System Metabolic Engineering Of Pichici Stipitis For Succinic Acid Production

In this study, Pichia stipitis FPL-UC7, a strain of uracil auxotroph, was used as startingstrain. The optimum synthesis pathway for succinate production in P. stipitis was achievedthrough aiding by genome-scale metabolic model. A series of metabolic engineering on keyregulatory points has been conducted, in order to improve the yield of succinic acid. The mainresults were described as follows:（1）The metabolic target genes was obtained for succinic acid production by GSMMiTL885simulation. In silico simulation predicted that: over-express the E. coli ppc gene orpck gene could increase the TCA anaplerotic pathway flux, and the succinic acid productionrate increased to0.02mmol/gDCW/h and0.03mmol/gDCW/h from9.0×10^-4mmol/gDCW/h,respectively; Over-expressing icl1gene could increase the glyoxylate pathway flux, and thesuccinic acid production rate increased to0.05mmol/gDCW/h; the deletion of sdh1in silicocould increase the glyoxylate pathway flux while breaking further metabolism of succinicacid in the TCA cycle, and succinic acid production rate reached to0.15mmol/gDCW/h;over-expressing icl1gene and deleting sdh1gene in silico could increase the succinic acidproduction rate to0.19mmol/gDCW/h. The optimum synthesis pathway for succinateproduction was: enhance the TCA-glyoxylate pathway to produce succinic acid byover-expressing icl1gene and deleting sdh1gene.（2） The TCA anaplerotic pathway of P. stipitis was studied to accumulate succinic acid.The strains FPLppc was obtained by over-expressing E. coli ppc gene, in which the PEPCenzyme activity was0.83U/mg. And the stain FPLpck was obtained by over-expressing pckgene, in which the PCK enzyme activity was0.35U/mg. Shake flask fermentationexperiments showed that: FPLppc could produce succinic acid at0.15g/L while FPLpckcould produce succinic acid at0.12g/L, increased275%and200%compared to the controlstrain FPLpy26, respectively. In addition, the accumulation of acetic acid was decreased to3.21g/L and3.52g/L, decreased39%and34%compared to the control strain. In this study,NaHCO3addition helps to constructed strains accumulation of succinic acid. The succinicacid production was increased to a titer of0.38g/L and0.2g/L by FPLppc and FPLpck in thepresent of5g/L NaHCO3. Effects of concentration of NaHCO3on succinic acid productionindicated that:5g/L NaHCO3was the best concentration for FPLppc producing succinic acidwhile20g/L was the best concentration for FPLpck.（3）Enhance the TCA-glyoxylate pathway in P. stipitis to increase succinic acidproduction. The genetic engineering stains FPLicl over-expressing icl1, FPL sdh deletingsdh1and FPL sdh-icl with sdh1-deleted and icl1over-expressed were constructed. The ICLenzyme activity increased about3.8times,16times and19times respectively，reached at1.6U/mg,5.6U/mg and6.6U/mg in the three strains, respectively. While the SDH enzymeactivity reduced22%in FPLicl and wasn’t detected in FPL sdh or FPL sdh-icl. Thefermentation parameters of the constructed strains was studied at5-L fermentor. As a result,0.3g/L,1.2g/L and1.6g/L succinate were produced by the three engineered strains on xyloseas carbon source, respectively. The fermentation parameters of the strain FPL sdh-icl on glucose as carbon source fermentation was studied. The growth rate increased65.9%, and theDCW increased22.6%, respectively, and the production of succinic acid reached to2.5g/L.