The biosynthetic pathway of the prenylated salicylaldehyde flavoglaucin and congeners in Aspergillus ruber was elucidated by genome mining, heterologous expression, precursor feeding, and biochemical characterization. The polyketide skeleton was released as alkylated salicyl alcohols, which is a prerequisite for consecutive hydroxylation and prenylation, before reoxidation to the final aldehyde products. Our results provide an excellent example for a highly programmed machinery in natural product biosynthesis.

The plant hormone auxin (indoleacetate) is anaerobically degraded by the Betaproteobacterium Aromatoleum aromaticum. We report here on a CoA ligase (IaaB) and a CoA-transferase (IaaL) which are encoded in the apparent substrate-induced iaa operon containing genes for indoleacetate degradation. IaaB is a highly specific indoleacetate-CoA ligase which activates indoleacetate to the CoA-thioester immediately after uptake into the cytoplasm. This enzyme only activates indoleacetate and some closely related compounds such as naphthylacetate, phenylacetate and indolepropionate, and is inhibited by high concentrations of substrates, and by the synthetic auxin compound 2,4-dichlorophenoxyacetate, which does not serve as substrate. IaaL is a CoA-transferase recognizing several C4-dicarboxylic acids, such as succinate, phenylsuccinate or benzylsuccinate and their CoA-thioesters, but only few monocarboxylic acids and no C3-dicarboxylic acids such as benzylmalonate. The enzyme shows no stereospecific discrimation of the benzylsuccinate enantiomers. Moreover, benzylsuccinate is regiospecifically activated to 2-benzylsuccinyl-CoA, whereas phenylsuccinate is converted to an equal mixture of both regioisomers (2- and 3-phenylsuccinyl-CoA). The identification of these two enzymes allows us to set up a modified version of the metabolic pathway of anaerobic indoleacetate degradation and to investigate the sequences databases for the occurrence and distribution of this pathway in other microorgansisms.

The echinulin family alkaloids can be grouped into three series depending on the number of the exo double bonds adjacent to the diketopiperazine core structure. Heterologous expression of the putative echinulin biosynthetic gene cluster from Aspergillus ruber in Aspergillus nidulans led to accumulation of echinulin without a double bond and neoechinulin A with one double bond (Δ10) as major products. Their analogues with a different number of prenyl moieties were detected as minor products. Neoechinulin B and analogues with two double bonds (Δ10,14) were not observed. Feeding experiments confirmed that the cytochrome P450 enzyme EchP450 only catalyzes the formation of the double bond between C10 and C11. Coincubation and substrate concentration dependent assays with the prenyltransferase EchPT2 revealed that the reversely C2-prenylated preechinulin without a double bond is a much better substrate than neoechinulin A. These results prove that preechinulin serves as a common substrate for the formation of echinulin by two regiospecific prenylation steps with EchPT2 or for EchP450 to introduce one double bond and subsequent prenylations with low regioselectivity.

Heterologous expression of secondary metabolite genes and gene clusters has been proven to be a successful strategy for identification of new natural products of cryptic or silent genes hidden in the genome sequences. It is also a useful tool to produce designed compounds by synthetic biology approaches. In this study, we demonstrate the potential usage of the gene locus pcr4401 in the fast-growing filamentous fungus Penicillium crustosum as an integration site for heterologous gene expression. The deduced polyketide synthase (PKS) Pcr4401 is involved in the dihydroxynaphthalene (DHN)-melanin pigment formation, and its deletion in P. crustosum PRB-2 led to an albino phenotype. Heterologous expression of pcr4401 in Aspergillus nidulans proved its function as the melanin precursor YWA1 synthase. To ensure gene expression after genomic integration and to easily identify the potential transformants by visualization, the gene locus of pcr4401 was chosen as an integration site. For heterologous expression in P. crustosum, the expression constructs were created by ligation-independent homologous recombination in Escherichia coli or Saccharomyces cerevisiae. A pyrG deficient strain was also created, so that both the pyrG and hph resistance gene can be used as selection markers. Successful expression in P. crustosum was demonstrated by using one uncharacterized PKS gene from Aspergillus and two from Penicillium strains. All three genes were successfully introduced, heterologously expressed, and their biosynthetic products elucidated. The results presented in this study demonstrated that P. crustosum can be used as a suitable host for heterologous expression of secondary metabolite genes.