Primary And Secondary Pathway Engineering Of FK506Producing Strain Based On Genome-scale Metabolic Flux Analysis

In the present work, FK506-producing Streptomyces tsukubaensis wasengineered to improve the titer based on a rational strain improvement approach,which was the―model construction-target prediction-experiment guidance‖strategy.Based on the genome-scale metabolic network of Streptomyces coelicolor andannotated genome and biochemical information, the genome-scale metabolic networkof S. tsukubaensis was accomplished after adding the biomass reactions and FK506biosynthetic reactions. The network was composed of865reactions and621metabolites. According to reconstruction of metabolic network, a series of targets thatcould improve the FK506production using the flux balance analysis andminimization of metabolic adjustment algorithms. Under the physiologicalmetabolism state, the potential genetic targets in the primary and secondary pathwayswere identified. The targets in the primary pathway contained knockout of gdhA andppc, overexpression of dahp, pntAB, accA2, zwf2; the targets in the secondarypathway contained overexpression of fkbO, fkbL, fkbP, fkbD, fkbM.In order to validate the prediction of model and enhance the FK506productionof strain, targets in the primary pathway were experimentally manipulated. Thedeletion of NADPH-dependent glutamate dehydrogenase gene gdhA andphosphoenolpyruvate carboxylase gene ppc, respectively, resulted in a37.8%and30%improvement of FK506, up to197.7±7.9mg/L and189.8±6.7mg/L, comparedwith the parent strain D852. In addition, the overexpression mutants were alsoexperimentally constructed for confirmation. The overexpression of3-deoxy-D-arabino-heptulosonate-7-phosphate synthase gene dahp, pyridinenucleotide transhydrogenase gene pntAB, acetyl-CoA carboxylase gene accA2,glucose-6-phosphate dehydrogenase gene zwf2, respectively, led to216.8±11.9mg/L,203.2±10.1mg/L,200.5±15.1mg/L,193.2±12.8mg/L of FK506,1.51-,1.46-,1.4-,1.35-fold of the value of D852. Moreover, the combined effect of the geneticmodifications was evaluated. Results showed that the strain HT-ΔGDH-DAZ withgdhA-deletion and dahp-, accA2-, zwf2-overexpression successfully directed carbonflux towards FK506, improving the titer by1.8-fold, up to398.9±14.8mg/L. Theexperimental results demonstrated the model-based approach could guide strain Guided by model prediction, the strain was rationally engineered in thesecondary pathway to improve FK506productivity. By amplifying fkbO, fkbL, fkbP,fkbD, fkbM, the resulted strain HT-FKBL, HT-FKBP, HT-FKBD, HT-FKBM produced200.1±9.3mg/L,186.3±7.2mg/L,161.9±4.1mg/L,173.1±10.8mg/L,178.3±9.9mg/L, a39.4%,29.8%,12.8%,20.6%,24.3%increase compared to the control D852.Furthermore, combinatorial overexpression of the five genes resulted in a1.46-foldincrease of the FK506titer to353.2±8.5mg/L at6d, in comparison with the value ofD852. In order to further improve the FK506production of the engineered strainHT-FKBOPLMD, various nutrients including soybean oil, lactate, succinate,shikimate, chorismate, lysine, pipecolate, ileucine and valine were supplemented. Byoptimizing the feeding concentrations and times, HT-FKBOPLMD was able toproduce457.5±10.3mg/L of FK506,29.5%more compared with unsupplementedcase, with44%and35%less of by-products FK520and37,38-dihydro-FK506,respectively. These results demonstrate the significance of model-based targetprediction to guide the production improvement of the rational metabolic engineeringfor the strain. Meanwhile, it emphasized the importance of exogenous feedingstrategies for FK506biosynthesis and reduce the by-products.