Dirk Schwartz

Biosynthetic Gene Cluster of the Herbicide Phosphinothricin Tripeptide from Streptomyces viridochromogenes Tü494

ABSTRACT The antibiotic phosphinothricin tripeptide (PTT) consists of two molecules of l-alanine and one molecule of the unusual amino acid phosphinothricin (PT) which are nonribosomally combined. The bioactive compound PT has bactericidal, fungicidal, and herbicidal properties and possesses a C—P—C bond, which is very rare in natural compounds. Previously uncharacterized flanking and middle regions of the PTT biosynthetic gene cluster from Streptomyces viridochromogenes Tü494 were isolated and sequenced. The boundaries of the gene cluster were identified by gene inactivation studies. Sequence analysis and homology searches led to the completion of the gene cluster, which consists of 24 genes. Four of these were identified as undescribed genes coding for proteins that are probably involved in uncharacterized early steps of antibiotic biosynthesis or in providing precursors of PTT biosynthesis (phosphoenolpyruvate, acetyl-coenzyme A, or l-alanine). The involvement of the genes orfM and trs and of the regulatory gene prpA in PTT biosynthesis was analyzed by gene inactivation and overexpression, respectively. Insight into the regulation of PTT was gained by determining the transcriptional start sites of the pmi and prpA genes. A previously undescribed regulatory gene involved in morphological differentiation in streptomycetes was identified outside of the left boundary of the PTT biosynthetic gene cluster.

A Glutamate Mutase Is Involved in the Biosynthesis of the Lipopeptide Antibiotic Friulimicin in Actinoplanes friuliensis

ABSTRACT Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family Enterobacteriaceae. The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.

The Phosphinomethylmalate Isomerase Gene pmi, Encoding an Aconitase-Like Enzyme, Is Involved in the Synthesis of Phosphinothricin Tripeptide in Streptomyces viridochromogenes

ABSTRACT Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197–222,in L. C. Vining and C. Stuttard, ed.,Genetics and Biochemistry of Antibiotic Production,1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced inStreptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnAmutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.

Phosphinothricin Tripeptide Synthetases in Streptomyces viridochromogenes Tü494

ABSTRACT The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from Streptomyces viridochromogenes, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor N-acetyl-demethylphosphinothricin (N-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both phsB and phsC appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene phsA, encoding PhsA, in the PTT biosynthesis gene cluster of S. viridochromogenes. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from S. viridochromogenes confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (the1 and the2) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.

Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis

Manipulation of secondary metabolite production in the rare actinomycete Actinoplanes friuliensis, the producer of the lipopeptide antibiotic friulimicin, is hampered by the lack of sophisticated genetic tools. Since no expression vectors have been developed from endogenous Actinoplanes plasmids and expression signals, engineering of antibiotic biosynthesis relies on the use of vector systems derived from Streptomyces. While PhiC31 derived vectors were shown to integrate efficiently into the chromosome of Actinoplanes, information on promoter activity is missing. The manuscript describes the investigation of several different promoter systems which are widely used in Streptomyces in A. friuliensis by promoter probe experiments using eGFP as a reporter. These experiments indicated that promoter strength in A. friuliensis did not correlate to activity in Streptomyces lividans. The ermE* promoter regarded as one of the strongest promoter in Streptomyces has only low activity in A. friuliensis. In contrast, the promoter of the apramycin resistance gene aac(3)IV, originating from the Gram-negative Escherichia coli had the highest activity. By real-time RT-PCR experiments the transcription activity of ermE* promoter in comparison to a native promoter of the friulimicin biosynthetic gene cluster was analysed. This confirmed the results of the promoter probe experiments that indicated quite weak promoter activity of P-ermE* in Actinoplanes.

Tricarboxylic acid cycle aconitase activity during the life cycle of Streptomyces viridochromogenes Tü494

Abstract. Previously, it was shown that inactivation of the tricarboxylic acid cycle aconitase gene acnA impairs the morphological and physiological differentiation of Streptomyces viridochromogenes Tü494, which produces the herbicide phosphinothricin tripeptide (PTT). In order to further characterize the role of the aconitase in the Streptomyces life cycle, aconitase activity was analyzed during growth of S. viridochromogenes in liquid culture. Two prominent maxima were measured in cell-free crude extracts. The first maximum was found at an early stage of growth, which is correlated with a decrease in pH when rapid glucose consumption is initiated. The second, lower maximum was detected at the beginning of the expression of the PTT-specific biosynthetic gene phsA, implying the onset of secondary metabolism. These results were confirmed by examining transcription of the acnA promoter in time-course experiments. The highest transcription rate was found during the early growth phases. In order to identify putative regulatory mechanisms, the transcriptional start site of the acnA transcript and subsequently the promoter were identified. Several putative, regulatory protein binding sites (e.g. regulators of oxygen stress or iron metabolism) were detected in the promoter region of acnA, which suggested complex regulation of acnA.

Complete genome sequence of the actinobacterium Actinoplanes friuliensis HAG 010964, producer of the lipopeptide antibiotic friulimycin.

Actinoplanes friuliensis HAG 010964 (DSM 7358) was isolated from a soil sample from the Friuli region in Italy and characterized as a producer of the antibiotic friulimycin. The complete genome sequence includes genomic information of secondary metabolite biosynthesis and of its lifestyle. Genbank/EMBL/DDBJ Accession Nr: CP006272 (chromosome).

Three Thioesterases Are Involved in the Biosynthesis of Phosphinothricin Tripeptide in Streptomyces viridochromogenes Tü494

ABSTRACT Phosphinothricin tripeptide (PTT) is a peptide antibiotic produced by Streptomyces viridochromogenes Tü494, and it is synthesized by nonribosomal peptide synthetases. The PTT biosynthetic gene cluster contains three peptide synthetase genes: phsA, phsB, and phsC. Each of these peptide synthetases comprises only one module. In neither PhsB nor PhsC is a typical C-terminal thioesterase domain present. In contrast, a single thioesterase GXSXG motif has been identified in the N terminus of the first peptide synthetase, PhsA. In addition, two external thioesterase genes, theA and theB, are located within the PTT biosynthetic gene cluster. To analyze the thioesterase function as well as the assembly of the peptide synthetases within PTT biosynthesis, several mutants were generated and analyzed. A phsA deletion mutant (MphsA) was complemented with two different phsA constructs that were carrying mutations in the thioesterase motif. In one construct, the thioesterase motif comprising 45 amino acids of phsA were deleted. In the second construct, the conserved serine residue of the GXSXG motif was replaced by an alanine. In both cases, the complementation of MphsA did not restore PTT biosynthesis, revealing that the thioesterase motif in the N terminus of PhsA is required for PTT production. In contrast, TheA and TheB might have editing functions, as an interruption of the theA and theB genes led to reduced PTT production, whereas an overexpression of both genes in the wild type enhanced the PTT yield. The presence of an active single thioesterase motif in the N terminus of PhsA points to a novel mechanism of product release.

Analysis of RegA, a pathway-specific regulator of the friulimicin biosynthesis in Actinoplanes friuliensis.

The rare actinomycete Actinoplanes friuliensis is the producer of the lipopeptide antibiotic friulimicin, which is active against a broad range of Gram-positive bacteria such as methicillin-resistant Enterococcus spec. and Staphylococcus aureus (MRE, MRSA) strains. Friulimicin consists of a decapeptide core and an acyl residue linked to an exocyclic amino acid. The complete biosynthetic gene cluster consisting of 24 open reading frames was characterized by sequence analysis and the transcription units were subsequently determined by RT-PCR experiments. In addition to several genes for biosynthesis, self-resistance and transport four different regulatory genes (regA, regB, regC and regD) were identified within the cluster. To analyse the role of the pathway-specific regulatory protein RegA in the friulimicin biosynthesis, the corresponding gene was inactivated resulting in friulimicin non-producing mutants. Furthermore, several protein-binding sites within the friulimicin gene cluster were identified by gel retardation assays. By real-time RT-PCR experiments, it was shown that the majority of the friulimicin biosynthetic genes is positively regulated by RegA.

Development of cultivation strategies for friulimicin production in Actinoplanes friuliensis

Actinoplanes friuliensis is a rare actinomycete which produces the highly potent lipopeptide antibiotic friulimicin. This lipopeptide antibiotic is active against a broad range of multi-resistant gram-positive bacteria such as methicillin-resistant Enterococcus sp. and Staphylococcus aureus (MRE, MRSA) strains. Antibiotic biosynthesis and regulation in actinomycetes is very complex. In order to study the biosynthesis of these species and to develop efficient production processes, standardized cultivation conditions are a prerequisite. For this reason a chemically defined production medium for A. friuliensis was developed. With this chemically defined medium it was possible to analyze the influence of medium components on growth and antibiotic biosynthesis. These findings were used to develop process strategies for friulimicin production. The focus of the project presented here was to develop cultivation strategies which included fed-batch and continuous cultivation processes. In fed-batch processes, volumetric productivities for friulimicin of 1-2 mg/l h were achieved. In a perfusion process, a very simple cell retention system, which works via sedimentation of the mycelial cell pellets, was used. With this system, stable continuous cultivations with cell retention were dependent on the dilution rate. With a dilution rate of 0.05 h(-1), cell retention worked well and volumetric productivity of friulimicin was enhanced to 3-5 mg/l h. With a higher dilution rate of 0.1 h(-1), friulimicin production ceased because cell retention was not possible any longer with this simple cell retention system. In order to support process development, cultivation data were used to characterize metabolic fluxes in the developed friulimicin production processes.