Optimization Of Bitter Acid Synthesis Process Based On Precursors Supply Enhancement Strategy

Hop bitter acids are key functional ingredients in hops,which have broad applications in brewing,food preservation,antibacterial,antiviral,anti-inflammatory,anti-cancer,anti-oxidation and et al.Due to the limitations of the extraction and chemo-synthesis methods,biosynthesis of bitter acids by engineered Escherichia coli become a candidate method in preparation of high-purity hop bitter acids.This study provides a technical reference for the downstream synthesis route,and is conducive to the large-scale industrial production of bitter acid.In this study,both strain and process optimization were carried out.The strain optimization utilized a fused enzyme PT1/2,which also delete the signal domain near the N terminal of the protein sequence.The fused enzyme together with other enzymes（IDI,CCL2,VPS and monooxygenase）were engineered in the optimized strains.The newly constructed strains present improved production of bitter acids.This study further optimized the fermentation process of the new strains and found that precursors supply significantly affect the production of bitter acids.After the analysis of the addition amount of MVA,isovaleric acid yeast extract under response surface design.The optimal addition amount were predicted to be 10.10 mL/L of MVA,15.05 mL/L of isovaleric acid,and 8.80 g/L of yeast extract.Finally,the study verified that higher amount of bitter acids were produced under these conditions,the titers of lupulone and humulone were improved to 0.85 g/L and 0.50 g/L.