莽草酸基因工程菌构建与发酵工艺研究-发酵工程专业论文.docxVIP

优秀毕业论文 精品参考文献资料 ABSTRACTShikimic ABSTRACT Shikimic acid is an important intermediate in the synthesis of aromatic amino acids and has emerged as a key chiral starting material for the synthesis of antiviral drug oseltamivir phosphatc(Tamiflu⑩)．Microbial fermentation production of shikimic acid has a variety of advantages，and Escherichia coli Was commonly applied in both large-scale fermentation andindustrial production．Metabolic engineering is one of the mainly technical methods to construct the industrialized high-yield shikimic acid strains． Besides short production cycle and low cost，microbial method also has less pollution on the environment．This paper focus on constructing shikimic acid high-yielding strains ．with genetic engineering methods，that experiments need to block shikimate metabolic pathways and branch pathways and strengthen the shikimate pathway metabolic flow． The finished research mainly included as following： (1)The gene aroL and aroK coding shikimic kinase was disrupted by Red recombination，constructed E．coli SHIKAaroL，E．coli SHIKAaroK,E．coli SHIKAaroLAaroK strains．Took shake flask fermentation under the same conditions，E coli SHIKAaroL had much higher shikimic acid production and had growth in good condition than the others． (2)To reduce metabolic flow consumption for metabolic branch，the gene ydiB coding quinic acid／shikimic acid dehydrogenase of E．coli SHIKAaroL was disrupted relying on Red recombination system，constructed strain E．coli SHIKAaroLAydiB， resulting on lessening the by—product content． (3)Strengthened the shikimate pathway metabolic flow by constructing plasmids： pTrc99A-aroG，pTrc99A-aroGE，pTrc99A—aroGED，pTrc99A-aroGEDB． (4)In order to prevent the plasmid pTrc99A—aroGEDB overload，we constructed a system of double expression plasmid，that plasmind pSTV28一aroB kep company with pTrc99A-aroG，pTrc99A—aroGE or pTrc99A-aroGED． (5)Establish shikimic acid genetic engineering bacteria by tansforming the plasmids the above mentioned into str