Metabolic Engineering Of Pichia Pastoris For Myo-inositol Production

Myo-inositol is a six-carbon compound and belongs to the B vitamins.It is widely used in food,medicine,feed and other industrial fields.At present,the main production methods of inositol include phytate hydrolysis,enzymatic catalysis and microbial fermentation.Among them,the microbial fermentation method is considered to be the most promising production method of inositol.Recently,it has been reported that Escherichia coli has been modified through metabolic engineering strategies for high-yield inositol production.However,E.coli is not a food-safe strain,which limits its application.Pichia pastoris is a generally recognized as safe（GRAS）strain certified by the US Food and Drug Administration（FDA）.It is currently widely used in the production of recombinant proteins and has been transformed into a high-value chemical production platform.Pichia pastoris itself has a synthesis pathway for inositol,so it is considered as a potential strain for inositol production.However,the synthesis of inositol in P.pastoris is subject to strict metabolic regulation,so wild-type strain cannot accumulate inositol in large quantities.It is necessary to construct an inositol cell factory through systematic metabolic engineering of P.pastoris.In this study,an efficient scarless gene editing tool was developed in P.pastoris.Based on the gene editing tool,an inositol producing P.pastoris strain was constructed by engineering the inositol synthesis pathway and the main carbon metabolism pathway of P.pastoris.The main results are as follows:First,a Pichia genome editing platform was established based on two selection markers:mazF from E.coli and zeo R from Streptoalloteichus hindustanus.Previous study demonstrated that the mazF-zeo R screening strategy is an efficient genome editing tool for P.pastoris.However,an unwanted scar would leave in the genome after each round of gene editing.In this study,the lengths of the homology arms were redesigned to control the two rounds of homogenous recombination events to achieve scarless gene editing.The long homology arm was about 1000-1200 bp long and mediated the first homologous recombination event;the short homology arm was about 100-300 bp long and mediated the second homologous recombination event.Results demonstrated that the modified mazF-zeo R screening strategy could achieve precise scarless gene editing with an efficiency of70%-100%.Furthermore,the induction medium was optimized.When 1%methanol was used as the sole carbon source in MY induction medium,the editing efficiency of the second round of homologous recombination increased from 0-30%to 80-100%.Secondly,the P.pastoris inositol production strain was obtained by engineering the P.pastoris inositol biosynthesis pathway and main carbon metabolism pathway.Using P.pastoris GS115 as the initial host,firstly the key gene IPS（Inositol-1-phosphate synthase）of its own inositol synthesis pathway was overexpressed and the inositol transporters Pp ITR1 and Pp ITR2 were knocked out to relieve the metabolic regulation for inositol production in P.pastoris.Secondly,by deleting the key gene pfk2（6-Phosphofructokinase）in the glycolysis pathway which would slow down the carbon flux of glycolysis,inositol accumulation was detected in the fermentation of the obtained strain JQ03.The results of shake flask fermentation showed that the production of inositol reached 0.72 g/L.Secondly,in order to further increase the production of inositol,two exogenous key inositol synthesis genes,Sc IPS from Saccharomyces cerevisiae and Ec IMP（Inositol monophosphatase）from E.coli,were selected for overexpression in P.pastoris,and a glycerol-inducible promoter was used to control the pgi,zwf and pfk1 genes expression which would further slowdown the carbon flux of glycolysis and pentose phosphate pathways,generating strain JQ08(Δpfk2,P_gut1-pgi,P_gut1-zwf,P_gut1-pfk1).The results of shake flask fermentation showed that the inositol production of JQ08 strain reached 5.04 g/L,which was 7times higher than that of JQ03 strain.Finally,high-density fermentation was carried out for the recombinant strain JQ08 in a 10-liter fermenter.The results showed that the highest production of inositol could reach 30.71 g/L under the conditions of 30oC,p H 5.0 and rotation speed 800 rpm.In summary,a scarless genome editing tool was established for P.pastoris in this study based on the two selection markers mazF and zeo R.This tool was used to strengthen the inositol biosynthesis pathway and weaken the glycolysis and pentose phosphate pathways in P.pastoris.Finally,the high inositol producing P.pastoris strain was obtained.This research provides a valuable reference for the construction of inositol-producing strains,and offers an essential guidance for metabolic engineering of P.pastoris for high-value chemicals production.