Metabolic Engineering Of Pichia Pastoris And Biosynthesis Of D-arabitol

D-arabitol is an important functional sugar alcohol with the properties of low-calorie,high sweetness,and anti-caries,which has versatile applications in the food and pharmaceutical industries.Biosynthesis of D-arabitol has gained tremendous attention due to its environmental friendliness,economic competitiveness,and mild conditions.At present,the research on Darabitol production using osmophilic yeasts to convert glucose,glycerol,and other subtracts has been reported extensively.However,most of the research focuses on screening high-yielding strains and optimizing fermentation,and the biosynthesis of D-arabitol through metabolic engineering and synthetic biology approaches has not been reported yet.In this study,Pichia pastoris GS115 which could naturally convert glucose to D-arabitol,was used as the host strain to explore the feasibility of improving the D-arabitol production using metabolism engineering,to realize the biosynthesis of D-arabitol in P.pastoris GS115 through enhancement of precursor pathway flux,promoter engineering optimization,multi-copy gene,and other synthetic biology strategies.This study would provide a reference for the biosynthesis of D-arabitol.The main research results are summarized below:(1)Exploration of overexpression of key enzyme in pentose phosphate pathway(PPP)affecting the synthesis of D-arabitol.The zwf1,gnd2 and araB gene coding glucose-6-phosphate dehydrogenase(G6PD),gluconate-6-phosphate dehydrogenase(G6PDH),and ribulokinase(AraB)was cloned and integrated into P.pastoris GS115 using the constitutive promoter GAP to obtain the recombinant strain PP01,PP02,and PP03,respectively.The yield of D-arabitol in PP01(overexpressed G6PD)and the titer of D-arabitol in PP02(overexpressed G6PDH)were 43.28%and 15.26%higher than that of the control strain PP,while there was no significant difference in titer and yield between PP03(overexpressed AraB)and PP.The results above indicate that G6PD or G6PDH overexpression alone was conducive to D-arabitol production,and the overexpression AraB may have no significant effect on the synthesis of D-arabitol.(2)Investigation on the effect of co-expression of G6PD and G6PDH on D-arabitol synthesis.First,the G6PD and G6PDH co-expression vector was constructed and integrated to P.pastoris GS115,obtaining the recombinant PP0A.The production of D-arabitol in this strain reached 12.39 g/L,compared with PP01 and PP02 increased by 90.32%and 43.90%,respectively.It was found that the co-expression of G6PD and G6PDH could enhance the synthesis of D-arabitol.Furthermore,G6PD and G6PDH expression levels were optimized by combining different strength promoters GAP,TEF1,and GCW14.Among recombinant strains,the PPOD produced the highest D-arabitol(15.01 g/L),and the promoters of its zwfl and gnd2 genes were TEF1 and GCW14,respectively.(3)Improvement of the production of D-arabitol by the strategy of ardh gene multi-copy.Firstly,the sequence of ardh gene from Candida sp.H2 was obtained and codon-optimized.The expression vector harboring single copy of ardh gene was constructed and integrated to PPOD,obtaining the recombinant PP0D-1H with the D-arabitol production of 15.75 g/L.Subsequently,the recombinant vectors harboring 2 to 6 copies of ardh gene expression cassettes were successfully generated via combing the "Bio-brick" method and "Gibson assembly".On this basis,the recombinant strains PP0D-xH(x=2,3…6)harboring 2 to 6 copies of ardh gene expression cassettes were then successfully obtained.Among them,the PP0D-4H showed the highest titers of D-arabitol(21.14 g/L),which was 34.23%higher than that of PPOD.(4)Optimization of the key factors for the fermentation of glucose to D-arabitol by recombinant strain PP0D-4H using the one-factor-at-a-time methodology.The optimal conditions were:initial glucose concentration 300 g/L,pH 6.0,and temperature 32℃.Under these conditions,the concentration of D-arabitol could reach 48.51 g/L,which exceeded the strain PP by 84.58%.Further,when the fermentation of strain PP0D-4H was scaled up to a 5 L bioreactor,78.07 g/L of D-arabitol production was achieved after 84 h,and the corresponding yield and productivity were 0.28 g/g and 0.93 g/L/h,respectively.