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

<ol>
	<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>

The authors have nothing to disclose.

有几个步骤是对演​​化过程的成功至关重要。首先，它的关键是选择合适的选择压力，带动人口发展走向所需要的成功应用所需的表型。下面的选择性压力被选为S.树干毕赤酵母的发展，并在适当的时间施加到引导富集所需表型:12％葡聚糖AFEX CSH（这迫使生长和多样的糖发酵在乙酸和呋喃醛和其它抑制剂的低含量的存在下）增加强度;木糖喂连续培养随着乙醇浓度（这迫使木糖酶诱导减少diauxic滞后）;并增加20％的固体负载PSGHL（这迫使生长和木糖的发酵在高乙酸，呋喃，和其它抑制剂的存在下）的强度。第二，通过冷冻甘油s到保持不断变化的酵母群体是重要人口样本tocks作为浓缩过程的进行。人口的这样的快照可以被存储用于周期性功能测试记录演化进度并根据需要，以允许随后的隔离，或裂孔后重新启动演化过程。进化过程中的第三个关键步骤是通过丰富上一个方便的选择培养基选择培养种群或甘油储备的人群（如含有一个应力梯度呈现一系列水解产物和/或乙醇的浓度的琼脂或微板），以回收呈分离。然后存活殖民地中最紧张的条件下生长，可采摘后保留用于表征。可以重复这三个基本步骤，以追求每个附加所需表型的选择压力，或可选择地，多重压力在合适的单个周期。当天然亲酵母群体暴露于各种胁迫，遗传多样性预计出现第<html>粗糙的天然或诱导的突变，并继续曝光将让自然选择，以丰富的最有益突变支持有竞争力的生存的人。据预计，所选择的表型将发生作为多个基因突变的结果。在上述协议中，突变可以UV处理期间或潜在酵母暴露于水解产物中被诱导。水解物已知含有三个类化合物的有害微生物:羧酸，醛和酚22活性醛，例如糠醛和5-羟甲基，可破坏细胞，引起活性氧的正视（ROS），通常在生成的线粒体。活性氧是公知的引起DNA突变的真核细胞。23,24存在于水解产物的酚醛树脂，虽然没有先前已知具有遗传毒性，可促进和协同醛和诱变活性氧的致突变作用。25 jove_content"&gt;逐步挑战性的环境的逐步战略预期建立的一系列表型的改善进化的菌株，有针对性和还未定位，这可能是某些非靶向的表型可能发生的是不期望的或不稳定的。为了捕捉到最高度运作和稳健的菌株，必须采取额外的关键一步是评估和比较选择的菌株为商业相关的特性，达到了有针对性和非针对性的表型。要做到这一点，隔离演出应该在比较各种应用型胁迫条件下，例如在具有不同的抑制剂的挑战和营养制剂，允许最佳的整体菌株是稳定的，坚固的，以广泛的工业木质纤维基材变异的精选水解物，此外，一个应变稳定性挑战可掺入的屏幕通过包括前期培养步骤involv在这个例子做在两个非选择性培养基，YM琼脂几代，接着用50g / L的木糖合成ODM额外6-7代，对于曝光之前所需培养性状不稳定在性能挑战给予机会ING初始酵母生长水解物。根据显著性能特征，如乙醇产量和木糖摄取率，分离然后排在每个不同的应力状态，如测试的每个水解产物的环境中，这可能是从分离的富集培养基不同。量纲RPI可用于计算分离的被比较的平均整体相对性能和等级。无量纲因素是适当的，以反映不同类型的水解产物的相对表现，并根据不同的性能评估，如收益率与利率。这个排名程序确定执行株一致各地在良好的生长条件和广阔HYDR变化olyzates和易于既稳健和遗传稳定的。 各种迭代和这些基本步骤的修改可能是必要的，以容纳能够对木质纤维素水解产物或感兴趣的其它基质特异性或独特性能特征的任何微生物菌株的进化。在S.树干毕赤酵母的例子，它要注意的养分，补充，包括低成本商业来源，以在高固体负载制备水解物是重要的被键筛选期间控制分离性能改进统计分离的动态范围。营养素浓缩抑制水解物的成功的发酵的重要性也已先前报道，并且被认为是由于升高的必要的氨基酸和细胞维持，氧化还原平衡，并且修复由于这种应力条件的结果所需的其他营养物，尤其是在木糖利用率。9,26,27此外，如果营养素过于稀疏或过于充沛，在分离演出的差异可能很难统计发现，由于所有菌株做非常不好，或都在做非常好，分别为。隔离能够执行以及在不同的条件下，预计是可靠的，还是稳健的工业条件的变化。 该协议的主要限制是，适应性进化和筛选是非常结局文字，在"一会得到什么人丰富和屏幕的"。因此，富集和屏幕条件需要被设计以扩增与表型分别识别个人，与所期望的变化，同时仍保持理想的预先存在的性状。进化可能影响非选择的是工业性能（如生长因子的要求）的关键特征。出于这个原因，对于一个特征人口富集进展需要定期监视埃德其他性状故障排除对不良变化的方法。例如，S的独特特征之一赤酵母是其天然生长并发酵木糖的能力。所有浓缩和筛选基材包括木糖作为抑制剂存在唯一或主要的贡献的碳源。因此，所有在本实施例中的富集培养物的一个共同特点是，它们直接选择用于更快速木糖利用菌株，其种群成为串行或连续培养越来越富集，因为他们更好的竞争对手。作为一个预期的结果，提高株都能够更快速的木糖吸收，但改进乙醇产量均高于木糖吸收率显着提高少得多。后者的趋势可能是约由于由亲本菌株的乙醇产率是相对高的在0.3左右克/克，或理论的60％（0.51克/克），细胞生长成为水解物静止，留下较少的空间改进。在文化中，较高的乙醇产量可能会选择可能对因为乙醇是一种生长抑制剂，因此有必要演进人口的性能监控此特征。保持低于对生长抑制的水平的乙醇浓度将趋向于消除这种作为选择的因素。对于S.赤酵母 NRRL Y-7124，64路同时g / L的阻止生长完全为40g / L乙醇半特定生长率28乙醇挑战连续培养喂木糖，通气，保持在较低水平和稀释率也保持足够低，以目前充裕木糖发酵培养而不是乙醇呼吸和防止富集不必要的乙醇使用属性。此外，营养补充剂在性能屏幕中使用的水解产物精心设计，以避免选择用于菌株需要昂贵的营养丰富供应，如酵母提取物。补充总是包含成本最低的商业来源availabl即，如大豆粉和尿素，虽然更丰富的组分，如在YM串联的培养物施加对比测试，例如在60％和75％PSGHL其通常消耗氮的制剂。 ODM的合成培养基，预培养和性能培养屏幕中使用，最初被设计为亲本菌株S的最佳性能树干毕赤酵母 NRRL Y-7124 7根据由商人蛋白20，该建议中定义的成分，包括维生素，矿物质，嘌呤和嘧啶，和氨基酸源供给兼容营养素具有成本效益的工业规模化使用中所述的培养基优化例行程序商业来源。为制定相关的菌株最后的建议，就是要保持尽可能预期的工业工艺条件相关的丰富和屏幕条件设计。 具有成本竞争力的可再生乙醇长期以来一直奉行与r得出对化石燃料的依赖。为了改善S的能力赤酵母 NRRL Y-7124耐受，生长和发酵木质纤维素水解产物抑制，一些研究人员已经使用了丰富的木糖白酒重复培养，从木质纤维素生物质稀酸预处理产生。13,14,16,17过石灰解毒仍需要抑制剂以允许对硬木和麦草预处理液早期适于菌株表现一个合理的水平。已应用紫外诱变和基因组改组的13,14-多幅重复几轮开发S的衍生物树干毕赤酵母 NRRL Y-7124具有改进抑制剂耐受性和在硬木生长花亚硫酸盐废液（HWSSL）16,17然而，通过在HWSSL这些菌株产生的乙醇浓度下，经过6〜7天10克/升。使用示出和描述这里的技术中，S的新发展的菌株赤酵母 NRRL Y-7124的开发是为了满足phenoty需要经济的商业用途PE目标:水解发酵，恕不另行解毒措施，如过石灰，这导致昂贵的垃圾处理pH值5-6;大大降低增长和diauxic滞后;乙醇积累商业蒸馏（&gt; 40克/升乙醇）足够高;更快速的增长和乙醇产比以前的原生酵母发酵undetoxified水解报告;并且在高固体负载多样水解物，包括适当的大豆氮补充SGH（它是否则氮差）和未补充AFEX CSH性能。使用新的激进做法体现在上面总结的关键步骤演变发展的优良品种，预计将在降低运营成本，资本成本，能源和水的投入，从农业生物质生产乙醇的经济中获益。这是在第一应变演进计划报告，得到S的适应株stipit为证明对undetoxified水解经济可采乙醇生产。 S的新菌株从进化过程（ 图1）的各相所产生的树干毕赤酵母是未来研究的候选，以确定与应用了特定的选择压力和机制提高水解产物的发酵的基础密钥的属性，如抑制剂耐受性和减少diauxic滞后相关的遗传变化。这些宝贵的新知识，将有助于新一代酵母生物催化剂和工艺的工程木质纤维素转化为生物燃料和其他产品。随着新的菌株衍生的，它很可能会是有益的再次通过类似于为了选择在水解商业用途中最有用的转化体在此描述的菌株演进协议传递衍生物人口。同样，随着新的微生物菌株是与潜在发现做出我的从可再生生物质实用的新产品里亚德的演变过程可以进一步应用到提高应变水解稳定性和生产效率，这可能允许进行新的生物催化工艺和产品可用。

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

<table 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.树干毕赤酵母 ，使用三个选择培养，其中包括AFEX CSH，PSGHL和乙醇挑战的木糖馈连续培养的组合演变而来的。 图1示出了具有分离株一起执行进化实验的示意图发现无论是最有效地执行整体，或最有效地对测试的水解物中的一个， 表3示出了这些优越菌株NRRL保藏号并总结在实现富集的群体，从每个菌株中分离的过程中施加的适应应力。有些菌株，被视为有一个或两个水解物类型相对优越的性能，但11 7株上的所有水解类型的表现不错，尽管大部分的这些暴露，并仅在进化过程中单一类型的挑战。各演变的成功办法在这里拍摄于图2展示了-图7和表4中 。 图2描绘了在其中应变NRRL Y-7124的健壮衍生物串行传输增加AFEX CSH（步骤2）的浓度中富集适应的第一阶段后见到的不同的改进。在文化上6％葡聚糖AFEX CSH增长，演进人口演示更迅速积累和乙醇比亲株较高的最终滴度。更快速的对葡萄糖的利用也与葡萄糖立即的枯竭以下更快速的木糖吸收一起看到。此外在图3中，改变的人口的稳定特征被证明在合成培养基的ODM为87克/升葡萄糖和66克/ L木糖的混合物。在这种情况下，无论是富集的群体（ 图3B）和分离的菌落（菌落1和菌落5， 图3C和3D，水库pectively）能够通过使用木糖更有效地制造乙醇更加迅速，从而减少时间由至少4天达到峰值乙醇胜过亲代菌株。显著更高的乙醇浓度通过相比于亲本（40-45克/升）上的ODM演进株（55-60克/升）积累。突变，而导致葡萄糖木糖过渡和更高效的木糖发酵过程中减少diauxy稳定的表型，如产生酵母人口AFEX CSH演变。 到菌落5进一步改进是通过在提高的乙醇水平高达50克/升的情况下将其提交给自然选择在木糖张纸连续培养所追求。在此条件下，保持在连续培养的酵母群必须能够诱导对木糖利用作为唯一碳源的酶，以便它能够在高到足以填充发酵罐的速度增长，尽管稳定稀释率。在亲本菌株，木糖酶的诱导开始被在乙醇浓度低至15-20克/升的抑制。菌落5 8衍生物在甘油储在连续培养的操作的早期和晚期时间点捕获和图4示出在菌落5的演变衍生物观察为40g / L乙醇的存在下与NRRL Y-7124亲和接种过程中的初始集落5相比，木糖利用成功的改进。 分离从图1的自适应方案的所有阶段所产生的在PSGHL筛选以鉴定具有优越能力发酵木糖（步骤10.1）。在图5所示的分离物上PSGHL表现最好选自在此主屏幕排〜150之间以及在性能所有辅助屏幕，如下所述。为了表明进化分离的改进相对于它们的亲本，每一分离物的性能被表示为分离的动力学参数值的给亲本株的比例。的比率值"一"时发生，如果分离性能相当于父代。 图5a和5b概括顶部隔离上PSGHL的60％和75％的优势与ODM或ODM + YM营养素补充剂表演。作为该水解物的浓度和粗糙度的增加，性能比下降。在60％的实力PSGHL，七前五名分离暴露PSGHL选择压力下进行的许多倍NRRL Y-7124更好（隔离1）。 4株进行比殖民地5（33分离），这已经暴露于AFEX CSH过程中放出，但有没有以前的选择性接触PSGHL更好。然而，在PSGHL的75％的实力，只有3株显著尽管补充营养素超过父进程和殖民地5（隔离33）。其中，卓越的隔离15和16分别前情Ÿ挑战随着PSGHL浓度作为选择压力进化的指导。隔离15，14和3只暴露于PSGHL，11，13和16具有多重曝光。分离3，11和13上PSGHL在进化过程中是从较早的时间点，因此不得不稍差开发耐受机会相比14，15和16虽然在图5中明显的是串行传输到PSGHL增加强度送达培养菌株健壮到其抑制环境中，也明显的是单独的亲本菌株NRRL Y-7124到AFEX CSH的曝光可能产生交叉耐受PSGHL菌株如集落5（33）。因此，有人指出，能够在多个水解执行稳健的菌株可以通过宽容富集于一体的水解产物，然后在另一个表现筛查发现。分离从木糖喂养连续培养获得的也被筛选与PSGHL 60％和75％的优势为了维持乙醇挑战和木糖测试diauxic滞后以下葡萄糖利用的营养物质和还添加葡萄糖在75克/升。而隔离27，28和30均优于其他从适应的这个阶段，产量和木糖摄取率性能比分别为类似于在75％PSGHL母体与加入的ODM和YM养分，这不一定是令人惊讶的，没有过先前接触这种水解产物（参见Slininger 等人 18为此处的培养物中未示出的附加数据提供75克/ L葡萄糖）。 从主屏幕上丰富的木糖PSGHL选择最佳的菌株随后筛选三个完整的水解产物。这些水解产物（ 表1）包括AFEX CSH和稀酸预处理SG与商业纤维素酶处理，在两个不同的氮水平（SGH-N1和SGH-N2）为辅，以确定最通用的ISO鲈相对于抑制剂和营养环境的变化。相对性能指数（RPI）提供计算出各发酵每个水解产物（ 图6A）内的隔离。 图6B示出对每个计算出的组合的总体性能指标隔离。 5株（3，14，27，28，33）有整体RPI 60岁以上，其中排名它们作为最强大的所有水解物结合变化和营养条件的。审查表3中 ，既隔离3和14中PSGHL进化而27,28，和33在AFEX CSH或AFEX CSH和木糖乙醇挑战连续培养物中演变而来。表现出优异的多种水解物使用的菌株没有被进化上的多个水解产物。 有些菌株在任SGH-N1 / 2或AFEX CSH"专家"表现更好。表现最好的作为SGH专家的菌株水解产物（11，16和9）是由进化上PSGHL作为最终还是只获得挑战。为隔离11和16，其最初通过增加AFEX CSH挑战富集，不是积极地在PSGHL冗长最终富集过程中选择以有效地利用AFEX CSH的能力，和支持它的使用关键遗传因素明显丢失。相反，隔离14，25，27，30和33是在AFEX CSH出众的表演，以及所有除隔离14人在AFEX CSH演变带或不带酒精的挑战。因此预期，直指上AFEX CSH进化或PSGHL倾向于选择酵母很好地适应选择水解物。在这方面的一个例外是分离14分离14源自PSGHL唯一挑战并且从最终PSGHL富集培养的YM琼脂稀释电镀分离。 Slininger 等人 18表明这个分离物具有中的存在在葡萄糖中生长的细胞木糖酶诱导的优越能力5-15克/升乙酸和ODM降低diauxic滞后用75克/升的每个葡萄糖和木糖的，尽管&gt; 30g / L的乙醇之前的葡萄糖木糖转变点发生。 进化的菌株的相对于亲本菌株S的优越动力学树干毕赤酵母 NRRL Y-7124进行了论证如表4所示，表示接种初始620 8.4±2.575毫升SGH-N2低电平曝气烧瓶培养物的结果，在25在125ml烧瓶孵育硅海绵帽（pH值6.2） ℃，150rpm下（1"轨道），并且在图7中 ，表示接种初始一个每50ml 620 0.5在23毫升SGH-N 2烧瓶适中曝气烧瓶培养物。18，这些条件代表由建议的两种不同类型的操作文学作为由S.赤酵母被用于乙醇生产的潜在的商业前景。8.22。在冷杉低水平曝气牛逼例如，高密度提供了快速发酵立即开始。而在第二个实例中，低细胞密度和更高级别的曝气导致对数生长建立人口和加快生长相关的糖转化为乙醇。这些数据表明，演进的菌株具有比亲本菌株显著动力学优势:更快速的葡萄糖摄取率（ 表4），更快速的具体木糖摄取率（ 表4），更快速的乙醇生产率上葡萄糖和木糖和整体（ 表4），更短的滞后前述生长（ 图7），以及更短的diauxic葡萄糖木糖过渡滞后。这些改进允许乙醇积聚至超过40克/升和峰值天前（ 图7）比被视为父NRRL Y-7124。更高的总乙醇生产率（ 表4）的混合物在1.5达到5时s表示取决于演进菌株与亲本菌株（ 表4），的。 <img alt="图1" src="/files/ftp_upload/54227/54227fig1.jpg" /> 图 1:Scheffersomyces树干毕赤酵母 适应流程图所示的图表示在适应过程中施加的应力的顺序和优越的菌株（括号内的数字）的回收点。另请参见表3株密钥作为应变身份的参考。提供时间方向，在红色的数字表示天的适应的每个阶段的数目。对于AFEX CSH和丰富的木糖PSGHL串行传输阶段，适应每一天代表大约2-4代。对于连续培养阶段（205总天），稀释率D组在0-0.1〜变小时-1时125 pH值驱动进给D操作的。下一个80天，操作是在在0.012小时-1的连续流以D，提供了一个产生时间（LN 2）58小时后，或在稳态每2.4 d 1的发电/（D）。下一个从205天连续培养的适于人口的一个样品用紫外线诱变和接种到在0.012小时-1的D操作的连续培养。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54227/54227fig2.jpg" /> 图2:6％葡聚糖AFEX CSH的改进的分批发酵 Scheffersomyces树干毕赤酵母 NRRL Y-7124亲代菌株发酵6％葡聚糖AFEX CSH（A）与比较适于菌落5的发酵6％葡聚糖AFEX预处理的玉米秸秆ħydrolyzate（B）。符号指定生物（红方），葡萄糖（黑色圆圈虚线），木糖（蓝色圆圈实线），乙醇（绿色三角形），木糖醇（紫钻）。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54227/54227fig3.jpg" /> 图3: 降低diauxic滞后确定的培养基中以混合糖发酵性能中的ODM比较66克/升葡萄糖和87克/ L木糖为亲本菌株S.。赤酵母 NRRL Y-7124（A），在AFEX CSH改编自Y型7124（B）衍生的人口，从适应S.分离的单个细胞集落1 赤酵母人口（C），单细胞科罗拉多州NY 5从适应人群（D）隔离。符号指定生物（红方），葡萄糖（黑色圆圈虚线），木糖（蓝色圆圈实线），乙醇（绿色三角形），木糖醇（紫钻）和阿东糖醇（金钻石有黑边）。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54227/54227fig4.jpg" /> 图4: 乙醇抗性菌落5.水解物宽容菌落5 的衍生物，通过在含有木糖作为唯一碳源和高水平的乙醇ODM连续培养选择得到了进一步发展。在选择过程中（2A.1.53R，橙色三角和虚线）早期得到了两个衍生甘油股票群和连续培养接种（2A.1.30R.2，紫色圆圈和虚线）的压脚提升紫外线照射显示与NRRL Y-7124母株（绿圈与实线）和AFEX CSH宽容殖民地5（黑色三角形与比较实线）。木糖吸收葡萄糖生长酵母密集的人口（620 = 50）的ODM与40g / L乙醇指出，所有适应株在诱导木糖代谢的能力超过了不适应的父母。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig4large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54227/54227fig5.jpg" /> 图 5: 宽容的分离的性能改进相比于母公司比上级宽容分离的性能相对于总结控制父株NRRL Y-7124的PSGHL的每个配方（A，B）。表演木糖吸收率方面进行了评估和乙醇产量每提供1糖（占分离的比率于母公司的绿线）（占分离于母公司的比蓝色条）。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig5large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54227/54227fig6.jpg" /> 图6: 基于RPI隔离排名 。相对性能指数（RPI）的概念被施加到二次筛选的性能结果以排序33分离株基于木糖摄取率和每供给糖乙醇收率每个水解产物类型内。 （A）相对跑任何给定的分离之王取决于水解型（P &lt;0.001）:SGH-N1（蓝条），SGH-N2（红色条）和AFEX CSH（绿色条）。 （ 二 ）在所有类型的水解产物计算的整体RPI（浅蓝色条）表示优异的菌株不同水解条件最强劲的性能。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig6large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54227/54227fig7.jpg" /> 图7:S.卓越的适应株比较SGH发酵树干毕赤酵母 。高级适于菌株及其亲代菌株NRRL Y-7124进行比较，在25℃在低的初始发酵稀酸预处理的柳枝稷（20％固体负载）的酶促水解产物和初始pH 6.2<html>细胞密度。生物量的时间进程（红色正方形），葡萄糖（黑色圆圈和虚线），木糖（蓝色圆圈和实线），和乙醇（绿色三角形）被示出。误差棒代表关于由符号标记的平均值的范围内。 （从Slininger 等 18转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227fig7large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 表1:在耕作中使用水解物成分 （ 从 Slininger 等[18]转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227table1.xlsx">请点击这里下载此表。</a> 表 2:Scheffersomyces赤酵母 NRRL Y-7124 的最优 7 中定义TPS://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227table2.xlsx"&gt;请点击这里下载此表。 表 3: 植物生物质水解发酵 优越的宽容 Scheffersomyces赤酵母 的菌株 摘要 （ 从 Slininger 等[18]转载） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227table3.xlsx">请点击这里下载此表。</a> 表4:柳枝水解SGH-N2分离的比较动力学接种到初始620 = 8.4±2.5汇率是葡萄糖，木糖消耗过程中每单位吸收率归。 （从Slininger 等转载。18）， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54227/54227table4.xlsx">请点击此处下载本TA</a>BLE。

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

用于将木质纤维素生物转化为乙醇的稳健戊糖发酵酵母的进化技术生物工程|视频

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

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

鲁棒戊糖发酵酵母的木质纤维素生物转化的演进技术乙醇

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

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

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

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

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

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

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

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

科研

生物工程

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

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

在芽殖酵母连续高分辨率显微观察复制性衰老

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

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 prosthesis, and 2) contrast the effects of the proposed technique and that of using intact limb inertial properties on joint kinetic estimates during walking in unilateral, transtibial amputees. An oscillation and reaction board system was validated and shown to be reliable when measuring inertial properties of known geometrical solids. When direct measurements of inertial properties of the prosthesis were used in inverse dynamics modeling of the lower extremity compared with inertial estimates based on an intact shank and foot, joint kinetics at the hip and knee were significantly lower during the swing phase of walking. Differences in joint kinetics during stance, however, were smaller than those observed during swing. Therefore, researchers focusing on the swing phase of walking should consider the impact of prosthesis inertia property estimates on study outcomes. For stance, either one of the two inertial models investigated in our study would likely lead to similar outcomes with an inverse dynamics assessment.

Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments.

对于膝下义肢的估算惯性振荡性质与反应板技术

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

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

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

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

隔离细胞脂滴:两个纯化技术从酵母细胞与人类的胎盘开始

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

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

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

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

制造业的鲁棒天然纤维预制体的利用细菌纤维素为粘结剂

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

细胞外囊储存稳定性的评价

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

用于向小鼠眼睛输送生物制剂的封装细胞技术

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...

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

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

在微流体进化加速器上生成跨癌症人群的异构药物梯度，用于实时观察

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