Study On Breeding Of Gsh High-production Strain By ARTP Mutagenesis And Its Fermentation With Sonication

Glutathione(GSH)is a small peptide including a thiol group and widely used in food and medicine due to its functions of anti-oxidation,anti-aging and detoxification.The production methods of GSH mainly included extraction,chemical synthesis,enzymolysis and microbial fermentation.In recent years,industrial production of GSH by microbial fermentation mainly concerned with yeasts or Escherichia coli,in which yeasts were the preferred strains due to their safety.However,the content of GSH in natural yeast is insufficient to meet the industrial demand.Therefore,it is essential to improve the production of GSH by yeast.Recently,The mutagenesis methods are always used to obtain high yield strain of GSH.Therefore,the purpose of this study was firstly to utilize the ARTP mutagenesis technology to increase the GSH production in S.cerevisiae and elucidate the mechanisms of high GSH production in the mutant strain by RNA-seq.Secondly,the fermentation media was optimized by single factor tests and response surface methodology.Finally a 7.5 L fermentation tank coupled with an in situ ultrasonic instrument was used to culture S.cerevisiae to explore the effects of low intensity ultrasound on the production of GSH.The research contents and results herein were as follows:(1)Screening of high GSH yield strains by ARTP mutagenesis and the preliminary study on the mechanism of high GSH production.First of all,a comparison of the intracellular GSH of two S.cerevisiae strains was preformed,the strain with high yield of intracellular GSH was selected.Finally,S.cerevisiae 1048 was selected as the starting strain for mutagenesis.Under the mutagenesis condition of power of 100 W,ventilation of 10 SLM,temperature of 30?and treatment time of 85 s,the lethal rate of S.cerevisiae was 90.1%.After mutagenesis treatment,the mutant evenly spread on solid media contained Zn Cl₂,and 188 mutants with large colony were selected.After fermentation by flask,17 positive mutants were screened,in which the mutant strain SCZ40 with the highest GSH production(728.46±7.66 mg/100g dry weight of yeast)increased by 32.74%compared to starting strain.Morphological observation by SEM showed that this mutant strain was longer and its surface was coarser than the starting strain.The SCZ40 was re-sequenced for better understanding the gene mutations related with the high production of GSH.The result showed that 311 InDels were detected.Among these mutants,185 mutant genes were involved in 78 KEGG pathways.Among these pathways,some were involved with the synthesis of GSH such as protein-serine/threonine kinase(path:k08286),deoxyhypusine monooxygenase(path:k06072),isocitrate dehydrogenase(NAD⁺)(path:k00030)and malate dehydrogenase(path:k00026).(2)The mechanism of the high yield GSH in S.cerevisiae obtained by ARTP was analyzed by RNA-seq.The results showed that positive effects on the yeast growth up-regulated expression of genes ASE1,CLB1/2/4/6,CDC5,LTE1 and MCD1.ARTP treatment improved the synthesis of cysteine,glycine and serine through up-regulated genes CYS3,GCV1/2 and SER2/3 and down-regulated ENO1/2,ERR1/2/3,FBA1,GPM1,PGI1,PGK1 and TDH1/2/3,which might be an important reason for decreasing the physiological functions of glycolysis/gluconeogenesis,and changing the distribution of carbon sources in the carbon metabolism network.(3)The fermentation media optimized,and cultured technology of S.cerevisiae for production of GSH in a 7.5 L fermentation tank coupled with an in situ ultrasonic instrument.After ARTP treatment,the intracellular GSH production increased.A combination of single factor tests and response surface methodology were employed to further increase the GSH production,and the optimal media conditions as follows:the34.98 g/L of tryptone,47.73 g/L of yeast,35 g/L of glucose,5 g/L of phosphate,4 g/L of(NH₄)₂SO₄ and 0.12 g/L of MgSO₄.The GSH production was 48.17 mg/L at the the optimal media conditions,which increase by 36.61%in comparison with control.In this study,an in situ ultrasonic fermentation tank was used to culture S.cerevisiae to explore the effects of low intensity ultrasound on the production of GSH.Ynder the ultrasonic conditions:frequency 28 k Hz,power density 28 W/L in sonication time of0?12 h and 280 W/L in 12?36 h respectively.Ultrasound treatment significantly increased total GSH production to a maximum value of 52.62 mg/L,which increase by15.6%compared with the control.However,the increase of intracellular and extracellular GSH production were different in the sonicated S.cerevisiae sample,which increased by 5.6%and 42.92%respectively.The changes of biomass,residual sugar,dissolved oxygen,pH and enzyme activity were detected during the ultrasonic assisted fermentation process.The results showed that sonication resulted in the biomass increased 7.19%and glucose consumption rate increased 53.08%,.At the end of fermentation,dissolved oxygen decrease by 41.67%,PK activity decreased by90.86%,HK activity increased by 270.58%and G6PDH activity increased by 203.97%respectively.It was found that the intracellular fluorescence intensity of Ca²⁺increased with fluorescent inverted microscope observation,which indicated that the sonication improved the permeability of yeast membrane.13 genes related to GSH synthesis and cell transport were selected for qRT-PCR quantitative analysis and the result showed that the expression of GSH1,CYS3 and SNQ2 up-regulated about 1.6,1.32 and 2.84times respectively,which indicated that ultrasound treatment changed the pathway of GSH synthesis and cell transporter.These mutant genes variations might be the reasons why GSH production increase.Logistic and Luedeking-Piret function were used to establish the kinetic model for biomass and GSH production.All models fitted well with Logistic or Luedeking-Piret regression,and the parameters of strain growth and GSH synthesis in sonication treatment models were both higher than those in non-sonication models...