Application Of Perturbation Factor For Breeding Lager Yeast With Lower Acetaldehyde Production And Related Mechanisms Analysis

Those carbonyl compounds metabolized by yeast not only lead to an unpleasant flavor but also boost the beer staling during storage and thereby affect the beer quality.As a result,the contents of aldehydes should be strictly controlled in beer.Acetaldehyde is the most quantitatively abundant aldehyde in beer,therefore decresing its content in beer is of great importance for beer industry,particularly when producing the light beer.Although great developments have been made in the breeding methods,it is still a great challenge to further improve the efficiency.Besides,the mechanisms of the acetaldehyde metabolism during fermentation remained unclear,which limit the application of breeding brewing yeast with low acetaldehyde production.Thus,developing new breeding methods for brewing yeast and unraveling the mechanisms for lower production of acetaldehyde during fermentation are of great interest.In this thesis,we carried out the study basing the Design-Build-Test-Learn(DBTL)cycle to develop the new method and strategy for breeding lager yeast with lower acetaldehyde level.The main results were described as follows:(1)Establishment of perturbation factor assisted rapid evolution method:The perturbation factor POL3/L612M,POL3-01 and POL3-01/L612M were constructed by site-directed mutagenesis and the introduction of these designed perturbation factor in vivo caused the mutation rates were increased by 21,53 and 237 fold,respectively.The application of these perturbation factor greatly improved the tolerance against acetic acid in yeasts with different genetic background,and higher mutation rate led to an increasingly remarkable change.Further,coupled with continuous culture and transfer under higher concentration of disulfiram,several mutants with lower acetaldehyde production were obtained.Mutant strain MGA produced 6-7mg/L acetaldehyde at the end of fermentation with good inherited stability and fermentation stability.The comparative genomics analysis indicated that the perturbation factor could cause genetic diversity in genome.Compared to the ARTP mutagenesis,perturbation factor induced less amounts of mutations but the similar mutation diversity and spectra.Therefore,the perturbation factor can serve as a complementary method to traditional methods for mutation-based strain improvements.(2)Unraveling the mechanism of the lower production of acetaldehyde in lager yeast:The genotypes and phenotypes of strain M14 and the evolved strain MGA during the fermentation were compared.According to the correlation and consistency among these genomic,transcriptional and metabolic data,we inferred that the mutations in MGA genomes,involving protein phosphorylation,transcription activity and regulation,and protein transport activity,resulted in the enhancements of respiration activity and reflected in the up-regulation of gene expressions and the over-production of metabolites in respiration metabolisms.As a cusal effect,the cellular reducing power(NADH/NAD~+ratio)was remarkably increased,thereby facilitating the reduction of acetaldehyde.Reverse engineering strategy proved that up-regulation of TCA cycle genes(mainly for CIT1,CIT2 and IDH1)could increase the cellular reducing power and decrease the production of acetaldehyde.In line with these results,decreasing the reducing power by overexpression FRD1 resulted in an increase in acetaldehyde production.Collectively,these results clearly demonstrate that regulation of TCA cycle in yeast could alter the cellular reducing power and thereby affect the final acetaldehyde production.Moreover,the final acetaldehyde production was well correlated with intracellular reducing power(r=-0.95).(3)The impacts of alcohol dehydrogenase activity and NADH availability on acetaldehyde reduction during fermentation:The fluctuations of acetaldehyde levels including peak value,reduction rate and final production were focused on during brewing with those constructed strains with higher ADH activity or higher cellular NADH availability.Both cytosolic and mitochondrial NADH could facilitate the reduction of acetaldehyde in yeast during the later phase of fermentation.Whereas,higher mitochondrial NADH level was more effective than higher cytosolic NADH level in reducing the accumulated acetaldehyde since acetaldehyde was the most important electron acceptor to maintain the mitochondrial redox balance while many other pathways like glycerol synthesis could contribute to alleviating the cytosolic redox imbalance.On the other side,ADH1 and ADH3 were the most essential ADH coding gene for the reduction of acetaldehyde during brewing.Higher ADH activity did not facilitate the reduction of acetaldehyde at the later fermentation phase when the acetaldehyde continuously declined,whereas it resulted in lower peak values.Since the higher ADH activity activated the ethanol metabolism untimely,a negative effect on cell growth and fermentation lag were observed.Collectively,these results indicated the dynamic of acetaldehyde would be regulated by different regulatory factors at different time course during fementation,and the regulation strategies for decreasing the final acetaldehyde production could take up from two aspects which were reducing the peak value and accelerating the reduction rate.(4)Evaluation and application of the strategy of breeding yeast with higher NADH availability:To further evaluate this concept,the concentrations of other flavor compounds and flavor stability of the fermented liquid produced by those higher cellular NADH level strains were compared to those without NADH perturbation.The results indicated that higher NADH strain exhibited greatly changed aroma profiles,e.g.higher alcohol production and less aldehyde production.In terms of flavor stability,the antistaling capacities and the shelf life of the beer samples produced by higher NADH strains were improved.Due to the more positive effects on beer flavor stability,this concept could be defined as a reasonable strategy for breeding lower acetaldehyde producing strain for industrial use.Then we proposed to use the chemical 2,4-dinitrophenol(DNP)as the selective pressure to obtain strain with higher NADH level.Coupled with the new established breeding method,we obtained the optimized yeast strain DNPR-17 which showed similar fermentation performance as the parental strain while whose acetaldehyde production was significantly decreased and the flavor stability was remarkably improved.These results indicated that DNP could be used as new selective pressure to breeding strain with lower acetaldehyde production.Finally,to meet the demand of current polices of industrialization,another strain XX-01 was obtained using atmospheric and room temperature plasma(ARTP)mutagenesis technology coupled with DNP selection,and its acetaldehyde production was approximately 6.32 mg/L.