Screening Of Flocculation Elements In Lager Yeast And Their Effects On Fermentation Performance

Lager yeast is the main strain of beer brewing in China.The flocculation of yeast provides an effective and convenient way to separate products and cells for industrial production,which greatly saves the production cost and is conducive to the recovery and reuse of yeast cells.Yeast flocculation is affected by various factors,such as genetic background,environmental stresses and so on.At present,studies on flocculation regulation mechanism of lager yeast during brewing process remains to be explored,which leads to the lack of efficient and directional screening method for flocculent yeast mutants for beer brewing.In this dissertation,industrial lager yeast G03 and its mutants with enhanced flocculation ability,G03-10 and G03-24,were taken as examples.By analyzing their differences at genomic level,the key genes related to flocculation phenotype variation were successfully identified,which laid a foundation for explaining the flocculation mechanism of lager yeast and provided a research example for the study of other industrial strains of with complex genetic background.The main results are as follows.1.In fermentation test of the parental strain G03 and its mutants G03-10 and G03-24,it was found that the fermentation ability of the two mutants was not significantly different from that of the parental strain,but the flocculation ability of the mutant was significantly improved at the end of fermentation relative to the parent.2.The non-synonymous or frameshift mutations in genes of G03-10 and G03-24 were analyzed by comparative genomics relative to the reference genome of G03,.It was found that the mutated genes were enriched in the pathways or biological processes related to cellular response to stresses including endoplasmic reticulum protein processing,membrane lipid metabolism,etc.This may be the result of adaptive evolution of mutant in order to adapt to the environmental stresses of long-term beer brewing.3.Nine candidate genes that were speculated to affect the flocculation performance of lager yeast G03 were further screened,and the recombinant strains deriving from G03 with candidate genes overexpression or disruption were constructed.The results showed that the absence of RIM101,RIM21,VPS36 and OSM1 improved the flocculation ability of lager yeast under beer brewing conditions.Except for vps36 disruption,the regulation of any other genes did not affect the fermentation performance of G03.Besides,overexpression of RIM101 and VPS36 decreased the flocculation potential of G03.4.Disruption of rim101 enhanced the tolerance of G03 to cell wall inhibitors.In the late stage of fermentation,the recombinant strain G03-rim101? with rim101 disruption had higher intracellular glycerol content than the original strain G03.Comparative transcriptomic analysis showed that the differentially expressed genes were mainly enriched in amino acid metabolism,sterol metabolism,nitrogen metabolism,redox,cellular response to stresses and other pathways or biological processes.It was hypothesized that the differences in expression of genes related to nitrogen metabolism repression pathway and sterol synthesis pathway might be the reason for the difference of flocculation performance between the recombinant strain and the original strain.