A Study On Construction Of L-phenylalanine Engineered Strain And Metabolic Regulation Of L-phenylalanine Biosynthesis Pathway

In order to keep the grain security, part of the grain would be stored after harvest. The grain quality together with the value would decrease, owing to metabolism of itself. Improper storage condition would accelerate the process. Deep processing of this kind of grain to promote its added value makes remarkable economic and social benefits. Utilizing of glucose from grain to produce L-phenylalanine could enhance the effectiveness of stale grain.L-phenylalanine (L-Phe) is an essential amino acid for humans and other animals, and therefore widely used as food additive, animal feed, infusion fluids, and substrates for chemical synthesis of active compounds. L-Phe with its derivatives’ annual sales has reached 1 billion US dollars. The consumer demand for L-Phe stems mainly from being a building block in the synthesis of aspartame, which is a low-calorie sweetener and is highly favored by consumers. China is a net exporter of aspartame and a net importer of L-Phe, due to the inefficiency technology used in production of L-Phe.The present study aimed to construct L-Phe overproducing strain and make it suitable for utilization of stale grain. Metabolic engineering was applied for modifying Escherichia coli strain NST74. After metabolic flux analysis, the supplement of PEP and E4P was enhanced, and the inhibition of key enzymes was released. The transport system of L-Phe was also modified. The main contents and results are as follows:(1) The ppsA and tktA genes, which are responsible for synthesis of PEP and E4P, were cloned and co-expressed in pQST plasmid. The results of SDS-PAGE and enzyme activity assay showed that the PpsA and TktA activities of E. coli NST74(pQST) were 7.4-fold and 8.1-fold of E. coli NST74, respectively. Moreover, the expression levels of the two enzymes were coordinated. Co-expression plasmid pQST was conducive to increase the availability of PEP and E4P, which were crucial for achieving the maximum flow of carbon into the shikimate pathway.(2) Based on pQT plasmid, three other genes, pheAfbr, aroGfbr, and tyrB, encoding the key enzymes of the shikimate pathway were cloned and co-expressed in pQSTERO plasmid. Enzymatic activity and feedback inhibition were compared between E. coli NST74(pQSTERO) and E. coli NST74. The AroG activity of E. coli NST74(pQSTERO) was 8.7-fold of E. coli NST74, the CM and PD activities of E. coli NST74(pQSTERO) were 8.2-fold and 7.4-fold of E. coli NST74, respectively, and the TyrB activity of E. coli NST74(pQSTERO) was 6.3-fold of E. coli NST74. When L-Phe concentration reached 10 mmol/L,68.3% of the AroGfbr activity was reserved,87.4% of the CMfbr activity was reserved and 84.3% of the PDfbr activity was reserved. Both AroGfbr and PheAfbr exhibited resistance to L-Phe inhibition.(3) The aroP gene of E. coli NST74 was knocked out to generate E. coli NST74△P, and the yddG gene was cloned and co-expressed in pQSTEROD plasmid. Shake flask fermentation and chromatography were carried out to compare L-Phe production capacity of the engineered strains, and E. coli NST74△P(pQSTEROD) was found to be the best one. Fed-batch fermentation was performed to compare the parameters between E. coli NST74△P(pQSTEROD) and E. coli NST74. After 48 h of cultivation, E. coli NST74AP(pQSTEROD) produced a L-Phe titer of 36.48 g/L, with a yield of 0.26 mol/mol, which represented improvements of 323.7% and 36.8%, respectively, over E. coli NST74. E. coli NST74△P(pQSTEROD) was demonstrated to have great potential for L-Phe production.(4) Three single-site mutant aroG alleles were constructed (aroG8, aroG15, and aroG29), which were then combined to generate three double-site aroGfbr mutant alleles (aroG8/15, aroG8/29, and aroG15/29). Enzymatic activity, feedback inhibition, and fermentation were analyzed in all of the mutants. All double-site mutants, except AroG15/29, showed higher enzymatic activity and greater resistance to feedback inhibition than their respective single-site mutants. The E. coli strain carrying the aroG8/15 allele produced an L-Phe titer of 26.78 g/1, a 116% improvement over the control strain (12.41 g/1). Our findings provide an effective method for modifying L-Phe biosynthetic genes, which may be applied to optimize the commercial manufacture of L-Phe.