Development Of Novel Flocculent Industrial Yeast And Investigation Of Ethanol Tolerance

Flocculation of Saccharomyces cerevisiae strains has raised great interest in industry due to its advantage in biomass recovery by sedimentation instead of centrifugation that is required by regular non-flocculating yeast, which consequently saves capital investment on centrifuges and energy consumption on centrifuge operation. However, constitutive flocculation of yeast cells has the disadvantage of slow growth and ethanol fermentation caused by mass transfer limitation. Therefore, inducible flocculation of yeast cells with the flocculation onset near the end of the fermentation is preferred. Moreover, improved ethanol tolerance ensures high yeast viability under high gravity fermentation conditions, and thus is important for fuel ethanol production to improve ethanol titer and save energy consumption for downstream processes such as ethanol distillation and stillage treatment. In this work, a platform for developing inducible flocculating yeast strains was established, and molecular mechanism underlying improved ethanol tolerance by zinc supplementation was investigated by transcriptomic and proteomic analysis, through which key genes involved in stress tolerance were proposed for developing robust industrial yeast strains for efficient ethanol fermentation.The flocculation phenotypes of S. cerevisiae were controlled by the expression of FLO genes, and thus the gene FLO1was cloned from the self-flocculating yeast SPSC01by PCR amplification, which was subsequently integrated into the chromosome of a non-flocculating industrial yeast Sc4126under the control of the trehalose-6-phosphate synthase1(TPS1) promoter from SPSC01, endowing the genetically modified strain an inducible flocculating phenotype in response to ethanol concentration. As a result, the flocculation of the recombinant strain was weak at low ethanol concentration, but was improved with ethanol accumulation. Almost all yeast cells flocculated at the end of the fermentation, which were recovered by sedimentation. Compared to the constitutive flocculation developed with the same host, no mass transfer limitation was observed with the inducible flocculating strain, which significantly improved its growth and ethanol fermentation performance. When TPS1cloned from the model yeast strain S288c with less STREs (stress response element) in the promoter region than that from SPSC01was employed, the response of the transformant’s flocculation to ethanol stress was attenuated, retarding the occurrence of yeast flocculation, and in the meantime the flocculation strength was weakened, which further improved its growth and ethanol fermentation. Ethanol tolerance of yeast cells is regulated by multiple genes, and thus can be improved by engineering global transcriptional factors. In the previous studies, it was found that zinc supplementation improved ethanol tolerance of the self-flocculating yeast SPSC01. Further study was performed in this work to reveal the mechanism underlying this phenomenon, providing potential targets in the key pathways for metabolic engineering. When continuous ethanol fermentation with the medium containing150g/L glucose was performed under0.05g/L zinc sulfate supplementation conditions, improved yeast viability to ethanol-shock treatment was observed, and production of by-products such as acetic acid and pyruvate acid was decreased. It was also found that the intracellular ROS level was down-regulated, and nitrogen and ergosterol contents were both increased. Transcriptomic and proteomic analysis revealed a total of330differentially expressed genes in transcriptional levels and71differentially expressed genes in translational levels, indicating the global effect of Zn2+on the metabolism of yeast cells. The inconsistency of the transcriptional and translational variations provides insights on the abundant post-transcriptional or post-translational regulations of genes affected by Zn2+, which include the synthesis of ribosome proteins, glycolysis, ergosterol synthesis and phospholipids metabolism. On the other hand, ROS response and intracellular redox balance, as well as zinc homeostasis were also identified to be impacted by the zinc supplementation. Activation or repression of several transcriptional factors was proposed as potential targets for engineering yeast cells with improved ethanol tolerance. Moreover,94genes with unknown functions associated with the response of Zn2were also identified, which will be further studied for their roles in ethanol tolerance.Finally, artificial transcription factor technique employing zinc finger protein was proved to efficiently alter the global network of yeast metabolism, which results in breeding stable industrial yeasts with improved ethanol tolerance, and provides basis for further developing robust industrial yeasts for more efficient ethanol fermentation.