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Abstract：
BACKGROUND: Saccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses-which is needed for reduced energy consumption of ethanol distillation at industrial scale-is yet to be achieved.
RESULTS: In the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present.
CONCLUSIONS: Our results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.
RESULTS: In the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present.
CONCLUSIONS: Our results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.
摘要：
背景：酿酒酵母是乙醇合成中的重要微生物，以甘蔗糖蜜为原料，乙醇正在可持续地合成，以满足日益增长的需求。然而,基于高浓度甘蔗糖蜜的高浓度乙醇发酵-这是工业规模上降低乙醇蒸馏能耗所需要的-尚未实现。
结果：在本研究中，为了确定这一过程的主要限制因素，应用了基于ARTP（大气和室温等离子体）诱变的适应性实验室进化和高通量筛选（Py-Fe3）。我们发现了高渗透压,高温,酒精含量高,和高浓度的K+，Ca2+,K+和Ca2+(K+&Ca2+),和甘蔗糖蜜为主要限制因子。NGT-F1、NGW-F1、NGC-F1、NGK+、NGCa2+NGK+&Ca2+-F1和NGTM-F1表现出对各自限制因子的高耐受性并表现出增加的产量。随后，乙醇合成,细胞形态学,比较基因组学，在含有250g/L总可发酵糖(TFS)的糖蜜肉汤中进行基因本体论(GO)富集分析。此外,酿酒酵母NGTM-F1与250g/L（TFS）甘蔗糖蜜一起在5L发酵罐中合成乙醇，产量为111.65g/L，糖向酒精的转化率达到95.53%。这是目前物理诱变产量的最高水平。
结论：我们的结果表明，K和Ca2离子主要限制了乙醇的有效生产。然后，随后的比较转录组GO和途径分析表明，K和Ca2的共存对有效的乙醇生产产生了最突出的限制。这项研究的结果可能通过促进糖蜜制造的绿色燃料的开发和利用而被证明是有用的。
结果：在本研究中，为了确定这一过程的主要限制因素，应用了基于ARTP（大气和室温等离子体）诱变的适应性实验室进化和高通量筛选（Py-Fe3）。我们发现了高渗透压,高温,酒精含量高,和高浓度的K+，Ca2+,K+和Ca2+(K+&Ca2+),和甘蔗糖蜜为主要限制因子。NGT-F1、NGW-F1、NGC-F1、NGK+、NGCa2+NGK+&Ca2+-F1和NGTM-F1表现出对各自限制因子的高耐受性并表现出增加的产量。随后，乙醇合成,细胞形态学,比较基因组学，在含有250g/L总可发酵糖(TFS)的糖蜜肉汤中进行基因本体论(GO)富集分析。此外,酿酒酵母NGTM-F1与250g/L（TFS）甘蔗糖蜜一起在5L发酵罐中合成乙醇，产量为111.65g/L，糖向酒精的转化率达到95.53%。这是目前物理诱变产量的最高水平。
结论：我们的结果表明，K和Ca2离子主要限制了乙醇的有效生产。然后，随后的比较转录组GO和途径分析表明，K和Ca2的共存对有效的乙醇生产产生了最突出的限制。这项研究的结果可能通过促进糖蜜制造的绿色燃料的开发和利用而被证明是有用的。
