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Studies on Coordinate Expression of Multienzyme-genes in Phenylalanine Biosynthesis Pathway

Author: LuJia
Tutor: GuoJun；HuangYingWu
School: Inner Mongolia Agricultural University
Course: Of Food Science
Keywords: L-phenylalanine Multi-enzyme gene Interval of regulatory sequences Coordinated expression Biosynthesis
CLC: TS201.2
Type: Master's thesis
Year: 2011
Downloads: 40
Quote: 1
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Abstract


Phenylalanine (phenylalanine, Phe), an essential amino acid needed by the body and have applications in medicine, nutrition, food processing and animal husbandry field. Phenylalanine or critical raw materials for the production of the sweetener aspartame (Aspartame), the increasing market demand. Of the lack of state-of-the-art and efficient L-phenylalanine (L-Phe) production technology mainly rely on imports, L-phenylalanine yield engineered bacteria using molecular biology techniques to build practical significance. This study design, optimizing the interval of regulation of the enzyme gene sequence (intergenic Regulatory Sequence), a key enzyme of the biosynthetic pathway of L-Phe Preparation gene pheA, aroF and the ppsA coordinated expression, so as to improve the yield of L-Phe: ⑴ respectively regulation sequences of the design gene pheA, aroF and ppsA constructed single-gene recombinant plasmid, which successfully constructed recombinant the sub pTrc-the 99a-pheA the pTrc-99a-aroF single gene enzyme protein for high expression; ⑵ to build pheA aroF gene the tandem recombinant plasmid pZE12-AF, change the two series of regulatory sequences of gene interval the the recombinant pZE12-RBS-AF The auxotrophic these two recombinant plasmids into to bacteria MG △ pheA ... aroF constructed successfully engineered bacteria MG △ pZE12-AF and MG △ pZE12-RBS-AF; ⑶ to build three gene pheA, aroF, and ppsA series the recombinant plasmid pZE12-AFP, adjust ppsA regulatory sequences recombinants pZE12-RBS-AFP, and successfully construct the corresponding the engineered bacteria MG △ pZE12-AFP and MG the △ pZE12-RBS-AFP. Observing the amount of enzyme protein by SDS-PAGE, to adjust the interval regulatory sequences of the multi-gene AF, the two recombinant plasmids pZE12-RBS-AF of the enzyme protein is higher than pZE12-AF expression amount; the same AFP three recombinant plasmid, pZE12 -RBS-AFP The plasmid to adjust the ppsA the interval regulation sequence, the enzyme protein than pZE12-AFP expression was significantly increased. Determination Fermentation culture supernatant of the content of L-Phe, the yield of L-Phe the the MG △ pZE12-RBS-AF engineering bacteria increased by 1.5 times than the MG △ pZE12-AF; yield of the MG △ pZE12-RBS-AFP ratio MG △ pZE12-AFP also increased 2-fold. MG △ pZE12-RBS-AFP bacteria L-Phe production is 1.2 times the MG △ pZE12-RBS-AF. To achieve a L-Phe biological synthesis key enzyme gene pheA and aroF and ppsA synergistic matching expression, significant metabolic flux flows final product L-Phe, indicate that adjustment tandem gene of the interval regulation sequence is changed metabolic flow and improve the L-Phe of the Yield is an effective method. In addition, the selection as the low copy pZE12 plasmid expression vector than the high copy plasmid (eg pTrc-99a) enzyme expression more moderate and stable, not prone to the formation of inclusion bodies and higher activity.

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