Systematic engineering enables efficient biosynthesis of L-phenylalanine in E. coli from inexpensive aromatic precursors

Abstract

Background: L-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant.

Results: The engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaE_P.p and threonine dehydratase A8H_B.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDH^V120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate.

Conclusion: Systematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.

Keywords: Aromatic precursors; Benzaldehyde; Benzyl alcohol; Engineering; Escherichia coli; L-phenylalanine.

Fig. 1

Design the artificial biosynthetic pathways for L-phenylalanine production from aromatic precursors (benzaldehyde or benzyl alcohol) and glycine. Gray arrows indicated the natural biosynthesis pathway of L-phenylalanine in E. coli. The green and red arrows demonstrated the novel pathway. ADH alcohol dehydrogenase, LTA threonine aldolase, LTD threonine dehydratase, PheDH phenylalanine dehydrogenase, FDH formate dehydrogenase. The dotted gray arrows indicate the repression and inhibition of the relevant genes in native pathway. Metabolites abbreviations: G6P glucose 6-phosphate, DAHP 3-deoxy-d-arabino- heptulosonate-7-phosphate, L-Glu L-glutamate, 2-KG 2-ketoglutarate

Fig. 2

Phenylpyruvate production using different combinations of threonine aldolase (LTA) and threonine dehydratase (LTD). a Phenylpyruvate production pathway from benzaldehyde and glycine catalyzed by LTA and LTD. b The effect of different threonine aldolases (LtaE_P.p, LtaE_E.c, LtaE_C.c) on phenylpyruvate production. c The effect of different L-phenylalanine dehydratase (A8H_B.t, IlvA_B.a, TAA_B.t) on phenylpyruvate production. Error bars are the standard deviation for three independent experiments

Fig. 3

Effect of reaction conditions on the phenylpyruvate production by the enzyme cascade of LtaE_P.p and A8H_B.t combination. a The effects of reaction temperatures on the phenylpyruvate production. b The effects of reaction pH on the phenylpyruvate production. The following buffer systems were used: 100 mM Tris-HCl for pH 6.5, 7.5, 8.0, and 8.5. Error bars are the standard deviation for three independent experiments

Fig. 4

The construction and evaluation of RidA strain M6. a Construction of strain M6 with plasmid pPLA-6 overexpressing RidA. b Effect of enzyme RidA on the conversion of benzaldehyde to phenylpyruvate. Error bars are the standard deviation for three independent experiments

Fig. 5

Biotransformation of phenylpyruvate into L-phenylalanine. a L-phenylalanine production from phenylpyruvate by coexpressing Bacillus badius phenylalanine dehydrogenase (PheDH) and Candida boidinii formate dehydrogenase (FDH^V120S). FDH was used for cofactor NADH regeneration. b The tolerance of the key enzymes PheDH and FDH^V120S to benzaldehyde. Error bars are the standard deviation for three independent experiments

Fig. 6

Biosynthesis of L-phenylalanine from aromatic precursor benzaldehyde. a Scheme showing the one-pot L-phenylalanine production from benzaldehyde with co-substrate formate for cofactor NADH regeneration. Production enzymes indicated the enzymes LtaE_P.p, A8H_B.t, and RidA that converted benzaldehyde to phenylpyruvate. b Biotransformation of the L-phenylalanine production in strain M8. Strain M8: BW25113 transformed with plasmids pPLA-6 and pPLA-7 to overexpress genes ltaE_P.p, A8H_B.t, ridA, pdh, and fdh. Error bars are the standard deviation for three independent experiments

Fig. 7

Biosynthesis of L-phenylalanine from aromatic precursor benzyl alcohol. a Scheme showing the one-pot L-phenylalanine production from benzyl alcohol with an engineering cofactor NADH self-regeneration system. Production enzymes indicated the LtaE_P.p, A8H_B.t, and RidA that converted benzaldehyde to phenylpyruvate. b Biotransformation of the L-phenylalanine production in strain M9. Strain M9: BW25113 transformed with plasmids pPLA-6 and pPLA-8 to overexpress genes ltaE_P.p, A8H_B.t, ridA, pdh, and xylB_P.p. Error bars are the standard deviation for three independent experiments