| L-Phe is one of the essential amino acids for human beings. It plays the important roles in human body. The market demanding for L-Phe has been significantly increased since the development of food and pharmacal industry especially in the production of aspartame, amino acid based anti-cancer drugs and Dietary Supplements. There are only several small industries of L-Phe in China and the yield of L-Phe is only about 8%. The quantity of L-Phe production could not fit the market demanding in China. The majority of L-Phe for China market need largely depends on the import from the overseas. To promote the development of the industries that use L-Phe as material and reduce the economic burden on our country. It is very necessary to improve the fermentation of L-Phe production technologies and realize large scale industrial production.Objectives1. To compare the effects of different carbon source on bacterial growth and the yield of L-Phe, the tyrosine auxotrophic strain having the same plasmid was cultivated in glycerol, glucose, the mixture of glucose and glycerol separately.2. To compare the yield of L-Phe in gene recombined E.coli and wild type E.coli at same cultivation condition, different genotypes of plasmid were expressed in tyrosine auxotrophic strain and wild type bacterial separately.Methods1. The auxotrophic strain MG1655△aroF was obtained by knocking out pheL, pheA, tyrA and aroF genes which were located in the chromosome of wild-type MG1655 usingλ-Red recombination.2. The different geneoytpes of plasmid were constructed by using pZE12 as the vector through the ways of site-directed mutation and multiplex PCR.3. The gene recombined and wild type E.coli were cultivated in glycerol, glucose, the mixture of glucose and glycerol (the mass ratio of glycerol and glucose was 3:2) separately. During the fermentation, the expression of proteins encoded by pheA, aroF and aroG was identified. Only those with high protein expression strains were selected for further fermentation.4. The rough yield of L-Phe was monitered using chromatography during the fermentation. The quantity of free L-Phe in the culture media was determined using amino acid analyzer at the end of fermentation.Results1. The same genotype E.coli was cultivated at different carbon sources environment including glucose, glycerol, glycerol and glucose mixtue separately. The results showed that the rate of bacterium growth was the lowest when the carbon source was glucose alone. The bacteria grown in the mixed carbon source reached to the log phase later than that of other carbon sources. The production of L-Phe in glycerol was higher than the other two kinds of carbon sources and it was even doubled compared to that in glucose alone.2. Six genotypes of E.coli constructed were expressed in the tyrosine auxotrophic strain MG1655△aroF and the wild-type E.coli MG1655 at the same time. Then the bacteria were cultivated in glycerol. The results showed that the plasmid pZE12AfbrGM produced the highest yield of L-Phe, while pZE12AG produced the lowest yeild. The synthetic capacity of L-Phe in pZE12AfbrF was higher than that in pZE12AF. The growth rate of pZE12AfbrGMMG1655 was faster than pZE12AfbrGMMG1655△aroF, but its production of L-Phe was lower than pZE12AfbrGMMG1655△aroF.Conclusions1. The growth rate of pZE12AF MG△aroF, pZE12AfbrF MG△aroF, pZE12AG MG△aroF, pZE12AGMMG△aroF, pZE12AfbrG MG△aroF and pZE12AfbrGMMG△aroF was lower than the wild type and their yield of L-Phe in glycerol were more than that in glucose.2. The feedback inhibition-resistance of pheA and aroG can enhance the synthetic capacity of L-Phe in E.coli.3. MG1655 could benefit the bacterial growth, but its synthesis capacity of L-Phe was less than MG1655△aroF.
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