Breeding Of Yeast With Improved Resistance To Lignocellulosic Inhibitory Compounds

The adverse effect of lignocellulosic inhibitors to microorganisms is one of the major bottlenecks in the conversion of lignocellulosic materials to bioethanol. Molecular biology methods and evolutionary adaption engineering process were used in this study to screen high lignocellulosic inhibitory compounds-resistance yeast. On one hand, genetically engineering methods were used to achieve the overexpression of membrane transporter genes in Saccharomyces cerevisiae for the construction of high inhibitor-resistance strains; on the other hand, directed evolutionary adaption was carried out to improve the inhibitor-resistance of yeast.Firstly, traditional genetic engineering technologies were used in this study for the construction of membrane transporters genes ADP1, STE6, MDR1, TPO1 overexpression strains S. cerevisiae 280/959-PGK-ADP1/MDR1/STE6/TPO1 and control strains S. cerevisiae 280/959-PGK. Serial dilution assay and fermentation experiments under the repression of inhibitors were carried out with the transformants in this study. The results indicated that S. cerevisiae 280-PGK-MDR1 and S. cerevisiae 959-PGK-ADP1 showed obvious advantages in growth rates, sugar consumption rates, ethanol productivities under the repression of formic acid, acetic acid and furfural when compared with the control strains.Secondly, evolutionary adaption engineering process was applied in S. cerevisiae 959 with the presence of formic acid and acetic acid, respectively. Inhibitor-resistance investigation and verification were implemented using the adapted strains with the comparison of original strains. The results showed that the adapted strains could grow, consume glucose and generate ethanol at a fast rate under the repression of specific inhibitors. In addition, the adapted strains in formic acid obtained high inhibitor-tolerance to acetic acid and mixed acids as well. However, no obvious differences were demonstrated in the enhanced tolerance to formic acid and mixed acid between the adapted strains in acetic acid and the original strain.Based on the above experiments, directed adaption was carried out to Kluyveromyces marxianus which possesses native ability to metabolise a variety of carbon sources including hexose and pentose. The adapted conditions simulated the cellulosic hydrolysate with multiple inhibitors. The adapted strains were used for inhibitor-tolerance investigation and fermentation experiments as well. In the results, the inhibitor-tolerance of adapted strains was significantly improved compared to that of the original strains. Moreover, two adapted strains, K. marxianus 1727-1 and K. marxianus 1727-5, not only had advantages in inhibitor-tolerance, but also showed obvious improvements in the consumption rates of xylose.Finally, K. marxianus 1727-5, which showed the most outstanding performance in every aspect, was chosen for whole genome resequencing. Sequencing results showed that the changes of adaptation not only exist in phenotype, but also in genotype. Particularly, results on SNP analysis focused on gene mutations related to sugar metabolism, transcriptional factors and transporters, which helped us to confirm the reasons that phenotype of K. marxianus 1727-5 changed. Above all, all these related mutations provide us with some significant references to figure out the clear mechanisms on inhibitory-resistance.