Construction Of PheA And TyrA Gene Deleted Escherichia Coli

As an essential amino acid for humans and animals, L-tryptophan (L-Trp) isgeneraly used in food, animal feed and pharmaceutical industries. Among all ways ofL-tryptophan production, microbial fermentation has been the main one owing to thevirtues of cheap sugar and simple extraction technology. However，due to the limitedresource of desirable random mutagenesis and complex metabolism of wildmutagenesis, it is hard to maintain a wild L-Trp hyper-production strains forindustrial production. Accompanied by development of biological technology, theL-Trp industry has been promoted by the application of metabolic engineering andgenetic engineering to strain improvement. According to the method of metabolicengineering, the basic strategies of engineering strain construction refer to increasingthe precursors, blocking the competitive metabolic pathway, or prohibiting thedegradation of expected substance. Gene deletion, which is always applied to genedecoration, consists of several methods with certain merits and demerits. The majorityof gene deletion methods are based on the λ prophage Red reconbinaition system.This study focused on basic work of constructing a pheA and tyrA gene deletedEscherichia coli according to the method of metabolic engineering and geneticengineering. On the basis of previous study on biosynthesis pathway of aromaticamino acids in Escherichia coli, a recombined strain could be constructed byshutting or reducing the L-phenylalanine/L-tyrosine branched pathway. In thiscompetitive pathway, the dual functional enzyme chorismate mutase-prephenatedehydratase (CM-PD) and chorismate mutase (CM-prephenate dehydrogenase) arespecified by gene pheA and tyrA, repectively. Two methods of pheA and tyrAdeletion，aiming at blocking the carbon flux of the L-pheA/L-tyrA branched pathwaywhile inproving L-Trp pathway，were explained. The main contents and results ofthis thesis are as following:1. The plasmid pUC57-Kan containing Kan gene and homologous sequences ofupstream pheA and tyrA downstream was constructed. A linearized DNA298-Kanflanked by homologous sequences for gen deletion was obtained by polymerase chainreaction (PCR) and transformed into a Escherichia coli K12strain habouring plasmid pKD46. Catalyzed by Red recombination enzymes, pheA and tyrA gene wereknocked out while Kan was knocked in.2. The plasmid pUC57-Kan-QDZ-SacB containing Kan geng, SacB gene, promoterand homologous sequences of upstream pheA and tyrA downstream was constructed.A linearized DNA casstte Kan-QDZ-SacB consists of Kan geng and SacB geng,flanked by homologous sequences for gene deletion was obtained by polymerasechain reaction (PCR). The pheA and tyrA were deleted without scar left by twice Redrecombination in a Escherichia coli K12strain habouring plasmid pKD46.3. The expression level of gene trpD and trpE, encoding the key enzymes of L-Trpbranched pathway, in pheA and tyrA gene deleted Escherichia coli K12strain wasdetermined and compared with that in wild Escherichia coli K12strain. The analysisindicated that recombinant strain obtained6times and10times enhancement thanwild strain in expression level of trpD and trpE, respectively.4. The influence of initial pH and loading volume on bacterial density on recombinantstrain fermentation was studied and analysied. The optimal conditions consists of pH6.5and loading200mL.