Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria.
Biotechnol Biofuels. 2014 Apr 3;7(1):49. doi: 10.1186/1754-6834-7-49.
Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2.
With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71 g/gCDW/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth.
Laboratory evolution of strain BP10001 resulted in effectively enhanced qXylose at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes.
木质纤维素水解物是最常用于发酵工业的微生物——酿酒酵母生产乙醇的困难底物。在原料预处理和水解过程中释放的抑制剂的高抗性以及对六碳糖和五碳糖的有效利用是开发用于生物质到乙醇工艺的酿酒酵母菌株的主要挑战。代谢工程和实验室进化单独或组合使用，以获得所需的菌株特性。然而，对于酿酒酵母在木质纤维素水解物转化中稳健性能的生理需求还不是很清楚。本文介绍的 IBB10A02 和 IBB10B05 菌株是 BP10001 菌株的后代，该菌株最初是通过向广泛使用的菌株 CEN.PK 113-5D 中引入一个主要氧化还原中性的木糖同化途径而衍生出来的。IBB 菌株是通过两步实验室进化获得的，该进化在厌氧生长之前（IBB10A02）和在重复木糖发酵适应后（IBB10B05）选择快速发酵木糖。酶解物是由高达 15%的干质量预处理（蒸汽爆炸）小麦秸秆制备的，其中葡萄糖和木糖的质量比约为 2。
所有菌株的总糖消耗的产率系数都很高，用于乙醇（0.39 至 0.40 g/g），用于发酵副产物（甘油：≤0.10 g/g；木糖醇：≤0.08 g/g；乙酸盐：0.04 g/g）的产率系数明显较低。与所有菌株相似的特定葡萄糖利用率（qGlucose ≈ 2.9 g/g 细胞干重（CDW）/h）相比，木糖消耗率提高了 11.5 倍（IBB10A02；qXylose = 0.23 g/gCDW/h）和 17.5 倍（IBB10B05；qXylose = 0.35 g/gCDW/h），而未经进化的菌株 BP10001 的 qXylose 则为 0.23 g/gCDW/h。在由 5%干质量制备的 50 g/L 木糖补充水解物中，菌株 IBB10B05 的 qXylose 为 0.71 g/gCDW/h，在 2 天内耗尽木糖，乙醇得率为 0.30 g/g。在使用的条件下，IBB10B05 也能够进行缓慢的厌氧生长。
BP10001 菌株的实验室进化导致 qXylose 得到了有效增强，同时几乎完全保留了代谢工程以前获得的发酵能力。菌株 IBB10B05 是强化木质纤维素到生物乙醇工艺的有力候选菌株。
