Florian Schauwecker

Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

ABSTRACT The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

BACKGROUND Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. RESULTS The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. CONCLUSIONS A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.

The Actinomycin Biosynthetic Gene Cluster of Streptomyces chrysomallus: a Genetic Hall of Mirrors for Synthesis of a Molecule with Mirror Symmetry

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.

Molecular characterization of the genes of actinomycin synthetase I and of a 4-methyl-3-hydroxyanthranilic acid carrier protein involved in the assembly of the acylpeptide chain of actinomycin in Streptomyces.

Actinomycin synthetase I (ACMS I) activates 4-methyl-3-hydroxyanthranilic acid, the precursor of the chromophoric moiety of the actinomycin, as adenylate. The gene acmA of ACMS I was identified upstream of the genes acmB andacmC encoding the two peptide synthetases ACMS II and ACMS III, respectively, which assemble the pentapeptide lactone rings of the antibiotic. Sequence analysis and expression of acmA inStreptomyces lividans as enzymatically active hexa-His-fusion confirmed the acmA gene product to be ACMS I. An open reading frame of 234 base pairs (acmD), which encodes a 78-amino acid protein with similarity to various acyl carrier proteins, is located downstream of acmA. TheacmD gene was expressed in Escherichia coli as hexa-His-fusion protein (Acm acyl carrier protein (AcmACP)). ACMS I in the presence of ATP acylated the purified AcmACP with radioactivep-toluic acid, used as substrate in place of 4-MHA. Only 10% of the AcmACP from E. coli was acylated, suggesting insufficient modification with 4′-phosphopantetheine cofactor. Incubation of this AcmACP with a holo-ACP synthase and coenzyme A quantitatively established the holo-form of AcmACP. Enzyme assays in the presence of ACMS II showed that toluyl-AcmACP directly acylated the thioester-bound threonine on ACMS II. Thus, AcmACP is a 4-MHA carrier protein in the peptide chain initiation of actinomycin synthesis.

Nonribosomal biosynthesis of microbial chromopeptides.

Nonribosomal chromopeptides and mixed chromopeptide-polyketides contain aromatic or heteroaromatic side groups which are important recognition elements for interaction with cellular targets such as DNA and proteins, resulting in the biological activities of these natural products. In the chromopeptide lactones and arylpeptide-siderophores from bacteria, the chromophore moiety--an aryl carboxylate amidated to the peptide chain--constitutes the formal amino terminus and is the starter residue of peptide assembly. Common to many arylpeptide systems is the activation by stand-alone adenylation domains and loading of the starter to discrete aryl carrier proteins (ArCPs) or ArCP domains which interact with the modules of the respective nonribosomal peptide synthetase (NRPS), assembling the next residues of the chain. Chain modification is another mechanism of nonribosomal chromopeptide synthesis where heteroaromatic rings such as thiazoles and oxazoles in peptides and polyketides are generated by heterocylizations of acyl- or peptidyl-cysteinyl or -serinyl/threonyl intermediates in each elongation step. In this review the basic mechanisms of chromophore acquisition in nonribosomal chromopeptide synthesis and mixed peptide/polyketide synthesis are illustrated by comparing the biosynthesis systems of various chromopeptides and chromopeptidic polyketide compounds.