Sengupta Priya, Mohan Ramya, Wheeldon Ian, Kisailus David, Wyman Charles E, Cai Charles M
Bourns College of Engineering, University of California Riverside (UCR), 900 University Avenue, Riverside, CA, 92521, USA.
Center for Environmental Research and Technology (CE-CERT), Bourns College of Engineering, University of California Riverside (UCR), 1084 Columbia Avenue, Riverside, CA, 92507, USA.
Biotechnol Biofuels Bioprod. 2022 Dec 6;15(1):134. doi: 10.1186/s13068-022-02232-9.
Simultaneous saccharification and fermentation (SSF) is effective for minimizing sugar inhibition during high solids fermentation of biomass solids to ethanol. However, fungal enzymes used during SSF are optimal between 50 and 60 °C, whereas most fermentative yeast, such as Saccharomyces cerevisiae, do not tolerate temperatures above 37 °C. Kluyveromyces marxianus variant CBS 6556 is a thermotolerant eukaryote that thrives at 43 °C, thus potentially serving as a promising new host for SSF operation in biorefineries. Here, we attempt to leverage the thermotolerance of the strain to demonstrate the application of CBS 6556 in a high solids (up to 20 wt% insoluble solid loading) SSF configuration to understand its capabilities and limitations as compared to a proven SSF strain, S. cerevisiae D5A. For this study, we first pretreated hardwood poplar chips using Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) to remove lignin and hemicellulose and to produce cellulose-enriched pretreated solids for SSF. Our results demonstrate that although CBS 6556 could not directly outperform D5A, it demonstrated similar tolerance to high gravity sugar solutions, superior growth rates at higher temperatures and higher early stage ethanol productivity. We discovered that CBS 6556's membrane was particularly sensitive to higher ethanol concentrations causing it to suffer earlier fermentation arrest than D5A. Cross-examination of metabolite data between CBS 6556 and D5A and cell surface imaging suggests that the combined stresses of high ethanol concentrations and temperature to CBS 6556's cell membrane was a primary factor limiting its ethanol productivity. Hence, we believe K. marxianus to be an excellent host for future genetic engineering efforts to improve membrane robustness especially at high temperatures in order to achieve higher ethanol productivity and titers, serving as a viable alternative to D5A.
同步糖化发酵（SSF）对于在生物质固体高固含量发酵生产乙醇过程中最大限度减少糖抑制作用是有效的。然而，SSF过程中使用的真菌酶在50至60°C之间活性最佳，而大多数发酵酵母，如酿酒酵母，不耐受37°C以上的温度。马克斯克鲁维酵母变种CBS 6556是一种耐热真核生物，在43°C时生长良好，因此有可能成为生物精炼厂中SSF操作的一种有前景的新宿主。在此，我们试图利用该菌株的耐热性，展示CBS 6556在高固含量（高达20 wt%不溶性固体负载）SSF配置中的应用，以了解其与成熟的SSF菌株酿酒酵母D5A相比的能力和局限性。在本研究中，我们首先使用共溶剂强化木质纤维素分级分离（CELF）预处理硬木杨木片，以去除木质素和半纤维素，并生产用于SSF的富含纤维素的预处理固体。我们的结果表明，尽管CBS 6556不能直接胜过D5A，但它对高浓度糖溶液表现出相似的耐受性，在较高温度下具有更高的生长速率和更高的早期乙醇生产率。我们发现CBS 6556的细胞膜对较高乙醇浓度特别敏感，导致其比D5A更早地停止发酵。对CBS 6556和D5A之间的代谢物数据进行交叉分析以及细胞表面成像表明，高乙醇浓度和温度对CBS 6556细胞膜的综合压力是限制其乙醇生产率的主要因素。因此，我们认为马克斯克鲁维酵母是未来基因工程努力的优良宿主，尤其是在高温下改善膜的稳健性，以实现更高的乙醇生产率和产量，作为D5A的可行替代方案。
