Using Global Transcription Machinery Engineering (GTME) and Site-Saturation Mutagenesis Technique to Improve Ethanol Yie
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sing Global Transcription Machinery Engineering (GTME) and Site-Saturation Mutagenesis Technique to Improve Ethanol Yield of Saccharomyces cerevisiae T. Kea, *, J. Liua, b, S. Zhaoa, b, X. Wanga, b, L. Wanga, b, Y. Lia, b, Y. Lua, and F. Huia, ** aCollege
of Life Science and Technology, Nanyang Normal University, Nanyang, 473061 China Tianguan Group Co. Ltd., State Key Laboratory of Motor Vehicle Biofuel Technology, Nanyang, 473000 Henan *e-mail: [email protected] **e-mail: [email protected]
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Received September 24, 2019; revised March 23, 2020; accepted April 22, 2020
Abstract—The SPT15 gene of Saccharomyces cerevisiae obtained using site-saturation mutagenesis was connected to the expression vector pYES2NTc. Then, the mutant library was constructed. Twelve single point mutant genes were obtained, and the recombinant plasmids were constructed with wild gene SPT15 and control gene. The S. cerevisiae strains were obtained by converting recombinant plasmids into the S. cerevisiae INVSC1 by lithium acetate method. Using glucose as substrate, the recombinant S. cerevisiae was inoculated and fermented to determine its ethanol yield. The ethanol yields of recombinant S. cerevisiae INVSC1-SPT15-K127M and S. cerevisiae INVSC1-SPT15-K127N were greatly increased by 43.0 ± 0.9 and 43.7 ± 0.2 g/L, i.e., 26.5 and 28.5%, respectively, compared with that of the control strain S. cerevisiae INVSC1-Neo. The site-saturation mutagenesis technique was used instead of random mutagenesis, and 2 mutant strains with phenotypic improvements were screened in the limited mutant library, which indicated that 127 amino acids had an important effect on the binding efficiency of Spt15 and TATA. Keywords: bioethanol, ethanol production, global transcription machinery engineering (gTME), site-saturation mutagenesis, SPT15, Saccharomyces cerevisiae DOI: 10.1134/S0003683820050087
The research on the genetic modification of yeast and the reconstruction of metabolic network to change the properties of ethanol yield and resistance has always been the focus of ethanol fermentation [1]. However, it is difficult to achieve the goal through traditional single-gene knockout. In addition, these traits are often related to multiple genes [2]. Therefore, the expression of the global transcriptional regulators changes the entire transcriptional regulation and then changes the transcription and expression of the target gene. This process is one of the most efficient methods to transform the fermentation performance of yeast and to obtain a strain with enhanced metabolic flow or specific phenotypic enhancement. Global transcription machinery engineering (gTME) [3] is used to establish a starting transcription factor mutation library by molecular biological methods, such as errorprone polymerase chain reaction (PCR) [4] and DNA shuffling [5] et al. In addition, directional screening was carried out to the target product or objective phenotype. Spt15, one of the starting transcription factors in S. cerevisiae, belongs to the transcription starting complex part. In
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