Optimization Of The Glucaric Acid Synthesis Pathway In Recombinant Pichia Pastoris

Glucaric acid?GA?,a top value-added chemical from biomass,has been used for prevention and control of diseases and the production of polymer materials.The synthesis pathway for the production of glucaric acid has been constructed through coexpression of the genes encoding uronate dehydrogenase?Udh?and myo-inositol oxygenase?MIOX?in microorganisms such as Escherichia coli and Pichia pastoris.This study optimizes the synthesis pathway of glucaric acid in the recombinant P.pastoris.The rate-limiting enzyme MIOX was mutated and screened by the constructed glucaric acid biosensor;the stability of MIOX was enhanced by fusing amphipathic peptides to it's N-terminal;the glucaric acid production was futher improved by overexpression of genes enconding enzymes in the synthesis pathway of the intermediate myo-inositol and optimization of cultivation conditions in the shake flasks.The main results were described as follows:?1?Construction of glucaric acid biosensor and screening of the MIOX mutants.MIOX catalyzes the rate-limiting step of glucaric acid synthesis pathway.In the present study,we constructed a glucaric acid biosensor based on green fluorescent reporter protein and a regulator protein CdaR.This high-throughput screening system combines the concentration of glucaric acid with the green fluorescent protein fluorescence intensity.It was found that the fluorescence intensity of the screening system has a good linear relationship with the concentration of glucaric acid in the range of 0-600 mg·L^-1.By applying this screening system,three positive variants?K59V/R60A,R171S and D276A?were screened from the mutant library.?2?The optimization of the fusion expressed gene MIOX and udh and reinforcement of glucaric acid production.In this study,Udh was fused to the N-terminal of MIOX using different amphipathic peptides as linker.Different peomoters were used to control the expression of the fusion gene MIOX and udh.It was found that the use of amphipathic peptide S1v1 enhanced the stability of MIOX and the application of the constitutive promoter GAP is most beneficial to the accumulation of glucaric acid.?3?Promoting the synthesis of myo-inositol.Separately overexpression of the genes encoding glucokinase?HXK?,inositol-1-phosphate synthetase?INO1?and inositol monophosphatase?INM1?can promote the synthesis of myo-inositol and then enhance the production of glucaric acid.Through coexpression of these genes in myo-inositol biosynthesis pathway,glucaric acid production in the medium with glucose as carbon source was increased from 331.9±12.3 mg·L^-1 to 435.7±7.0 mg·L^-1.?4?OptimizingtheshakeflaskcultureconditionofP.pastoris GS115-UsvM?R171S?-HNM.Through optimization of shake flask fermentation,the optimum medium components was determined to be 10 g·L^-11 yeast extract as additional nitrogen source,60 g·L^-1 glucose and 20 g·L^-1 glycerol as mixed carbon source,supplying 0.5mmol·L^-1 Fe²⁺and removing Cu²⁺from the medium.The optimum culture condition was determinded to be 500 mL shake flask filled with 100 mL broth,maintaining the temperature at 34°C and the pH value at 6.0 during the fermentation process.After optimization,the production of glucuronic acid reached 1189.9±154.1 mg·L^-1,an increase of 55%compared with the pre-optimization titer of 766.3±94.3 mg·L^-1.According to the results of the above-mentioned shake flask fermentation optimization,the fermentation in a 3-L fermentor was performed.After 96 hours of culture,the production of glucaric acid reached 8.47 g·L^-1.