One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli

Abstract

Background: L-tryptophan is an aromatic amino acid widely used in the food, chemical and pharmaceutical industries. In Escherichia coli, L-tryptophan is synthesized from phosphoenolpyruvate and erythrose 4-phosphate by enzymes in the shikimate pathway and L-tryptophan branch pathway, while L-serine and phosphoribosylpyrophosphate are also involved in L-tryptophan synthesis. In order to construct a microbial strain for efficient L-tryptophan production from glucose, we developed a one step tryptophan attenuator inactivation and promoter swapping strategy for metabolic flux optimization after a base strain was obtained by overexpressing the tktA, mutated trpE and aroG genes and inactivating a series of competitive steps.

Results: The engineered E. coli GPT1002 with tryptophan attenuator inactivation and tryptophan operon promoter substitution exhibited 1.67 ~ 9.29 times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated 1.70 g l(-1) L-tryptophan after 36 h batch cultivation in 300-mL shake flask. Bioreactor fermentation experiments showed that GPT1002 could produce 10.15 g l(-1) L-tryptophan in 48 h.

Conclusions: The one step inactivating and promoter swapping is an efficient method for metabolic engineering. This method can also be applied in other bacteria.

Figure 1

The strategies for constructing the L-tryptophan producing strain GPT1002. The shaded boxes represent genetic modification, and the gray bars indicate the genes that were deleted. Dotted lines indicate feedback inhibition. The black X indicates that the inhibition is removed. The thick black arrows indicate the increased flux or activity by directly overexpressing the corresponding genes in plasmids. Glc glucose, G6P glucose-6-phosphate, E4P erythrose-4-phosphate, PEP phosphoenolpyruvate, DAHP 3-deoxy-D-arabino-heptulosonate, CHA chorismate, ANTA anthranilate, L-Phe L-phenylalanine, L-Tyr L-tyrosine, L-Trp L-tryptophan, tktA transketolase, aroG 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phenylalanine repressible), trpE component I of anthranilate synthase, trpR trp operon repressor, tnaA tryptophanase, ptsG glucose-specific PTS enzyme IIBC components, manXYZ mannose-specific PTS enzyme IIABCD components, galP D-galactose transporter, PP pathway pentose phosphate pathway.

Figure 2

Outline of plasmid pKMT construction and promoter swapping.

Figure 3

RT-PCR analysis of constructed E. coli. (A) Relative gene expression of E. coli GPT1002 to the control GPT1001. (B) Relative gene expression of E. coli GPT101 to the control GPT100. gapA transcripts was selected as standard and each measurements were repeated three times. The error bars indicate standard deviations.

Figure 4

Batch cultivation of E. coli GPT1001 and GPT1002 in 300-mL shake flasks. For E. coli GPT1001, (filled square) growth curves; (filled circle) glucose consumption; (filled triangle) L-tryptophan yield. For E. coli GPT1002, (open square) growth curves; (open circle) glucose consumption; (open triangle) L-tryptophan yield. The error bars represent standard deviations from three replicate fermentations.

Figure 5

Fed-batch fermentation of GPT1002 in 5-L fermentator. (Filled square) growth curves; (filled circle) glucose consumption; (filled triangle) L-tryptophan yield. The error bars represent standard deviations from three measurements.