Genetic engineering of Escherichia coli to improve L-phenylalanine production

Abstract

Background: L-phenylalanine (L-Phe) is an essential amino acid for mammals and applications expand into human health and nutritional products. In this study, a system level engineering was conducted to enhance L-Phe biosynthesis in Escherichia coli.

Results: We inactivated the PTS system and recruited glucose uptake via combinatorial modulation of galP and glk to increase PEP supply in the Xllp01 strain. In addition, the HTH domain of the transcription factor TyrR was engineered to decrease the repression on the transcriptional levels of L-Phe pathway enzymes. Finally, proteomics analysis demonstrated the third step of the SHIK pathway (catalyzed via AroD) as the rate-limiting step for L-Phe production. After optimization of the aroD promoter strength, the titer of L-Phe increased by 13.3%. Analysis of the transcriptional level of genes involved in the central metabolic pathways and L-Phe biosynthesis via RT-PCR showed that the recombinant L-Phe producer exhibited a great capability in the glucose utilization and precursor (PEP and E4P) generation. Via systems level engineering, the L-Phe titer of Xllp21 strain reached 72.9 g/L in a 5 L fermenter under the non-optimized fermentation conditions, which was 1.62-times that of the original strain Xllp01.

Conclusion: The metabolic engineering strategy reported here can be broadly employed for developing genetically defined organisms for the efficient production of other aromatic amino acids and derived compounds.

Keywords: AroD; L-phenylalanine; Metabolic engineering; Proteomics; TyrR.

Fig. 1

Growth curves and fermentation results of the recombinant strains combinational modulating the galp and glk genes with different promoters. a Growth curves of the recombinant strains; b The fermentation results of the recombinant strains in shake flasks. The experiment was repeated three times, and measurements are represented as means with their standard deviation

Fig. 2

Fermentation results and transcriptional analysis of the TyrR mutant strains. a Fermentation results of the TyrR mutant strains in shake flasks. Black columns stands for OD₆₀₀ and gray columns stands for the L-Phe production. b Real-time PCR analysis consequences of the genes regulated by the TyrR protein in Xllp08 contrast to Xllp04. Experiments were conducted in triplicate

Fig. 3

Gene ontology analysis of the proteins identified by the proteomics

Fig. 4

Proteomic analysis of the genes in Xllp08 and wild-type E. coli W3110. a Comparison of central carbon metabolism between wild-type W3110 and Xllp08. Two precursors (PEP and E4P) of the L-Phe synthesis were located in the EMP pathway and PP pathway respectively, the distribution of which would directly affect the L-Phe production and yield. The red data meant upregulation and green data meant downregulation. b The great disparity between two studied strains in the aromatic amino acid synthesis pathway. This figure showed comparion of enzymes in the L-Phe generating pathway between Xllp08 strain and wild-type E. coli W3110

Fig. 5

Fermentation results of the Xllp08 strains and its derivative strains. Xllp16, Xllp17, and Xllp18 were obtained by overexpressing aroD, ydiB and aroC in Xllp08 strains, respcetively. The black column meant OD₆₀₀ and the gray column meant L-Phe titer. Experiments were conducted in triplicate

Fig. 6

Fermentation results of the recombinant strains overexpressing aroD gene with different promoters in Xllp08 strain. The black column meant OD₆₀₀ and the gray column meant the L-Phe titer. The expreiments were repeated three times

Fig. 7

Relative transcription level of genes in Xllp21 and Xllp08 by real-time PCR analysis. Experiments were conducted in triplicate, and measurements are represented as means with their standard deviation

Fig. 8

Time course of L-Phe production by strain Xllp01 (a) and Xllp21 (b) in a 5 L fermenter. Dots meant residual glucose concentration, blocks meant the OD₆₀₀ and triangles meant the L-Phe concentration. The final L-Phe yield and titer was basically constant among the three replicates and one of the three times was given as an example to show the fermentation results