら、発酵阻害物質の生成を最小限に抑えます。 PSGHLは、前処理後に残ったセルロースから分離可能であり、特徴的に加水分解されたヘミセルロースからキシロースが豊富ではなく、グルコースが低いです。 AFEX CSHとPSGHL組成物は、進化のプロセスを管理するために悪用された重要な側面が互いに異なります。 AFEX CSHはPSGHL（ 表1）と比較して、アミノ酸およびアンモニア態窒素源でフランアルデヒド及び酢酸インヒビターで低いが高いです。 PSGHLが優勢糖が利用可能であるキシロースの新たな課題が発生します。したがってPSGHLは、具体的には、加水分解に改善されたキシロースの利用のために利用可能な酵母の商用利用を防止弱点を豊かにすることが適切です。でもネイティブペントース発酵酵母の中で、次善の砂糖キシロオリゴへの依存SEは、細胞の成長をサポートすると修復はさらに難しいため、様々な理由のの加水分解物で次のようになります。による酸化還元不均衡への代謝に構造的完全性、および中断をセルに広範囲の損傷を引き起こす栄養不足、阻害剤9窒素の補給を、特にの形でアミノ酸は、発酵のためにかなりの運転コストを表すことができます。分離株のスクリーニングとランキングに窒素補給の影響は、スイッチグラス加水分解物で調査しました。 改善された個体はSの天然の遺伝的多様性に依存し、複数の選択圧を使用して進化する集団に濃縮しました2さまざまな加水分解物、エタノールまたはUV放射への曝露によって誘発される人口と突然変異stipitis。選択圧は、Sの進化の進行状況を調査するために並列に直列に適用されました成長し、加水分解において効率的に発酵することが目的のデリバティブに向かってstipitis（ 図1）。ますます困難な加水分解物中の官能集団の反復培養は固形分20％の負荷で調製12％グルカンAFEX CSHあるいはPGSHLのいずれかの希釈系列を用いたマイクロプレートで達成しました。連続培養におけるキシロースに対するエタノールの不自由な成長のアプリケーションさらに向上AFEX CSHは、キシロース利用の抑制をエタノールに少ない感受性を実証する表現型を濃縮することにより、集団を適応しました。後者の特徴は、最近、グルコース発酵以下の菌株NRRL Y-7124によってペントース利用に問題が示された。PSGHL上の8濃縮は次の加水分解物の機能性を広げるために調査しました。 推定は、Sの誘導体を向上しましたstipitis NRRL Y-7124は、最も一般的な集団からコロニーを選択するストレス状態と希釈平板の下で目標と濃縮を使用して、進化過程の各段階から単離しました。無次元相対性能指数（RPIS）は、速度論的挙動は、印加異なる加水分解物の種類および栄養補助食品を評価した全体的なパフォーマンスに基づいて、歪みをランク付けするために使用しました。様々な適応手順の成功は、Sの機能性を向上させるがリグノセルロース加水分解物でstipitisが以前undetoxified加水分解物に経済的なエタノール生産を実証する菌株は、以前に報告されていない、文書化されている。13-17進化の手順を使用して、ここでより詳細に可視化するために、Slininger ら 18を大幅に超える改善された株を開発します親株NRRL Y-7124とAFEXのCSHで&gt; 40グラム/ Lのエタノールを生産し、適切な窒素源を補充した糖化スイッチグラス加水分解物（SGH）を酵素することができます。これらの新規株は、エタノール産業の発展リグノセルロースにし、追加のゲノミクス研究棟の対象として今後注目されています以前に配列決定株NRRL Y-11545のもので、図1に図解さ進化のさまざまな段階の間に生成トップの株の19ゲノミクス研究はさらに菌株改良研究の前置きとして、開発中に発生した遺伝的変化の歴史を解明することになります。

<table border="0" cellpadding="0" cellspacing="0" width="1252">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cellic Ctec, Contains Xylanase (endo-1,4-)</td>
			<td style="width:251px;">Novozymes</td>
			<td style="width:180px;">No product number</td>
			<td style="width:467px;">www.novozymes.com, 1-919-494-3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cellic Htec, Contains Cellulase and Xyalanase</td>
			<td style="width:251px;">Novozymes</td>
			<td style="width:180px;">No product number</td>
			<td>www.novozymes.com, 1-919-494-3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Toasted Nutrisoy Flour</td>
			<td style="width:251px;">Archer Daniels Midland Co. (ADM)</td>
			<td style="width:180px;">63160</td>
			<td>ADM, 4666 Faries Parkway, Decatur, IL&#160; 1800-37-5843</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Pluronic F-68 (Surfactant)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">P1300</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Difco Vitamin Assay Casamino Acids</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
			<td style="width:180px;">228830</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D,L-tryptophan&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:180px;">T3300</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-cysteine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:180px;">C7352</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Becton Dickinson and Company</td>
			<td style="width:180px;">214010</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bacto Malt Extract</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
			<td style="width:180px;">218630</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Bacto Yeast Extract</td>
			<td style="width:251px;">Becton Dickinson and Company</td>
			<td style="width:180px;">212750</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Peptone Type IV from soybean</td>
			<td style="width:251px;">Fluka</td>
			<td style="width:180px;">P0521-500g</td>
			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Adenine, &#62; 99% powder</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">A8626</td>
			<td>CAS 73-24-5,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Cytosine, &#62; 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">C3506</td>
			<td>CAS 71-30-7,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Guanine, SigmaUltra</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">G6779</td>
			<td>CAS 73-40-5,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Thymine, 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">T0376</td>
			<td>CAS 65-71-4,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Uracil, 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">U0750</td>
			<td>CAS 66-22-8,&#160; Could use other brands.&#160; Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Dextrose (D-Glucose), Anhydrous, Certified ACS</td>
			<td style="width:251px;">Fisher Chemical</td>
			<td style="width:180px;">D16-500</td>
			<td>CAS 50-99-7, Could use other brands.&#160; Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">D-Xylose, assay &#62; 99%</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td style="width:180px;">X1500</td>
			<td>CAS 58-86-6, Could use other brands.&#160; Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">96-well, flat bottom plates</td>
			<td style="width:251px;">Becton Dickinson Falcon</td>
			<td style="width:180px;">351172</td>
			<td>multiple suppliers:&#160; e.g. Thermo-Fisher, VWR, Daigger</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Wypall L40 Wiper</td>
			<td style="width:251px;">Kimberly-Clark</td>
			<td style="width:180px;"></td>
			<td style="width:467px;">towel in microplate boxes to absorb water for humidification;&#160; multiple suppliers:&#160; e.g. Thermo-Fisher, uline, Daigger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Corning graduated pyrex flask, 125-mL, narrow opening (stopper #5)</td>
			<td style="width:251px;">Corning Life Science Glass</td>
			<td style="width:180px;">4980-125</td>
			<td>multiple suppliers:&#160; e.g. Thermo-Fisher, VWR, Daigger</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Innova 42R shaker/incubator, 2.5 cm (1&#34;) rotation</td>
			<td style="width:251px;">New Brunswick Scientific (1-800-631-5417)</td>
			<td style="width:180px;">M1335-0016</td>
			<td>multiple suppliers:&#160; e.g. Eppendorf, Thermo-Fisher. Other shaker/incubators with a 2.5 cm (1&#34;) throw could be used.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Duetz Cover clamp for 4 deepwell MTP plates</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">Z365001700</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Duetz System sandwich cover for 96 deepwell plates</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">Z365001296</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Duetz System silicone seal (0.8mm black low evap) for 96 deep well plate cover</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">V0W1040027</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Blue microfiber layer for Duetz system sandwich cover</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">V0W1040001</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">96 well, 2 mL square well pyramid bottom plates, natural popypropylene</td>
			<td style="width:251px;">Applikon Biotechnology</td>
			<td style="width:180px;">ZC3DXP0240</td>
			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:355px;">Bellco 32mm silicon sponge plug closures, pk of 25 for 125-mL flasks</td>
			<td style="width:251px;">Bellco</td>
			<td style="width:180px;">1924-00032</td>
			<td>Thomas Scientific, their Catalog number is 1203K27</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:355px;">Bellco Spinner Flask, 1968-Glass Dome, Sealable Flange Type, 100-mL&#160; working volume.&#160; This design no longer manufactured.</td>
			<td style="width:251px;">Bellco</td>
			<td style="width:180px;">1968-00100 (original Cat. No.)</td>
			<td style="width:467px;">Jacketed vessels have lower inlet &#38; upper outlet ports for temp. control with circulating water bath. Vessels are 75mm in outer diam and 200mm in height. There are four side ports at ~45o angles and one top port. Port openings appropriate size for size 0 neoprene stoppers (21-22mm inner diameters on ports).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Mathis Labomat IR Dryer Oven</td>
			<td style="width:251px;">MathisAg</td>
			<td style="width:180px;">Typ-Nbr BFA12 215307</td>
			<td>Werner Mathis U.S.A. Inc. usa@mathisag.com, 704-786-6157</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dual Channel Biochemistry Analyzer</td>
			<td>YSI Life Sciences</td>
			<td>2900D-UP</td>
			<td style="width:467px;">www.ysi.com, robotic system for rapid sugars assay in 96-well microplate format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PowerWave XS Microplate Spectrophotometer</td>
			<td>Bio-Tek Instruments, Inc</td>
			<td>MQX200R</td>
			<td><a href="http://www.biotek.com/">www.biotek.com</a></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.のstipitisは AFEX CSH、PSGHL、およびエタノールチャレンジキシロース給電連続培養を含めた3つの選択培養液、の組み合わせを使用して発生した。 図1は、分離株と一緒に行わ進化実験の模式図は、最も効果的に実行するかがわかっ示し全体的に、または最も効果的にテストした加水分解物のいずれかで。 表3は、これらの優れた分離株のNRRL受託番号...

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

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

適応進化と分離技術は、ヘキソースおよび酵素でペントース混合糖がundetoxified加水分解物を糖化し、40以上のグラム/ Lのエタノールを蓄積するために急速に消費することができますScheffersomyces stipitis株NRRL Y-7124の誘導体を得るために説明し、実証されています。

エタノールへのリグノセルロースの生物変換のための堅牢なペントース発酵酵母の進化のための技術

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

Research

Education

JoVE Journal

JoVE Core

Biology

Bioengineering

Evolution

Natural Selection

SCIENTISTS IN ACTION

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

出芽酵母の老化複製の連続高分解能顕微鏡観察

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis

膝下プロテーゼの推定慣性特性に関する振動及び反応ボードテクニック

The purpose of this study was two-fold: 1) demonstrate a technique that can be used to directly estimate the inertial properties of a below-knee ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

堅牢、マイクロ流体細胞内送達プラットフォームとしての細胞絞る

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

細胞の脂質滴の分離：酵母細胞とヒト胎盤から2精製技術

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Harmonic Nanoparticles for Regenerative Research

回生研究ハーモニックナノ粒子

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

リアルタイム観察のためのマイクロ流体進化加速器上の癌集団全体にわたる異種薬物勾配の生成

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

液滴ベースのRNAシーケンシングによるハイスループット酵母株フェノタイピング

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

スクリーニング用途のための線維芽細胞由来ヒト工学的結合組織

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

微生物微小液滴培養システム(MMC)を用いた微生物の自動培養と適応進化

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...