Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix espanaensis

Abstract

The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-pcoumaric acid is first activated before the 3-hydroxylation of its ring takes place.Saccharothrix is a genus of gram-positive bacteria belonging to the well-known order Actinomycetales. Most agents used at present for the treatment of bacterial infections were discovered in members of the Actinomycetales. Saccharothrix espanaensis produces the two heptadecaglycoside antibiotics saccharomicins A and B, which represent a new class of antibiotics (15, 18). They exhibit potent antibacterial activity both in vitro and in vivo against multiply-resistant strains of Staphylococcus aureus as well as vancomycin-resistant enterococci (25). The saccharomicins consist of an oligosaccharide portion and the intriguing aglycon N-(m,p-dihydroxycinnamoyl)taurine (Fig. 1), in which caffeic acid is linked to the amino sulfonic acid taurine via an amide bond.Enzymes belonging to the group of ammonia-lyases catalyze the conversion of ␣-amino acids into ␣,␤-unsaturated acids by elimination of ammonia. Ubiquitous in plants and fungi, phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) catalyzes the nonoxidative deamination of the primary amino acid L-phenylalanine to trans-cinnamic acid (trans-cinnamate), which is the first reaction of the so-called general phenylpropanoid pathway in plants (8). Phenylpropanoids include several important natural product classes, for example, flavonoids, lignins, and coumarins. In monocotyledons, PAL utilizes L-tyrosine in addition to L-phenylalanine (resulting in trans-p-coumaric acid), whereas the enzyme from dicotyledons converts only L-phenylalanine sufficiently. Both PAL and tyrosine ammonia-lyase (TAL) activity are very rare in bacteria (17, 30).The next reactions of the three-step general phenylpropanoid pathway are catalyzed by the enzymes trans-cinnamate 4-monooxygenase (also called cinnamate 4-hydroxylase; EC 1.14.13.11), leading to trans-p-coumaric acid (trans-4-coumarate), and 4-coumarate-coenzyme A (CoA) ligase (EC 6.2.1.12), leading to 4-coumaroyl-CoA (8).In this report, we describe the cloning and identification of two genes from S. espanaensis w...

New Aminocoumarin Antibiotics Derived from 4-Hydroxycinnamic Acid are Formed after Heterologous Expression of a Modified Clorobiocin Biosynthetic Gene Cluster

“…Our present findings stimulated us to re-examine the genome sequence of S. coelicolor [24]. Blast search revealed one gene with 32% identity to the tyrosine ammonia-lyase Sam8 of Saccharothrix espanaensis [21]. This gene, SCO4932, is currently annotated as histidine ammonia-lyase.…”

“…In Actinomycetes, however, these compounds are relatively rare, and only recently it was demonstrated that several Actinomycetes possess phenylalanine ammonialyase (PAL) and tyrosine ammonia-lyase (TAL) genes for the biosynthesis of cinnamic and coumaric acid [21,22]. However, neither in the biosynthetic gene cluster of clorobiocin, nor in the completely sequenced genome of S. coelicolor, PAL or TAL genes have been described so far.…”

Heterologous Production of Curcuminoids

“…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.…”

“…Berner et al (154) studied the caffeic acid biosynthetic pathway in the actinomycete Saccharothrix espanaensis and heterologously expressed it in Streptomyces fradiae XKS, a polyketide synthase-defective mutant strain that is considered to be a very convenient host for expression experiments (166). Streptomyces, applied in the production of a wide range of secondary metabolites, including polyketides, can be another candidate for the heterologous production of plantspecific polyketides (167).…”

Hyperpigmentation Analysis of Tal Enzyme on Danio Rerio Obtained From Basella Alba

“…[22] Hence a depigmenting enzyme - [7] and Saccharothrixespanaensis (Gen-Bank accession number: ABC88669). [8] This variety reflects the enzyme's specialized role in p-coumaric acid biosynthesis.Tyrosine ammonia-lyase (TAL, EC 4.3.1.5) has recently been described as a member of the aromatic amino acid lyase family of enzymes, which also includes phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and histidine ammonia-lyase (HAL, EC 4.3.1.3). [4] TAL is required for deamination of the amino acid L-tyrosine to p-coumaric acid and releases ammonia.…”