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
Summary
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。
Abstract
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.
Introduction
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>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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Cellic Ctec, Contains Xylanase (endo-1,4-) | Novozymes | No product number | www.novozymes.com, 1-919-494-3000 |
| Cellic Htec, Contains Cellulase and Xyalanase | Novozymes | No product number | www.novozymes.com, 1-919-494-3000 |
| Toasted Nutrisoy Flour | Archer Daniels Midland Co. (ADM) | 63160 | ADM, 4666 Faries Parkway, Decatur, IL 1800-37-5843 |
| Pluronic F-68 (Surfactant) | Sigma-Aldrich | P1300 | Sigma-Aldrich |
| Difco Vitamin Assay Casamino Acids | Becton Dickinson and Company | 228830 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
| D,L-tryptophan | Sigma-Aldrich | T3300 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
| L-cysteine | Sigma-Aldrich | C7352 | multiple suppliers: e.g. Fisher Scientific, Sigma-Aldrich |
| Bacto Agar | Becton Dickinson and Company | 214010 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
| Bacto Malt Extract | Becton Dickinson and Company | 218630 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
| Bacto Yeast Extract | Becton Dickinson and Company | 212750 | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
| Peptone Type IV from soybean | Fluka | P0521-500g | multiple suppliers: e.g. Fisher Scientific, VWR, Daigger |
| Adenine, > 99% powder | Sigma-Aldrich | A8626 | CAS 73-24-5, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
| Cytosine, > 99% | Sigma-Aldrich | C3506 | CAS 71-30-7, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
| Guanine, SigmaUltra | Sigma-Aldrich | G6779 | CAS 73-40-5, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
| Thymine, 99% | Sigma-Aldrich | T0376 | CAS 65-71-4, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
| Uracil, 99% | Sigma-Aldrich | U0750 | CAS 66-22-8, Could use other brands. Multiple suppliers: e.g. Sigma-Aldrich, Acros Organics, MP Biomedicals LLC |
| Dextrose (D-Glucose), Anhydrous, Certified ACS | Fisher Chemical | D16-500 | CAS 50-99-7, Could use other brands. Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich |
| D-Xylose, assay > 99% | Sigma-Aldrich | X1500 | CAS 58-86-6, Could use other brands. Multiple suppliers: e.g. Acros Organics, Fisher Scientific, MP Biomedicals, Sigma-Aldrich |
| 96-well, flat bottom plates | Becton Dickinson Falcon | 351172 | multiple suppliers: e.g. Thermo-Fisher, VWR, Daigger |
| Wypall L40 Wiper | Kimberly-Clark | towel in microplate boxes to absorb water for humidification; multiple suppliers: e.g. Thermo-Fisher, uline, Daigger | |
| Corning graduated pyrex flask, 125-mL, narrow opening (stopper #5) | Corning Life Science Glass | 4980-125 | multiple suppliers: e.g. Thermo-Fisher, VWR, Daigger |
| Innova 42R shaker/incubator, 2.5 cm (1") rotation | New Brunswick Scientific (1-800-631-5417) | M1335-0016 | multiple suppliers: e.g. Eppendorf, Thermo-Fisher. Other shaker/incubators with a 2.5 cm (1") throw could be used. |
| Duetz Cover clamp for 4 deepwell MTP plates | Applikon Biotechnology | Z365001700 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
| Duetz System sandwich cover for 96 deepwell plates | Applikon Biotechnology | Z365001296 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
| Duetz System silicone seal (0.8mm black low evap) for 96 deep well plate cover | Applikon Biotechnology | V0W1040027 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
| Blue microfiber layer for Duetz system sandwich cover | Applikon Biotechnology | V0W1040001 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
| 96 well, 2 mL square well pyramid bottom plates, natural popypropylene | Applikon Biotechnology | ZC3DXP0240 | applikon-biotechnology.com (U.S.), 1-650-578-1396 |
| Bellco 32mm silicon sponge plug closures, pk of 25 for 125-mL flasks | Bellco | 1924-00032 | Thomas Scientific, their Catalog number is 1203K27 |
| Bellco Spinner Flask, 1968-Glass Dome, Sealable Flange Type, 100-mL working volume. This design no longer manufactured. | Bellco | 1968-00100 (original Cat. No.) | Jacketed vessels have lower inlet & 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). |
| Mathis Labomat IR Dryer Oven | MathisAg | Typ-Nbr BFA12 215307 | Werner Mathis U.S.A. Inc. usa@mathisag.com, 704-786-6157 |
| Dual Channel Biochemistry Analyzer | YSI Life Sciences | 2900D-UP | www.ysi.com, robotic system for rapid sugars assay in 96-well microplate format |
| PowerWave XS Microplate Spectrophotometer | Bio-Tek Instruments, Inc | MQX200R | www.biotek.com |
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