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. Assays के लिए सामग्री और उपकरण शुरू तैयार <ol><li> रैंकिंग प्रक्रियाओं विकास, अलगाव में उपयोग और के लिए pretreatment प्रतिक्रिया में 18 से 20% प्रारंभिक बायोमास सूखी वजन का उपयोग कर hydrolyzates तैयार करें। Slininger एट अल। विस्तृ?...

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कई कदम विकास की प्रक्रिया की सफलता के लिए महत्वपूर्ण थे। सबसे पहले, यह वांछित phenotypes कि सफल आवेदन के लिए आवश्यक हैं की ओर जनसंख्या विकास ड्राइव करने के लिए उचित चयन के दबाव का चयन करने के लिए महत्वपूर्ण है?...

एक अनुमान के अनुसार वार्षिक 1.3 अरब lignocellulosic बायोमास की सूखी टन इथेनॉल उत्पादन का समर्थन और अमेरिका के 30% द्वारा अपने पेट्रोलियम की खपत को कम करने के लिए अनुमति दे सकता है। 1 हालांकि बायोमास संयंत्र हाइड्रोलिसिस पैदावार चीनी ग्लूकोज और सिलोज़ में अमीर मिश्रण, किण्वन अवरोधकों आवश्यक रासायनिक pretreatment द्वारा उत्पन्न कर रहे हैं नीचे hemicellulose तोड़ने और enzymatic हमले के लिए सेल्यूलोज को बेनकाब करने के लिए। एसिटिक एसिड, furfural, और hydroxymethylfurfural (HMF) कई अवरोधकों कि pretreatment के दौरान फार्म के बीच प्रमुख घटक माना जाता है। आदेश lignocellulosic इथेनॉल उद्योग आगे बढ़ने के लिए, अनुसंधान और प्रक्रियाओं खमीर जीवित और कुशलता से इस तरह के निरोधात्मक यौगिकों की उपस्थिति में दोनों hexose और pentose शर्करा का उपयोग करने की जरूरत है कार्य करने में सक्षम उपभेदों के विकास की अनुमति है। ऐसे Saccharomyces cerevisiae के रूप में पारंपरिक औद्योगिक खमीर में तनाव, का एक महत्वपूर्ण अतिरिक्त कमजोरी, असमर्थता कुशलतापूर्वक fermen करने के लिए हैसिलोज़ बायोमास संयंत्र की hydrolyzates में उपलब्ध टी। Pichia stipitis प्रकार के तनाव NRRL वाई-7124 (CBS 5773), हाल ही में नाम बदलकर Scheffersomyces stipitis, एक देशी pentose fermenting खमीर कि अच्छी तरह से इथेनॉल के लिए सिलोज़ विक्षोभ के लिए जाना जाता है तनाव NRRL वाई-7124 के विकास के लिए यहां अपनाई थी क्योंकि यह करने के लिए दस्तावेज किया गया है। 2,3 इष्टतम मीडिया, एस में थोड़ा xylitol प्रतिफल के साथ 40 ग्राम / एल से अधिक आर्थिक रूप से वसूली इथेनॉल जमा करने के लिए मूल निवासी खमीर उपभेदों की सबसे बड़ी क्षमता है। 4,5,6 stipitis तनाव NRRL वाई-7124 0.41 ± 0.06 छ उच्च घनत्व सेल संस्कृतियों (6 ग्राम / एल कोशिकाओं) में / जी। 7,8 प्रतिरोध की उपज में 40 घंटा (1.75 छ / एल / घंटा) में 70 ग्राम / एल इथेनॉल का उत्पादन किण्वन अवरोधकों इथेनॉल, furfural, और HMF को भी सूचना दी गई है, 9 और एस stipitis सबसे होनहार मूल निवासी pentose-fermenting व्यावसायिक पैमाने इथेनॉल productio के लिए उपलब्ध yeasts के बीच में स्थान दिया गया हैn lignocellulose से। 10 हमारा उद्देश्य औद्योगिक अनुप्रयोगों के लिए उपयुक्त तनाव NRRL वाई-7124 की एक और अधिक मजबूत व्युत्पन्न की ओर विकास के लिए मजबूर करने के लिए विविध undetoxified lignocellulosic hydrolyzates और इथेनॉल के चयन के दबाव लागू करने के लिए किया गया था। मांग की बेहतर सुविधाओं के बीच कुंजी केंद्रित hydrolyzates में तेजी से चीनी तेज दर, अधिक कुशल मिश्रित चीनी उपयोग के लिए कम diauxy, और इथेनॉल और अवरोधकों के उच्च tolerances थे। एस के आवेदन undetoxified hydrolyzates करने के लिए इस तरह के stipitis overliming रूप हायड्रोलायसेट विषहरण प्रक्रियाओं, के साथ जुड़े जोड़ा परिचालन खर्च को खत्म करने के लिए अनुसंधान के एक प्रमुख ध्यान केंद्रित किया गया था। दो औद्योगिक रूप से होनहार hydrolyzates विकास के लिए मजबूर करने के लिए लागू किया गया:। Saccharified अमोनिया फाइबर विस्तार-pretreated मकई पशुओं का चारा हायड्रोलायसेट (AFEX CSH) एंजाइम और तनु अम्ल-pretreated switchgrass हायड्रोलायसेट शराब (PSGHL) 11,12 AFEX pretreatment प्रौद्योगिकी के लिए विकसित किया जा रहा हैकिण्वन अवरोधकों के उत्पादन को कम करते हुए तनु अम्ल pretreatment वर्तमान सबसे कम लागत प्रौद्योगिकी का प्रतिनिधित्व सबसे अधिक एंजाइमी शर्करीकरण के लिए cellulosic बायोमास को बेनकाब करने का अभ्यास किया। PSGHL सेल्यूलोज pretreatment के बाद शेष से वियोज्य है और विशेषतया hydrolyzed hemicellulose से सिलोज़ में समृद्ध है, लेकिन ग्लूकोज में कम है। AFEX CSH और PSGHL रचनाओं महत्वपूर्ण पहलुओं जो विकास की प्रक्रिया का प्रबंधन करने के लिए इस्तेमाल किया गया था में एक-दूसरे से भिन्न होते हैं। AFEX CSH PSGHL (तालिका 1) के साथ तुलना furan एल्डीहाइड और एसिटिक एसिड अवरोधकों में कम है, लेकिन अमीनो एसिड और अमोनिया नाइट्रोजन स्रोतों में अधिक है। PSGHL प्रमुख चीनी उपलब्ध होने सिलोज़ के अतिरिक्त चुनौती प्रस्तुत करता है। इस प्रकार PSGHL विशेष रूप से, hydrolyzates में सिलोज़ उपयोग में सुधार के लिए संतुष्ट करने के लिए एक कमजोरी उपलब्ध खमीर के वाणिज्यिक उपयोग को रोकने के लिए उचित है। यहाँ तक कि मूल निवासी pentose fermenting खमीर बीच, सबऑप्टिमल चीनी जाइलो पर निर्भरताएसई सेल के विकास का समर्थन करने के लिए और मरम्मत के लिए और भी अधिक कारणों की एक किस्म की वजह से hydrolyzates में चुनौतीपूर्ण हो जाता है। पोषक तत्वों की कमी, अवरोधकों संरचनात्मक अखंडता, और चयापचय के लिए व्यवधान सेल व्यापक क्षति के कारण redox असंतुलन के कारण 9 नाइट्रोजन पूरकता, खासकर के रूप में अमीनो एसिड, fermentations के लिए एक महत्वपूर्ण परिचालन लागत का प्रतिनिधित्व कर सकते हैं। अलग स्क्रीनिंग और रैंकिंग पर नाइट्रोजन पूरकता का प्रभाव switchgrass hydrolyzates साथ पता लगाया था। सुधार व्यक्तियों एस के प्राकृतिक आनुवंशिक विविधता पर निर्भर एकाधिक चयन के दबाव का उपयोग कर एक उभरती आबादी में समृद्ध थे stipitis जनसंख्या और म्यूटेशन दो विविध hydrolyzates, इथेनॉल या पराबैंगनी विकिरण के लिए जोखिम द्वारा प्रेरित किया। चयन के दबाव में समानांतर और श्रृंखला में लागू किया गया एस के विकास प्रगति का पता लगाने के लिए वांछित बढ़ने और hydrolyzates में कुशलता विक्षोभ करने में सक्षम डेरिवेटिव की ओर stipitis(चित्रा 1)। तेजी से चुनौतीपूर्ण hydrolyzates में कार्यात्मक आबादी का दोहराव संवर्धन या तो 12% Glucan AFEX CSH के कमजोर पड़ने श्रृंखला रोजगार वरना PGSHL 20% ठोस लोडिंग पर तैयार microplates में पूरा किया गया। निरंतर संस्कृति में सिलोज़ पर इथेनॉल को चुनौती दी विकास के आवेदन के आगे कम संवेदनशीलता का प्रदर्शन सिलोज़ उपयोग का दमन इथेनॉल के लिए phenotypes के लिए समृद्ध AFEX CSH अनुकूलित आबादी में सुधार हुआ। उत्तरार्द्ध सुविधा हाल ही में ग्लूकोज किण्वन के बाद तनाव NRRL वाई-7124 से pentose उपयोग करने के लिए समस्याग्रस्त दिखाया गया था। PSGHL पर 8 संवर्धन अगले हायड्रोलायसेट कार्यक्षमता को व्यापक बनाने का पता लगाया था। एस के ख्यात में सुधार डेरिवेटिव stipitis NRRL वाई-7124 विकास प्रक्रिया तनाव की स्थिति और कमजोर पड़ने चढ़ाना के तहत लक्षित संवर्धन का उपयोग करते हुए सबसे अधिक प्रचलित आबादी से कालोनियों लेने के लिए के प्रत्येक चरण से अलग थे। आयामरहित रिश्तेदारप्रदर्शन सूचकांक (RPIs) समग्र प्रदर्शन, जहां गतिज व्यवहार अलग हायड्रोलायसेट प्रकार और पोषक तत्वों की खुराक लागू किया पर मूल्यांकन किया गया था पर आधारित उपभेदों पद के लिए इस्तेमाल किया गया। विभिन्न प्रक्रियाओं अनुकूलन की सफलता एस की कार्यक्षमता में सुधार करने के लिए यद्यपि lignocellulosic hydrolyzates में stipitis पहले से प्रलेखित किया गया है, undetoxified hydrolyzates पर किफायती इथेनॉल उत्पादन का प्रदर्शन उपभेदों पहले से नहीं बताया गया है। 13-17 विकास प्रक्रियाओं का उपयोग और अधिक विस्तार में यहाँ देखे जा करने के लिए, Slininger एट अल। 18 विकसित उपभेदों कि काफी अधिक सुधार कर रहे हैं माता पिता के तनाव NRRL वाई-7124 और AFEX CSH में&gt; 40 ग्राम / एल इथेनॉल का उत्पादन और saccharified switchgrass हायड्रोलायसेट (SGH) उचित रूप से नाइट्रोजन स्रोतों के साथ पूरक एंजाइम करने में सक्षम हैं। ये उपन्यास उपभेदों इथेनॉल उद्योग को विकसित करने के lignocellulose को भविष्य में ब्याज की और अतिरिक्त जीनोमिक्स के अध्ययन के निर्माण के विषय के रूप में कर रहे हैं19 पहले से अनुक्रम तनाव NRRL वाई-11545 के उन लोगों पर। चित्रा 1 में diagramed आनुवंशिक परिवर्तन है कि एक प्रस्तावना तनाव सुधार अनुसंधान और आगे के रूप में विकास के दौरान हुई के इतिहास को स्पष्ट होता है विकास के विभिन्न चरणों के दौरान उत्पादन शीर्ष उपभेदों के एक अध्ययन जीनोमिक्स।

<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">
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			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
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			<td style="width:251px;">Applikon Biotechnology</td>
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			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
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			<td>applikon-biotechnology.com (U.S.), 1-650-578-1396</td>
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			<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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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.

एस stipitis तीन चयन संस्कृतियों, जो AFEX CSH, PSGHL, और इथेनॉल को चुनौती दी सिलोज़ खिलाया निरंतर संस्कृति शामिल के संयोजन का उपयोग कर विकसित किया गया था। चित्रा 1 से पता चलता वियोजन के साथ प्रदर्शन किया विका?...

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

अनुकूली विकास और अलगाव की तकनीक का वर्णन किया और Scheffersomyces stipitis तनाव के डेरिवेटिव NRRL वाई-7124 है कि सक्षम हैं तेजी से उपभोग करने के लिए hexose और एंजाइम में pentose मिश्रित शर्करा undetoxified hydrolyzates saccharified और 40 ग्राम / एल इथेनॉल पर जमा करने के लिए उपज के लिए प्रदर्शन कर रहे हैं।

इथेनॉल के लिए Lignocellulose की bioconversion के लिए मजबूत pentose-fermenting खमीर के विकास के लिए तकनीक

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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