Constructing The L-tyrosine Metabolic Platform And Its Application On The Production Of Danshensu

L-tyrosine, one of the aromatic amino acids, is widely used in food,pharmaceutical and chemical fields. Due to the low ability to accumulate L-tyrosine innatural microbes, maximizing the metabolic flux of the whole pathway from biomasscarbon source such as glucose to L-tyrosine, constructing the L-tyrosine highproduction metabolic platform and appling it fro the biosynthesis of derivatives havesignificance meanings.Applying the modular engineering approach to target on broad metabolicpathways including both L-tyrosine biosynthetic pathway,glucose utilization, TCAcycle and acid tolerence，12genes were preferred and assembled to four modules viarationally designed BldgBrick system. Combining and adjusting the suitability amongthe four modules，we found that vector pYBT5with combined expression of module1consisting of aroGfbr-tyrAfbr-aroE and module2consisting of ppsA-tktA-glk couldefficiently accumulate L-tyrosine. Furthermore, the regulatory gene tyrR andalternative genes including ptsG, pykA, pykF and pheA involved in competingpathways were deleted from the E. coli chromosome generating5chassisstrains.Screening the different combinations of pYBT5and chassis strains，weselected the strain BKT5with all the inactivation was the best. Utilizeing glucose ascarbon source, strain BKT5produced702mg/L L-tyrosine in72h with a19.5%theory yield. What is more, by designing and screening sRNAs sequence of csrA inE.coli, rational design of anti-csrA sRNA2successfully knocked down the expressionof csrA and lead to a further1.2-fold improvement over the L-tyrosine production.Introducing the dasnhensu biosynthetic module combined with hapBC andmutated d-ldhY52Ato the constructed L-tyrosine metabolic platform, a danshensuproduce strain, BKD5, was finally constructed. Using fed-batch fermentation strategy,strain BKD5achieved high productivity of danshensu (7.1g/L) with a yield of0.47mol/mol glucose in72h.Based on synthetic biology, we optimized the global pathway of L-tyrosine andimproved the production of L-tyrosine via modular engineering and chassis strainsconstruction. Subsequently, the introduction of unprecedented artificial biosynthetic pathway realized the effective production of danshensu. The constructed L-tyrosinemetabolic platform possesed high stability and effecticy and couled be applied on thebiosynthesis of other derivatives.