Combinatorialization of Fungal Polyketide Synthase–Peptide Synthetase Hybrid Proteins

The programming of the fungal polyketide synthase (PKS) is quite complex, with a simple domain architecture leading to elaborate products. An additional level of complexity has been found within PKS-based pathways where the PKS is fused to a single module nonribosomal peptide synthetase (NRPS) to synthesize polyketides conjugated to amino acids. Here, we sought to understand the communication between these modules that enable correct formation of polyketide-peptide hybrid products. To do so, we fused together the genes that are responsible for forming five highly chemically diverse fungal natural products in a total of 57 different combinations, comprising 34 distinct module swaps. Gene fusions were formed with the idea of testing the connection and compatibility of the PKS and NRPS modules mediated by the acyl carrier protein (ACP), condensation (C) and ketoreductase (KR) domains. The resulting recombinant gene fusions were analyzed in a high-yielding expression platform to avail six new compounds, including the first successful fusion between a PKS and NRPS that make highly divergent products, and four previously reported molecules. Our results show that C domains are highly selective for a subset of substrates. We discovered that within the highly reducing (hr) PKS class, noncognate ACPs of closely related members complement PKS function. We intercepted a pre-Diels–Alder intermediate in lovastatin synthesis for the first time, shedding light on this canonical fungal biochemical reaction. The results of these experiments provide a set of ground rules for the successful engineering of hr-PKS and PKS-NRPS products in fungi

Biosynthesis of the Tetramic Acids Sch210971 and Sch210972

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (<b>1a</b>) and Sch210972 (<b>1b</b>) from <i>Hapsidospora irregularis</i>, was characterized by reconstitution and heterologous expression in <i>Fusarium heterosporum</i>. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor

Two Related Pyrrolidinedione Synthetase Loci in <i>Fusarium heterosporum</i> ATCC 74349 Produce Divergent Metabolites

Equisetin synthetase (EqiS), from the filamentous fungus <i>Fusarium heterosporum</i> ATCC 74349, was initially assigned on the basis of genetic knockout and expression analysis. Increasing inconsistencies in experimental results led us to question this assignment. Here, we sequenced the <i>F. heterosporum</i> genome, revealing two hybrid polyketide-peptide proteins that were candidates for the equisetin synthetase. The surrounding genes in both clusters had the needed auxiliary genes that might be responsible for producing equisetin. Genetic mutation, biochemical analysis, and recombinant expression in the fungus enabled us to show that the initially assigned EqiS does not produce equisetin but instead produces a related 2,4-pyrrolidinedione, fusaridione A, that was previously unknown. Fusaridione A is methylated in the 3-position of the pyrrolidinedione, which has not otherwise been found in natural products, leading to spontaneous reverse-Dieckmann reactions. A newly described gene cluster, <i>eqx</i>, is responsible for producing equisetin

Biosynthesis of the Tetramic Acids Sch210971 and Sch210972

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (<b>1a</b>) and Sch210972 (<b>1b</b>) from <i>Hapsidospora irregularis</i>, was characterized by reconstitution and heterologous expression in <i>Fusarium heterosporum</i>. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor

Biosynthesis of para-Cyclophane-Containing Hirsutellone Family of Fungal Natural Products

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene

Native Promoter Strategy for High-Yielding Synthesis and Engineering of Fungal Secondary Metabolites

Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus <i>Fusarium heterosporum</i> natively synthesizes the polyketide equisetin at >2 g L^–1 in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native <i>eqxS</i> promoter and <i>eqxR</i> regulator in <i>F. heterosporum</i>, synthesizing heterologous natural products in yields of ∼1 g L^–1. As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L^–1, leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites

An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites

An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites

Identification of Cyclic Depsipeptides and Their Dedicated Synthetase from <i>Hapsidospora irregularis</i>

Seven cyclic depsipeptides were isolated from <i>Hapsidospora irregularis</i> and structurally characterized as the calcium channel blocker leualacin and six new analogues based on the NMR and HRESIMS data. These new compounds were named leualacins B–G. The absolute configurations of the amino acids and 2-hydroxyisocaproic acids were determined by recording the optical rotation values. Biological studies showed that calcium influx elicited by leualacin F in primary human lobar bronchial epithelial cells involves the TRPA1 channel. Through genome sequencing and targeted gene disruption, a noniterative nonribosomal peptide synthetase was found to be involved in the biosynthesis of leualacin. A comparison of the structures of leualacin and its analogues indicated that the A₂ and A₄ domains of the leualacin synthetase are substrate specific, while A₁, A₃, and A₅ can accept alternative precursors to yield new molecules

Isolation of Pyrrolocins A–C: <i>cis</i>- and <i>trans</i>-Decalin Tetramic Acid Antibiotics from an Endophytic Fungal-Derived Pathway

Three new decalin-type tetramic acid analogues, pyrrolocins A (<b>1</b>), B (<b>2</b>), and C (<b>3</b>), were defined as products of a metabolic pathway from a fern endophyte, NRRL 50135, from Papua New Guinea. NRRL 50135 initially produced <b>1</b> but ceased its production before chemical or biological evaluation could be completed. Upon transfer of the biosynthetic pathway to a model host, <b>1</b>–<b>3</b> were produced. All three compounds are structurally related to equisetin-type compounds, with <b>1</b> and <b>3</b> having a <i>trans</i>-decalin ring system, while <b>2</b> has a <i>cis</i>-fused decalin. All were active against <i>Mycobacterium tuberculosis</i>, with the <i>trans-</i>decalin analogues <b>1</b> and <b>3</b> exhibiting lower MICs than the <i>cis</i>-decalin analogue <b>2</b>. Here we report the isolation, structure elucidation, and antimycobacterial activities of <b>1</b>–<b>3</b> from the recombinant expression as well as the isolation of <b>1</b> from the wild-type fungus NRRL 50135