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. Tahliller için Malzemeler ve Ekipmanları Başlangıç ​​hazırlayın <ol><li> evrim, izolasyon kullanımı ve sıralama işlemleri için ön reaksiyonunda 18 ila% 20 ilk biyokütle kuru ağırlık kullanılarak hydrolyzates hazırlayın. Slininger ve ark. Detaylı yöntemleri için 2015 18 evrim, izolasyon veya sıralamasında kullanılan azot takviyesi N1 veya N2 ile AFEX CSH, PSGHL ve SGH hazırlamak bakın. Her hidrolizat Çeşidi kompozisyon için Tablo 1&#39;e bakın?...

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Birkaç adım evrim sürecinin başarısı için kritik idi. Birincisi, başarılı bir uygulama için gerekli olan istenilen fenotip doğru nüfus evrimi götürmek uygun seçim baskıları seçmek için anahtardır. Aşağıdaki seçici gerilmeleri S. seçildi gelişimi stipitis ve istenen fenotipleri için zenginleştirme yol uygun zamanlarda uygulanır:% 12 (asetik asit ve furan aldehitler ve diğer inhibitörleri düşük seviyelerde mevcudiyetinde büyüme ve çeşitli şekerlerin fermentasyonu k...

Lignoselülozik biyokütle tahmini yıllık 1,3 milyar kuru ton etanol üretimini desteklemek ve ABD% 30, petrol tüketimini azaltmak için izin verebilir. 1 glikoz ve ksiloz zengin bitki biyokütle hidroliz verimleri şeker karışımları, fermantasyon inhibitörleri gerekli kimyasal ön tarafından oluşturulan rağmen hemiselüloz yıkmak ve enzimatik saldırı için selülozu ortaya çıkarmak. Asetik asit, furfural ve (HMF) hidroksimetilfurfural ön-muamelesi sırasında oluşturan birçok önleyicileri arasında temel bileşenleri olduğu düşünülmektedir. İleri lignoselülozik etanol endüstriyi için, araştırma ve prosedürler hayatta kalan ve etkili bir şekilde gerekli olan bu tür önleyici bileşikler varlığında heksoz ve pentoz şekerleri her ikisini de kullanmak fonksiyon görme yeteneğine sahip maya suşları evrimini izin vermek. Saccharomyces cerevisiae gibi geleneksel sanayi maya suşları, önemli bir ilave zayıf verimli fermen yetersizlikBitki biyokütle hidrolizatları kullanılabilir ksiloz t. Pichia stipitis tip suşu NRRL Y-7124 (5773 CBS), son zamanlarda yeniden Scheffersomyces stipitis iyi etanol ksiloz fermente bilinen bir yerli pentoz fermantasyon mayası olduğunu. 2,3 burada izlenmiştir NRRL Y-7124 evrimi bu belgelenmiş oldu çünkü küçük ksilitol yan ürün ile birlikte, 40 g / l&#39;den fazla ekonomik olarak etanol birikmesine ana maya kökü büyük bir potansiyele sahiptir. 4,5,6 uygun ortam, S. olarak stipitis NRRL-Y-7124 0.41 ± 0.06 g / g yüksek hücre yoğunluklu kültürleri (6 g / L hücreleri). 7,8 direnç bir verimle 40 saat (1.75 g / L / saat) &#39;de 70 g / L etanol üretir fermantasyon inhibitörleri, etanol, furfural ve HMF de 9 ve S. bildirilmiştir stipitis ticari ölçekli etanol Bluz mevcut en umut verici yerli pentoz fermente mayalar arasında yer olmuşturn lignoselüloz gelen. 10 Amacımız endüstriyel uygulamalar için uygun NRRL Y-7124 daha sağlam bir türevi doğru evrimi zorlamak için çeşitli undetoxified lignoselülozik hydrolyzates ve etanol seçilim baskıları uygulamak oldu. aranan geliştirilmiş özellikleri arasında anahtar konsantre hidrolizatları daha hızlı şeker alım hızları, daha etkin bir karışık şeker kullanımı için daha az diauxy ve etanol ve önleyicilerinin yüksek toleransları edildi. S. uygulanması undetoxified hidrolizatları için stipitis gibi overliming olarak hidrolizat detoksifikasyon süreçleri ile ilişkili ilave işletme giderini ortadan kaldırmak için araştırma önemli bir odak noktası oldu. İki endüstriyel umut verici hidrolizatları evrimi zorlamak için uygulanmıştır. (PSGHL) şekerleştirilmiş amonyak fiber genişletme ön tedavi mısır koçanı hidrolisat (AFEX CSH) enzim ve asit-ön tedavi darı hidrolizat içki sulandırmak 11,12 AFEX ön teknolojisi geliştiriliyorseyreltilmiş asit ön en yaygın güncel en düşük maliyet teknolojiyi temsil eden enzimatik sakarifikasyon için selülozik biyokütle maruz uygulanan ederken, fermantasyon inhibitörlerinin üretimini en aza indirmek. PSGHL ön sonrası kalan selüloz ayrılabilir ve glikoz karakteristik hidrolize hemiselüloz gelen ksiloz zengin, ama düşüktür. AFEX CSH ve PSGHL kompozisyonlar evrim sürecini yönetmek için istismar edildi kilit yönlerini birbirinden farklıdır. AFEX CSH PSGHL (Tablo 1) ile karşılaştırıldığında, amino asitler ve amonyum azot kaynakları furan aldehit ve asetik asit inhibitörleri düşük ancak daha yüksektir. PSGHL baskın şeker mevcut olan ksiloz ek bir meydan okuma sunuyor. Böylece PSGHL özellikle hidrolizatları geliştirilmiş ksiloz kullanımı için mevcut maya ticari kullanımını engelleyen bir zayıflık zenginleştirmek için uygundur. Hatta yerli pentoz fermantasyon mayaları arasında optimal şeker Xylo dayanmakse hücre büyümesini desteklemek ve onarım daha da zorlu nedenlerden dolayı çeşitli hidrolizatları kazanmaktadır. sebebiyle redoks dengesizlikler metabolizmaya yapısal bütünlüğünü ve bozulma hücre yaygın hasara neden besin eksiklikleri, inhibitörleri 9 Azot takviyesi, özellikle şeklinde amino asitler, fermantasyonlar için önemli bir işletme maliyetini temsil edebilir. izole tarama ve sıralama azot takviyesi etkisi darı hidrolizatları ile araştırılmıştır. Geliştirilmiş bireyler S. doğal genetik çeşitliliği dayanan çoklu seçim baskıları kullanarak gelişmekte popülasyonda zenginleştirildi İki farklı hidrolizatları, etanol veya UV ışınlarına maruz kalma tarafından uyarılan stipitis nüfus ve mutasyonlar. Seçim basınçları S. evrimi ilerlemesini keşfetmek için paralel ve seri olarak uygulanmıştır büyümek ve hidrolizatları verimli fermente edebilir istenen türevleri doğru stipitis(Şekil 1). giderek daha zor hidrolizatları fonksiyonel popülasyonlarının tekrar kültür ya da% 12 glukan AFEX csh&#39;ın bir seyreltme serisi kullanılarak ya da başka bir PGSHL% 20 katı yüklemede hazırlanan mikro-plakalar içinde gerçekleştirilmiştir. Sürekli kültüründe ksiloz üzerinde etanol-meydan büyüme uygulama daha ksiloz kullanımı baskıyı etanol daha az etkileniyor gösteren fenotipleri için zenginleştirerek AFEX CSH adapte popülasyonları düzeldi. İkinci özellik son zamanlarda glikoz fermantasyonu aşağıdaki NRRL Y-7124 ile pentoz kullanımına sorunlu gösterilmiştir. PSGHL 8 Zenginleştirme sonraki hidrolizat işlevselliği genişletmek için araştırılmıştır. S. varsayılan gelişmiş türevleri stipitis NRRL Y-7124 en yaygın nüfus gelen koloniler almak için stres koşulları ve seyreltme kaplama kapsamında hedeflenen zenginleştirme kullanarak evrim sürecinin her aşamasında izole edilmiştir. boyutsuz akrabaperformans endeksleri (RPIs) kinetik davranış uygulanan farklı hidrolizat türleri ve besin takviyeleri değerlendirildi genel performansa dayalı suşları sıralamak için kullanıldı. Çeşitli adaptasyon prosedürlerinin başarıları S. işlevselliğini artırmak için olsa lignoselülozik hidrolizatları bölgesindeki stipitis önce undetoxified hidrolizatları, ekonomik etanol üretimi gösteren suşlar, daha önce rapor edilmemiş, belgelenmiştir. 13-17 evrimi prosedürler kullanılarak burada daha ayrıntılı olarak görüntülenmiştir için Slininger ve ark., 18 belirgin olarak üzerinde geliştirilmiş olup soyları geliştirilmiştir üst NRRL-Y-7124 ve AFEX CSH&gt; 40 g / L etanol üretmek ve uygun nitrojen kaynakları ile takviye edilmiş şekerleştirilmiş switchgrass hidrolizatı (SGH) enzim edebiliyoruz. Bu yeni suşlar etanol endüstrisine gelişmekte olan lignoselüloz gelecekteki ilgi ve ek genomik çalışmalar binanın konular gibidirDaha önce sıralı NRRL Y-11545 olanlar üzerinde. 19 suş geliştirme araştırmaları ilerletmek için bir başlangıcı olarak gelişimi sırasında meydana gelen genetik değişikliklerin geçmişini aydınlatmak istiyorum Şekil 1&#39;de diagramed evrimin çeşitli aşamalarında üretilen üst soyların bir genomik çalışması.

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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Becton Dickinson and Company</td>
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			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:355px;">Peptone Type IV from soybean</td>
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			<td>multiple suppliers:&#160; e.g. Fisher Scientific, VWR, Daigger</td>
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		<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>
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			<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>
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		<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>
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			<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. stipitis AFEX CSH, PSGHL ve etanol-meydan ksiloz beslenen sürekli kültürü dahil üç seçim kültürlerin kombinasyonları kullanılarak gelişti. Şekil 1 izolatların birlikte gerçekleştirilen evrim deneyleri şeması ya en etkin gerçekleştirmek için bulundu gösterir Genel olarak ya da en etkili test hidrolizatlarının seçebilir. Tablo 3, bu birçok izolatların NRRL erişim numaraları gösterir ve her suş izole edildiği zenginleştirilmiş popülasyonu elde...

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

Adaptif gelişimi ve izolasyon teknikleri tarif hızla heksoz ve enzimin pentoz karışık şekerler undetoxified hidrolizatlarını şekerleştirilir ve 40 g / L etanol içinde birikir tüketmek mümkün Scheffersomyces stipitis suşunun türevleri NRRL-Y-7124 elde etmek üzere gösterilmektedir.

Etanol ile lignoselüloz biyo dönüşümünü için Sağlam Pentoz-fermente Maya Evrimi Teknikleri

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

The overall goal of this adaptive evolution procedure is to derive robust strains of the native xylose fermenting yeast scheffersomyces stipitis NRRL Y-7124 that can more rapidly ferment the hexose and pentose sugars in undetoxified hydrolysates to economically recoverable ethanol. This method moves the lignocellulose to ethanol industry forward by providing a road map to strains with complex modifications, allowing fermentation of sugars in hydrolysates which contain microbial inhibitors. Here, the main advantage of targeted evolution is that robust strains are molded from a native yeast able to ferment xylose, eliminating the need to engineer industrial saccharomyces strains.We realized that any single yeast strain was unlikely to be robust in different types of hydrolysates, such as AFEX pretreated corn stover or dilute acid pretreated switchgrass. The ability of scheffersomyces stipitis to undergo a sexual reproductive cycle is beneficial to further exploitation in the present work because the various strains developed can be combined into a single, more robust strain through mating of strains having these various traits. Demonstrating the procedure will be coauthors Stephanie Thomson and Maureen Shea-Andersh who are biological science technicians in my laboratory.Prior to starting this enrichment procedure, prepare a preculture of strain NRRL Y-7124 in a 125 milliliter flask and a dilution series of the enzyme saccharified ammonia fiber expansion pretreated corn stover hydrolysate, or AFEX CSH. Use a repeating pipette to fill a 96 well microplate with 15 microliters per well, and eight wells per hydrolysate dilution. Inoculate with a few microliters of preculture per well to allow an initial absorbance at 620 nanometers, or A620 of greater than or equal to 0.1.Place the microplate in a plastic box with a wet paper towel for humidity and incubate statically for 24 to 28 hours at 25 degrees Celsius. Next, use a microplate reading spectrophotometer to identify the cultures that physically grew to an A620 greater than 1.0 in the most concentrated hydrolysate dilution. Pool the identified wells in a microfuge tube, and distribute to a fill plate.Transfer one to five microliters from the fill plate to each well of a new hydrolysate dilution series to achieve an initial A620 of greater than or equal to 0.1 and incubate the plate as before. Monitor the growth of the cultures in the microplate with a microplate reading spectrophotometer. Prepare glycerol stocks of adaptation cultures regularly.Pool four wells of the greatest hydrolysate concentration colonized in a cryofile, and mix one to one with 20%sterile glycerol. Freeze cells at minus 80 degrees Celsius. Repeat this enrichment procedure until growth in 12%glucan AFEX CSH is consistently visible at 24 hours.To begin this procedure, streak selected glycerol stocks of adaptation cultures to yeast malt peptone dextrose, or YM agar. Then after growth, use streaks to inoculate 50 microliters of 3%or 6%glucan AFEX CSH in each of three microplate wells. Incubate the microplate for 24 hours at 25 degrees Celsius.Pool replicate colonized wells at the highest hydrolysate strength with strong growth. And dilution plate to YM or 6%glucan AFEX CSH agar. Incubate the plates in a 25 degree Celsius incubator for 24 to 48 hours.Pick five single colonies from the highest dilution plate showing growth to freeze as glycerol stock cultures. Streak each colony to a YM plate and incubate for 24 hours. With a sterile loop, transfer a developed streak of cells to a small volume of 10%glycerol mixed to suspend the cells and distribute to two to three cryofiles for freezing at minus 80 degrees Celsius.To test isolates in simple 6%glucan AFEX CSH batch cultures, transfer cells from 48 hour plates streaked from glycerol stocks to pH 7 potassium phosphate buffer to prepare cell suspensions. After measuring the optical density of each cell suspension, adjust the volume with additional buffer to reach an A620 of ten. Use one microliter of each suspension to inoculate each of four wells of 50 microliters of 3%glucan hydrolysate to an initial A620 of 0.2.Incubate the microplate for 24 hours. Next, transfer two 24 hour microplate wells to inoculate 25 milliliter precultures of 6%glucan AFEX CSH at pH 5. Close the flasks with silicon sponge closures and incubate for 24 hours at 25 degrees Celsius and 150 RPM.Use the 6%glucan AFEX CSH precultures to inoculate similar 25 milliliter growth cultures in duplicate to an initial A620 of 0.1. Incubate the growth cultures for 24 hours at 25 degrees Celsius and 150 RPM. To test dioxic lack of isolates in batch cultures of optimal defined medium, or ODM, with mixed sugars, inoculate precultures of 75 milliliters ODM with 150 grams per liter of xylose in 125 milliliter flasks with silicone sponge closures.Incubate for 24 hours at 25 degrees Celsius and 150 RPM. Use the precultures to inoculate to an A620 of 0.1 similar duplicate 75 milliliter test cultures with ODM containing 75 grams per liter of glucose and 75 grams per liter of xylose at pH 6.5. Shake flasks at 25 degrees Celsius and 150 RPM.24 hours prior to inoculating a continuous culture, prepare a preculture of a representative AFEX CSH tolerant colony in ethanol-free ODM in a 125 milliliter flask. Use five to 10 milliliters of the AFEX CSH tolerant isolate preculture to inoculate 100 milliliters of ODM in a jacketed 100 milliliter spinner flask to obtain an initial A620 of 0.5. Maintain the culture holding volume at 25 degrees Celsius.Stir it at 200 RPM and outfit it with a sterilizable pH electrode, a pH controller, and a temperature controlled circulating water bath. Initially, since cell growth will be slow due to the ethanol exposure, set up a pH actuated pump so that when the culture fermentation decreases the pH to 5.4, the pump will feed pH 6.3 ODM with 100 grams per liter of xylose and 50 grams per liter of ethanol to stop further pH decreases. Maintain the volume at 100 milliliters with an effluent pump continuously skimming the culture surface.Measure the effluent volume collected and draw samples from continuous cultures every 48 to 72 hours for analysis of viable cell concentration, products, and substrates as described in the manuscript. Save glycerol stocks regularly by isolating colonies from viability plates of buffered sample dilutions forming 30 to 100 colonies, representing the most prevalent robust colonies at that time in enrichment. Flood the plates with five milliliters of 10%glycerol to allow preparation of duplicate cryofiles.If cultures can grow steadily at specific rates of greater than 0.01 per hour on xylose in the presence of more than 25 grams per liter of ethanol, start the continuous culture feed at a low dilution rate of about 0.01 per hour to the 100 milliliter holding volume. Over time, raise the ethanol in the feed toward 50 grams per liter. Ultraviolet irradiation of inocula for restarting continuous cultures is an option to hasten the mutation rate.Capture advanced populations in glycerol stocks. Prior to isolating single cell colonies, glycerol stock populations with improved xylose fermentation in the presence of ethanol, are identified as described in the text protocol. Use these superior populations to streak plates.The streaked plates are used to prepare dense buffered cell suspensions of an A620 equal to five. Then cell suspensions are used to inoculate precultures in 96 deep well plates. Incubate for 48 hours at 25 degrees Celsius, 400 RPM and one inch orbit.Next, enrich tolerant colonies by using each preculture to inoculate 16 one milliliter replicate cultures to an initial A620 of 0.5 in acid pretreated switchgrass hydrolysate liquor, mixed one to one with ODM plus 50 grams per liter of xylose, and enough ethanol to provide a 20, 30 or 40 grams per liter of ethanol challenge. Incubate for 48 hours at 25 degrees Celsius, 400 RPM and one inch orbit. For each different glycerol stock, harvest the culture wells with the highest ethanol concentration allowing growth and xylose use.Plate each cell line to YM agar to obtain prevalent single colonies to preserve in glycerol. Pick 10 colonies per cell line and streak each to a YM agar plate for glycerol stock preparation. After adaptation on AFEX CSH, a comparison of fermentation performances in ODM between the parent strain and adapted populations reveal that the adapted populations use xylose more efficiently to make ethanol more rapidly and in higher amounts.A hydrolysate tolerant colony was further developed for improved ethanol tolerance by continuous culture selection on ODM containing xylose and high levels of ethanol. All adapted populations surpassed the parent in the ability to induce xylose metabolism in the presence of 40 grams per liter of ethanol. These graphs summarize the performances of superior tolerant isolates assessed in terms of xylose uptake rates and ethanol yields relative to the parent strain for each formulation of PSGHL.The best strains selected from the primary screen on xylose rich PSGHL were subsequently screened on three complete hydrolysates and the relative performance index, or RPI, was calculated for the fermentation of each isolate within each hydrolysate. Next, the combined overall performance indices for each isolate were calculated. Five isolates, three, 14, 27, 28 and 33, had overall RPIs above 60, which ranked them as the most robust to all of the variations of hydrolysate and nutrient conditions combined.After watching this video, you should know how to challenge a xylose fermenting yeast like scheffersomyces stipitis NRRL Y-7124 in a targeted evolution process, enrich, isolate, and evaluate more robust derivatives which more effectively ferment mixed sugars in undetoxified hydrolysates. Once mastered, this technique can be used with a daily time investment averaging about an hour per day. Significant changes in the evolution process can be documented in as little as four months.While attempting this procedure, it's important to save advancing populations regularly by preparing glycerol stocks to freeze at minus 80 degrees Celsius. This allows population recovery, periodic evaluation, isolation or restarting the evolution process. To manage various mutations, remember to regularly screen the yeast under process specific conditions for an array of phenotypes.Calculation of overall dimensionless relative performance indices, or RPIs, based on kinetic parameters, allows assessment of strain rank and robustness to variations in fermentation conditions like hydrolysate type and nutrient supplementation. Superior yeasts arising from each phase of the evolution process are candidates for genome sequencing so that the nature of the improvements such as inhibitor tolerance are reduced and dioxic lag can be understood and used for design of next generation biocatalytic strains.

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Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Cihazlar arasında iki klinik öncesi görüntüleme modalitesinin etkili bir şekilde birlikte kaydedilmesi

Multimodal Cross-Device and Marker-Free Co-Registration of Preclinical Imaging Modalities

Yazar Spotlight: Birden Fazla Preklinik Görüntüleme Modalitesinin Otomatik Füzyonu için Verimli ve Sağlam Bir Yazılım

Preklinik Görüntüleme Modalitelerinin Multimodal Cihazlar Arası ve İşaretleyicisiz Ortak Kaydı

Integrated preclinical multimodal imaging systems, such as X-ray computed tomography (CT) combined with positron emission tomography (PET) or magnetic ...