| Glutathione?GSH?is widely used in the food,cosmetics and pharmaceutical industries because of its good nutritional value,antioxidant properties and detoxification function.At present,microbial fermentation is the main method for producing glutathione.Compared with traditional chemical synthesis and enzymatic methods to produce GSH,this method is lower cost,simpler operation,and it needs no more extra ATP and cofactors.Due to the advantages such as high protein expression and easy high-density culture of recombinant Pichia pastoris,it has initially shown its great value in industrial production of glutathione.In this study,the recombinant P.pastoris GS115 that heterologously expressed the glutathione enzyme genes?gsh1 and gsh2?derived from Saccharomyces cerevisiae S288c was used as the starting strain.In order to improve the synthesis efficiency of glutathione,we selected strains from mutation,screened copy number of specific genes,deleting and integrating target genes and optimized fermentation conditions.The main research contents and conclusions are as follows:?1?Recombinant P.pastoris GS115 was subjected to ultraviolet and ethyl methanesulfonate,and then it was screened by ethionine which is analogous to glutathione.A stable hereditary strain was obtained after 40 s of the UV mutagenesis,0.4 mg·mL-1 of the ethyl methanesulfonate and 0.8 g·L-1 of the ethionine.The GSH yield of the positive mutant GS115-M7 was up to 1124 mg·L-1,which is 37.5%higher than the starting strain.And its GSH yield per unit cell reaches 100.6 mg·g-1,which is 43.7%higher than the starting strain.?2?A recombinant expression plasmid containing the genes of glutathione synthetase?gsh1 and gsh2?derived from S.cerevisiae S288c was constructed,and it was integrated to the genome of target strain at the site of 5'AOX1 through homologous recombination.In order to get high-copy recombinant strains,the gsh1 and gsh2 genes was overexpressed and the strains were screened by the increasing concentration of G418 antibiotics.Finally,a stable inheritance strain GS115-M710 was obtained and its GSH yield reached 1613 mg·L-1,which is 45.5%higher than the control GS115-M7.The unit cell yield of this strain reached 150.75 mg·g-1,increasing 49.9%.?3?We respectively knocked out three genes?dug1/2/3?in the glutathione catabolism pathway by CRISPR/Cas9 technology,and then investigated the growthand GSH production capacity of knock out strains.The results showed that deleting the dug genes in yeast increased the GSH content of per unit mass,but impacted its growth.The GSH yields of the dug1,dug2and dug3 knockout strains were 1510 mg·L-1,1669 mg·L-1,1620 mg·L-1,respectively,and the unit cell yields were 206 mg·g-1 and 215.6 mg·g-1,223.1 mg·g-1,which were 35.5%,41.9%,and 47%higher than the control.?4?A yeast integrated expression plasmid containing the Vitreoscilla hemoglobin gene was constructed,and it was integrated into the genome of the strain GS115-M710 at the site of 3'AOX1 by homologous recombination.Then the recombinant strain GS115-M710V containing the vgb gene was obtained after screened by hygromycin resistance.When the filling volume of the shake flask was 20/250 mL,the recombinant yeast grew significantly better than the control strain,and the production of GSH reached 1824 mg·L-1.When the filling volume was 40/250 mL,the growth of the recombinant strain is still slightly better than the control,and its unit cell yield reached 157.4 mg·g-1.?5?Finally,we optimized the fermentation medium and environmental conditions.And the recombinant strain GS115-M710V was used as the production strain.After optimization,the initial addition amount of three precursor amino acids in the fermentation medium was determined to be glutamic acid 81 mmol·L-1,glycine 27 mmol·L-1,cysteine 13.5 mmol·L-1,and the substrate supplement was determined to be glutamic acid:glycine:cysteine=9:9:20mmol·L-1.Then we investigated the influence of environmental conditions on the fermentation of recombinant P.pastoris was,and the optimal fermentation conditions at the shake flask level were determined:the inoculation volume was 10%,the feeding time point was at 40 h,the initial temperature was 30?and adjusted to 26°C after 40 h fermentation.After fermentation optimization,the production of GSH and the production of each cell reached1884 mg·L-1 and 166.7 mg·g-1,respectively. |
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