Construction And Fermentation Optimization Of Engineered Pichia Pastoris For Improved Glutathione Production

Glutathione（GSH）,a kind of tripeptide substances containing aγ-amide bond and sulfhydryl groups,has a wide range of applications in the medicine,food and health products industries owing to its function of protecting and regulating the redox balance in cells.With a variety of advantages such as low price of raw materials,mild reaction conditions and environmental friendliness,microbial fermentation has become the main production method of GSH.In this study,we first overexpressedγ-glutamylcysteine synthase Sc GSH1 and glutathione synthase Sc GSH2 in Pichia pastoris GS115,the original strain,to accumulate GSH.On this basis,the supply of intracellular energy was enhanced by adding additional auxiliary energy substrate sodium citrate and overexpressing adenosine kinase Sc ADK1.Then,we explored the effect of bifunctional enzyme St Gsh F on GSH synthesis and realize the dual-pathway synthesis of GSH in yeast.Finally,after optimizing of the fermentation conditions on the shake flask and 5 L fermentor,the final optimized synthesis of GSH was realized.The main content and conclusions are as follows:（1）We used P.pastoris GS115 as the original strain.By overexpressing Sc GSH1 derived from Saccharomyces cerevisiae and co-expressing Sc GSH1 and Sc GSH2,strains G2 and G3were successfully constructed respectively.Under the condition of adding 10 m M precursor substances（glutamic acid,glycine,cysteine）,the accumulation of GSH production of G2 and G3 reached 261.66±21.70 mg·L^-1 and 302.27±5.06 mg·L^-1,which were 2.50 times and 2.88times higher than the control strain,the yields of GSH of G2 and G3 reached 39.82±2.93mg·g^-1 and 45.35±1.35 mg·g^-1,respectively,which were 2.20 times and 2.51 times higher than that of the control strain.We realized the initial accumulation of GSH so far.（2）In order to enhance the energy supply in GSH synthesis,the conditions for adding sodium citrate,a co-energy substrate in G3 fermentation,were explored.The results proved that energy is the key point that affects the excessive accumulation of GSH,therefore,we further explored the effects of adenosine kinase ADK1 and Vitreoscilla hemoglobin Vs VHB on the accumulation of GSH in yeast,found that the adenosine kinase Sc ADK1 from S.cerevisiae can enhance the synthesis of GSH as well.We named this constructed strain G3-SA.The GSH production and yield of G3-SA at 30 h fermentation were 387.95±10.47 mg·L^-1 and60.40±1.16 mg·g^-1,respectively,which were 25.56%and 19.80%higher than G3.Afterwards,it was found that when 4 g·L^-1 sodium citrate was added at 12 h,the highest GSH production and yield were obtained 465.5±29.90 mg·L^-1 and 64.87±2.46 mg·g^-1after the G3-SA sodium citrate addition conditions were optimized.（3）The bifunctional enzyme Gsh F has the activities of GSH1 and GSH2,therefore,in this study,St Gsh F from Streptococcus thermophiles was codon-optimized and transferred to P.pastoris to explore the effect of Gsh F on GSH synthesis.The results showed that:by introducing St Gsh F into G3,the production and yield of GSH produced by G3-F obtained were 411.69±10.37 mg·L^-1 and 59.56±1.26 mg·g^-1,respectively,which were 36.17%and33.81%higher than those of the control,the dual-path synthesis synthesis of GSH in yeast has been realized as well.In order to improve the expression of St Gsh F,the St Gsh F promoter was replaced by P_GAP,P_OCA1,P_GCW14,P_TEF1 and P_TPI1based on the promoter engineering.The results showed that the P_GAP is the most suitable promoter for St Gsh F.Finally,by co-expressing St Gsh F and Sc ADK1 in the G3 strain,the G3-SF was obtained.After adding 4g·L^-1 sodium citrate at 12 h of fermentation,the GSH production and yield can reach527.14±17.92 mg·L^-1 and 76.79±2.13 mg·g^-1,respectively.（4）Fermentation conditions of G3-SF were optimized on the shake flask and 5 L fermentor.On the shake flask,when the glucose concentration is 60 g·L^-1,the initial p H is 7,15%of the inoculum amount is transferred to the fermentation medium after the seed is cultivated to 21 h,and 10 m M precursor is added at 4 h of fermentation,the GSH production and yield can reach 998.05±29.56 mg·L^-1 and 92.97±1.68 mg·g^-1,respectively.High cell density fermentation was carried out on a 5 L fermentor by using the modified BSM basic inorganic salt medium(the glycerol was replaced with 60 g·L^-1 glucose)and adding 10 m M precursor substances at 36 h for fermentation.The GSH production,yield and productivity reached 2229.23 mg·L^-1,18.85 mg·g^-1 and 46.44 mg·（L·h）^-1,respectively.Then,the effect of addition conditions of the precursor substances on the synthesis of GSH was investigated,and it was determined that when 40 m M precursor were added in the fermentation at 40 h,the GSH production,yield and productivity were respectively reached 5680 mg·L^-1,45.13 mg·g^-1and 113.60 mg·（L·h）^-1.