Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain

Abstract

BackgroundThe phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms.ResultsWe develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation.ConclusionsWe achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

Heterologous Production of Curcuminoids

“…coli and S. cerevisiae lack the CoA-ester starter substrates needed for curcuminoid production, and the naturally produced amino acids (phenylalanine and tyrosine) that are converted to the phenylpropanoic acids are not produced in sufficient amounts, which represents a limiting step (126,140,141). Although the amino acids and phenylpropanoic acids can be supplemented to the culture medium, the development of strains capable of converting glucose or other simple carbon sources to curcuminoids represents an important strategy for the engineering process.…”

“…Some studies related to production of phenylpropanoic acids (140,141,(150)(151)(152) and flavonoids (153) have used tyrosine-or phenylalanine-overproducing strains, and all obtained better results with these strains than with the wild type. All of them used similar approaches, in which they overexpressed aroG fbr and tyrA fbr , and in some cases ppsA and tktA, and deleted tyrR (⌬tyrR).…”

“…In the last years, several studies have used the enzymes involved in the phenylpropanoid pathway to produce compounds other than curcuminoids, such as phenylpropanoic acids (coumaric acid, caffeic acid, and ferulic acid) (140,141,151,152,(154)(155)(156)(157), stilbenoids (155,158), and flavonoids (116,117,150,155,(159)(160)(161)(162). In the design and construction of those pathways, some enzymes from other organisms were used that could also be considered for the optimization of the heterologous production of curcuminoids.…”

“…Then, after reaching the exponential phase, the cells are harvested and transferred to minimal medium, such as M9 medium, where the substrates are added and the curcuminoids are produced. This strategy was pursued in curcuminoid production in vivo (106,121,122) and also for other phenolic compounds (116,117,137,138,141,151,153,155,192,193). The use of plasmids imposes a metabolic burden on the host strain that usually reduces the growth rate of the cell due to the expression of plasmid-borne resistance and replication of the plasmids.…”

Engineering

“…For example, the construction of codon-optimized heterologous gene clusters with a wide span of strengths in promoter and ribosome binding sequences (RBS) has allowed the generation of E. coli strains capable of producing phenylpropanoic acids such as caffeic acid, coumaric acid and ferulic acid [103][104][105] (Figure 2), as well as hydroxycinnamoyl anthranilates [87] and other derivatives, such as hydroxytyrosol [106] (Figure 3). Another combinatorial Salvianic acid or danshensu (3,4-dihydroxyphenyllactic acid) A naturally occurring plant polyphenolic acid, considered as a superior antioxidant.…”