Breeding Of Efficient Saccharomyces Cerevisiae Strain For Biomass-based Protein Synthesis

Biomass-based protein refers to a type of alternative protein isolated from microorganisms with high protein content,and it has significant economic value.Among them,biomass-based protein derived from Saccharomyces cerevisiae has been widely used in various fields such as food additives,animal feed,and cosmetic raw materials.However,the current production of biomass-based protein from S.cerevisiae faces deficicency such as low efficiency in obtaining biomass and low overall protein.Therefore,the selection of S.cerevisiae strains with high efficiency in synthesizing biomass-based protein is of great significance for improving the production efficiency of alternative proteins.This study first targeted high biomass as the screening goal.It employed an adaptive evolution approach to screen high biomass brewing yeast mutants.Subsequently,the impact of key genes on yeast biomass growth was analyzed through transcriptomic analysis.The genetic stability of the mutants was improved by continuously passaging them in culture media with a fixed pressure.Finally,the synthesis efficiency of biomass-based protein in the mutant strains was further enhanced through optimization of fermentation media and fermentation conditions.The main research findings of this study are as follows:（1）Establishment of adaptive evolution methods and screening of high-biomass mutant strains.Starting with a wild-type yeast strain S2 with a higher initial biomass-based protein titer,we established an adaptive evolution method for obtaining high-biomass mutant strains by adding growth inhibitors during the passaging process.The evolution concentration of cycloheximide B was increased from 50 mg·L^-1 to 400 mg·L^-1,and the evolution concentration of G418 was increased from 40 mg·L^-1 to 640 mg·L^-1.Subsequently,the strains of the two inhibitors with different evolutionary concentrations were screened,and a high-biomass mutant strain 2G160-1 was preliminarily screened,which dry weight increased by 21.2%compared with the original strain,but the protein content decreased by 13.2%.（2）Verification of the effects of key genes on S.cerevisiae growth based on comparative transcriptomics and improvement of the genetic stability of mutant strains.By conducting transcriptome sequencing on the high-biomass mutant strain 2G160-1 and the original strain,the differences in metabolic pathways and genes were compared and analyzed.Using the S.cerevisiae CEN.PK2-1C model strain as the research object,by overexpressing the highest up-regulated degree genes LEU1,LEU2,BAP2（which encoding enzymes related to leucine synthesis pathway）and HXT15（which encoding the hexose transporter）at genome,the results showed no effect on yeast growth.By CRISPRi inhibition of the highest down-regulated degree genens THI4 and MCH5（which encode enzymes related to the B vitamins synthesis pathway）,GNP1（which encodes amino acid permeability enzymes）,and PES4（which encodes RNA binding proteins）,the results showed that the OD₆₀₀ of yeast growth increased by 34.1%,29.4%,22.2%,and 11.9%,respectively than the original strain after 16 hours of shake flask fermentation.To address the poor genetic stability of the high-biomass mutant strain 2G160-1,a high-biomass and genetically stable mutant strain 2G160-3 was selected after continuous subculture in a medium containing evolutionary pressure for 20 days.During a 10-days genetic stability test,the high biomass characteristics of mutant strain 2G160-3 can be stably passaged for more than 120 generations,and OD₆₀₀ can be stably higher than that of the original strain by 22-26%（3）Fermentation optimization of the high-biomass mutant strain 2G160-3 to enhance the biomass-based protein synthesis efficiency.By selecting the best suitable fermentation carbon source and nitrogen source at the shake flask level,the best suitable carbon-nitrogen ratio of the feeding medium at the 3-L fermenter level,the high-biomass mutant strain 2G160-3 can synthesize biomass-based protein more efficiently.The optimization results show that when the carbon source and nitrogen source are glucose and urea,respectively,and the carbon-nitrogen ratio of the feeding medium is 8,the synthesis efficiency of biomass-based protein in the high-biomass mutant strain 2G160-3 is the highest.After 16 h of fermentation,the OD₆₀₀ can reach94.4,which is a 13.1%increase compared to the starting strain S2,and the biomass-based protein titer is 13.6 g·L^-1,which is a 9.61%increase compared to the starting strain.