Genetic engineering of Escherichia coli to improve L-phenylalanine production

Abstract

BackgroundL-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.ResultsWe 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0418-1) contains supplementary material, which is available to authorized users.

Expanding the repertoire of aromatic chemicals by microbial production

“…23 W3110, such as replacing PTS with the GalP-Glk system, relieving feedback inhibition of aroF, pheA and tyrR, and overexpressing aroD, the L-Phe titer of the Xllp21 strain reached 72.9 g L −1 in a 5 L fermenter after 52-h cultivation. 37 L-Phe production by C. glutamicum also has been widely reported. Through combinational expression of the key genes of the L-Phe biosynthesis pathway, modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways, the titer of L-Phe reached 15.76 g L −1 under fed-batch fermentation conditions.…”

Molecular control of gene expression by Brucella BaaR, an IclR-type transcriptional repressor

“…Such a reaction has been previously described in an engineered E. coli strain producing isopropanol (69) and in acetone production in Clostridium acetobutylicum (70). In these strains, a CoAtransferase was involved in the conversion of acetoacetyl-CoA into acetoacetate, using butyrate or acetate as CoA acceptors (69,70). Such a reaction in B. abortus may explain why the bab2_0278-bab2_0282 ABC transport system is downregulated when the bab2_0213-bab2_0217 locus is overexpressed.…”

“…Instead, as a last step, we propose that the CoAtransferase family III enzyme (Bab2_0217) present in the bab2_0213-bab2_0217 locus may catalyze a reversible transfer of coenzyme A from CoA-thioesters to free acids ( Figure S8) (53,68), leading to the formation of ketones. Such a reaction has been previously described in an engineered E. coli strain producing isopropanol (69) and in acetone production in Clostridium acetobutylicum (70). In these strains, a CoAtransferase was involved in the conversion of acetoacetyl-CoA into acetoacetate, using butyrate or acetate as CoA acceptors (69,70).…”

Development of a high‐efficiencytrans‐cinnamic acid bioproduction method by pH‐controlled separation technology

“…Recently, there has been great interest in using PAL in clinical, industrial and biotechnological applications, because it contains a radical‐generating cofactor 4‐methylidene‐imidazole‐5‐one (MIO) . All studies have shown that l ‐phe can be produced with a high yield from glucose in a fermentation system, but the concentration of t ‐CA has proven too low by the fermentation of sugars (Table ) . Vargas‐Tah et al produced 530 μmol L –1 t ‐CA with the PAL enzyme from Arabidopsis thaliana .…”