Polyketide Biosynthesis Research Articles

Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides.

Aromatic polyketides are natural polyphenolic compounds with a broad spectrum of pharmacological activities. Production of those metabolites in the model organisms Escherichia coli and Saccharomyces cerevisiae has been limited by the extensive cellular engineering needed for the coordinated biosynthesis of polyketides and their precursors. In contrast, the amoeba Dictyostelium discoideum is a native producer of secondary metabolites and harbors a wide, but largely unexplored, repertoire of genes for the biosynthesis of polyketides and terpenoids. Here we present D. discoideum as an advantageous chassis for the production of aromatic polyketides. By expressing its native and cognate plant polyketide synthase genes in D. discoideum, we demonstrate production of phlorocaprophenone, methyl-olivetol, resveratrol and olivetolic acid (OA), which is the central intermediate in the biosynthesis of cannabinoids. To facilitate OA synthesis, we further engineered an amoeba/plant inter-kingdom hybrid enzyme that produced OA from primary metabolites in two enzymatic steps, providing a shortcut in a synthetic cannabinoid pathway using the D. discoideum host system.

Structural Insight into the Reaction Mechanism of Ketosynthase-Like Decarboxylase in a Loading Module of Modular Polyketide Synthases.

Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular polyketide synthases (PKSs) and are proposed to catalyze the decarboxylation of a malonyl or methylmalonyl unit for the construction of the PKS starter unit. KSQ domains have high sequence similarity to ketosynthase (KS) domains, which catalyze transacylation and decarboxylative condensation in polyketide and fatty acid biosynthesis, except that the catalytic Cys residue of KS domains is replaced by Gln in KSQ domains. Here, we present biochemical analyses of GfsA KSQ and CmiP4 KSQ, which are involved in the biosynthesis of FD-891 and cremimycin, respectively. In vitro analysis showed that these KSQ domains catalyze the decarboxylation of malonyl and methylmalonyl units. Furthermore, we determined the crystal structure of GfsA KSQ in complex with a malonyl thioester substrate analogue, which enabled identification of key amino acid residues involved in the decarboxylation reaction. The importance of these residues was confirmed by mutational analysis. On the basis of these findings, we propose a mechanism of the decarboxylation reaction catalyzed by GfsA KSQ. GfsA KSQ initiates decarboxylation by fixing the substrate in a suitable conformation for decarboxylation. The formation of enolate upon decarboxylation is assisted by two conserved threonine residues. Comparison of the structure of GfsA KSQ with those of KS domains suggests that the Gln residue in the active site of the KSQ domain mimics the acylated Cys residue in the active site of KS domains.

Isolation and Characterization of Bacillus velezensis Strain P2-1 for Biocontrol of Apple Postharvest Decay Caused by Botryosphaeria dothidea.

Botryosphaeria dothidea causes apple ring rot, which is among the most prevalent postharvest diseases of apples and causes significant economic loss during storage. In this study, we investigated the biocontrol activity and possible mechanism of Bacillus velezensis strain P2-1 isolated from apple branches against B. dothidea in postharvest apple fruit. The results showed strain P2-1, one of the 80 different endophytic bacterial strains from apple branches, exhibited strong inhibitory effects against B. dothidea growth and resulted in hyphal deformity. B. velezensis P2-1 treatment significantly reduced the ring rot caused by B. dothidea. Additionally, the supernatant of strain P2-1 exhibited antifungal activity against B. dothidea. Re-isolation assay indicated the capability of strain P2-1 to colonize and survive in apple fruit. PCR and qRT-PCR assays revealed that strain P2-1 harbored the gene clusters required for biosynthesis of antifungal lipopeptides and polyketides. Strain P2-1 treatment significantly enhanced the expression levels of pathogenesis-related genes (MdPR1 and MdPR5) but did not significantly affect apple fruit qualities (measured in fruit firmness, titratable acid, ascorbic acid, and soluble sugar). Thus, our results suggest that B. velezensis strain P2-1 is a biocontrol agent against B. dothidea-induced apple postharvest decay. It acts partially by inhibiting mycelial growth of B. dothidea, secreting antifungal substances, and inducing apple defense responses.

Engineered biosynthesis of alkyne-tagged polyketides.

Polyketides have demonstrated their significance as therapeutics, industrial products, pesticides, and biological probes following intense study over the past decades. Tagging polyketides with a bioorthogonal functionality enables various applications such as diversification, quantification, visualization and mode-of-action elucidation. The terminal alkyne moiety, as a small, stable and highly selective clickable functionality, is widely adopted in tagging natural products. De novo biosynthesis of alkyne-tagged polyketides offers the unique advantage of reducing the background from feeding the biorthogonal moiety itself, leading to the accomplishment of in situ generation of a clickable functionality for bioorthogonal reactions. Here, we introduce several engineering strategies to apply terminal alkyne biosynthetic machinery, represented by JamABC, which produces a short terminal alkyne-bearing fatty acyl chain on a carrier protein, to functions with different downstream polyketide synthases (PKSs). Successful results in engineering type III and type I PKSs provide engineering guidelines and strategies that are applicable to additional PKSs to produce targeted alkyne-tagged metabolites for chemical and biological applications.

Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis.

Review covering up to mid-2021The structure of polyketide and non-ribosomal peptide natural products is strongly influenced by how they are released from their biosynthetic enzymes. As such, Nature has evolved a diverse range of release mechanisms, leading to the formation of bioactive chemical scaffolds such as lactones, lactams, diketopiperazines, and tetronates. Here, we review the enzymes and mechanisms used for chain release in polyketide and non-ribosomal peptide biosynthesis, how these mechanisms affect natural product structure, and how they could be utilised to introduce structural diversity into the products of engineered biosynthetic pathways.

Enzymology of standalone elongating ketosynthases.

The β-ketoacyl-acyl carrier protein synthase, or ketosynthase (KS), catalyses carbon–carbon bond formation in fatty acid and polyketide biosynthesis via a decarboxylative Claisen-like condensation. In prokaryotes, standalone elongating KSs interact with the acyl carrier protein (ACP) which shuttles substrates to each partner enzyme in the elongation cycle for catalysis. Despite ongoing research for more than 50 years since KS was first identified in E. coli, the complex mechanism of KSs continues to be unravelled, including recent understanding of gating motifs, KS–ACP interactions, substrate recognition and delivery, and roles in unsaturated fatty acid biosynthesis. In this review, we summarize the latest studies, primarily conducted through structural biology and molecular probe design, that shed light on the emerging enzymology of standalone elongating KSs.

Biosynthesis of fungal polyketides by collaborating and trans-acting enzymes.

Covering: 1999 up to 2021Fungal polyketides encompass a range of structurally diverse molecules with a wide variety of biological activities. The giant multifunctional enzymes that synthesize polyketide backbones remain enigmatic, as do many of the tailoring enzymes involved in functional modifications. Recent advances in elucidating biosynthetic gene clusters (BGCs) have revealed numerous examples of fungal polyketide synthases that require the action of collaborating enzymes to synthesize the carbon backbone. This review will discuss collaborating and trans-acting enzymes involved in loading, extending, and releasing polyketide intermediates from fungal polyketide synthases, and additional modifications introduced by trans-acting enzymes demonstrating the complexity encountered when investigating natural product biosynthesis in fungi.

An isotopic probe to follow the stereochemical course of dehydratase reactions in polyketide and fatty acid biosynthesis

Four stereoisomeric and isotopically labelled probes that are suitable to easily follow the stereochemical course of dehydratases have been synthesised.

Delineating transcriptional crosstalk between Mycobacterium avium subsp. paratuberculosis and human THP-1 cells at the early stage of infection via dual RNA-seq analysis

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic debilitating disease in ruminants. To control this disease, it is crucial to understand immune evasion and the mechanism of persistence by analyzing the early phase interplays of the intracellular pathogens and their hosts. In the present study, host–pathogen interactions at the transcriptomic level were investigated in an in vitro macrophage infection model. When differentiated human THP-1 cells were infected with MAP, the expression of various genes associated with stress responses and metabolism was altered in both host and MAP at 3 h post-infection. MAP upregulates stress-responsive global gene regulators, such as two-component systems and sigma factors, in response to oxidative and cell wall stress. Downstream genes involved in type VII secretion systems, cell wall synthesis (polyketide biosynthesis proteins), and iron uptake were changed in response to the intracellular environment of macrophages. On the host side, upregulation of inflammatory cytokine genes was observed along with pattern recognition receptor genes. Notably, alterations in gene sets involved in arginine metabolism were observed in both the host and MAP, along with significant downregulation of NOS2 expression. These observations suggest that the utilization of metabolites such as arginine by intracellular MAP might affect host NO production. Our dual RNA-seq data can provide novel insights by capturing the global transcriptome with higher resolution, especially in MAP, thus enabling a more systematic understanding of host–pathogen interactions.

Fluvirucins B7-B10, new antifungal macrolactams from a marine-derived Nonomuraea sp. MYH522.

Marine rare actinomycetes are an important source of secondary metabolites. From a marine-derived actinomycete Nonomuraea sp. MYH522, four new macrolactams, fluvirucins B7–B10, together with known fluvirucin B6 were isolated. Their structures were determined based on comprehensive analysis of HRESIMS and NMR spectroscopic data as well as by comparing 13C NMR resonances and optical rotation values with those for related congeners. Fluvirucins are characterized by a 14-membered macrolactam attached by an aminosugar moiety. The discovery of fluvirucins B6–B10 enriched the N-acetylated derivatives of fluvirucins. The diverse alkyl substituents at C-2 and C-6 implied substrate promiscuity in fluvirucin polyketide biosynthesis. These compounds didn't exhibit any antibacterial or antifungal activities when used alone, which suggested the importance of the free amino group in the antimicrobial activity of fluvirucins. However, fluvirucins B6, B9, and B10 showed synergistic antifungal activity with fluconazole against fluconazole-resistant isolates of Candida albicans.

Engineering Modular Polyketide Biosynthesis in Streptomyces Using CRISPR/Cas: A Practical Guide.

The CRISPR/Cas system, which has been widely applied to organisms ranging from microbes to animals, is currently being adapted for use in Streptomyces bacteria. In this case, it is notably applied to rationally modify the biosynthetic pathways giving rise to the polyketide natural products, which are heavily exploited in the medical and agricultural arenas. Our aim here is to provide the potential user with a practical guide to exploit this approach for manipulating polyketide biosynthesis, by treating key experimental aspects including vector choice, design of the basic engineering components, and trouble-shooting.

An acetyltransferase controls the metabolic flux in rubromycin polyketide biosynthesis by direct modulation of redox tailoring enzymes.

The often complex control of bacterial natural product biosynthesis typically involves global and pathway-specific transcriptional regulators of gene expression, which often limits the yield of bioactive compounds under laboratory conditions. However, little is known about regulation mechanisms on the enzymatic level. Here, we report a novel regulatory principle for natural products involving a dedicated acetyltransferase, which modifies a redox-tailoring enzyme and thereby enables pathway furcation and alternating pharmacophore assembly in rubromycin polyketide biosynthesis. The rubromycins such as griseorhodin (grh) A are complex bioactive aromatic polyketides from Actinobacteria with a hallmark bisbenzannulated [5,6]-spiroketal pharmacophore that is mainly installed by two flavoprotein monooxygenases. First, GrhO5 converts the advanced precursor collinone into the [6,6]-spiroketal containing dihydrolenticulone, before GrhO6 effectuates a ring contraction to afford the [5,6]-spiroketal. Our results show that pharmacophore assembly in addition involves the acetyl-CoA-dependent acetyltransferase GrhJ that activates GrhO6 to allow the rapid generation and release of its labile product, which is subsequently sequestered by ketoreductase GrhO10 and converted into a stable intermediate. Consequently, the biosynthesis is directed to the generation of canonical rubromycins, while the alternative spontaneous [5,6]-spiroketal hydrolysis to a ring-opened pathway product is thwarted. Presumably, this allows the bacteria to rapidly adjust the biosynthesis of functionally distinct secondary metabolites depending on nutrient and precursor (i.e. acetyl-CoA) availability. Our study thus illustrates how natural product biosynthesis can be enzymatically regulated and provides new perspectives for the improvement of in vitro enzyme activities and natural product titers via biotechnological approaches.

Discovery of Mycothiogranaticins from Streptomyces vietnamensis GIMV4.0001 and the Regulatory Effect of Mycothiol on the Granaticin Biosynthesis.

Granaticins are benzoisochromanequinone polyketides with remarkable antibacterial and anticancer activities. Three sulfur-containing granaticin congeners, mycothiogranaticins A (1), B (2) and granaticin MA (3) were discovered from a granaticin-producing strain of Streptomyces vietnamensis GIMV4.0001. Two of them were structurally determined with mycothiol or N-acetylcysteine moieties and found to be bio-actively reluctant. Disruption of the mshA gene (SVTN_RS20640) that encodes the D-inositol-3-phosphate glycosyltransferase crucial for mycothiol biosynthesis, fully abolished the production of mycothiogranaticins. The result substantiated that the newly discovered mycothiogranaticins are consequences of the combination of the granaticin and mycothiol biosynthetic pathways. The overall granaticin production of the ΔmshA mutant strain was unexpectedly decreased by at least more than 50%, while similar production level of granaticins to that of the wild type strain was observed in an mycothiol-S transferase gene (SVTN_RS22215) disruptant Δmst. These results indicated that the mycothiol deficiency was responsible for the decreased production of granaticins. Mycothiol may positively regulate the biosynthesis of granaticin possibly by maintaining the cellular redox balance. To the best of our knowledge, this is the first report that mycothiol can not only be a direct building block of polyketides but also play a regulatory role in the polyketide biosynthesis.

Unexpected Role of a Short‐Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis

AbstractWe investigated the biosynthetic pathway of type II polyketide murayaquinone. The murayaquinone biosynthetic cluster contains genes for three putative short‐chain dehydrogenase/reductase family enzymes including MrqF and MrqH with known functions and MrqM with unclear function. We report the functional characterization of MrqM for its role in murayaquinone biosynthesis. Our gene deletion experiment and structural elucidation of the accumulated intermediates revealed that MrqM is related with the second polyketide ring cyclization, because the inactivation of mrqM resulted in the accumulation of an off‐pathway intermediate SEK43 and disrupted the second and third ring cyclization. Site‐directed mutagenesis studies showed that two conserved residues in MrqM and homologous proteins Y151 and K155 are essential for its activity. The previously proposed second/third ring cyclase, MrqD, might instead play another important role in the chain releasing step of the murayaquinone biosynthesis.

Unexpected Role of a Short-Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis.

We investigated the biosynthetic pathway of type II polyketide murayaquinone. The murayaquinone biosynthetic cluster contains genes for three putative short-chain dehydrogenase/reductase family enzymes including MrqF and MrqH with known functions and MrqM with unclear function. We report the functional characterization of MrqM for its role in murayaquinone biosynthesis. Our gene deletion experiment and structural elucidation of the accumulated intermediates revealed that MrqM is related with the second polyketide ring cyclization, because the inactivation of mrqM resulted in the accumulation of an off-pathway intermediate SEK43 and disrupted the second and third ring cyclization. Site-directed mutagenesis studies showed that two conserved residues in MrqM and homologous proteins Y151 and K155 are essential for its activity. The previously proposed second/third ring cyclase, MrqD, might instead play another important role in the chain releasing step of the murayaquinone biosynthesis.

Streptomyces tsukubensis VKM Aс-2618D-an Effective Producer of Tacrolimus.

The Streptomyces sp. VKM Ac-2618D strain has been identified, and its morphological and physiological features have been studied in relation to the production of the immunosuppressant tacrolimus. The phenotypic variability of the strain was analyzed, and a dissociant with a high level of tacrolimus production was selected. Based on a comprehensive study of morphological, physiological, and chemotaxonomic properties and on phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis are shown. The results broaden the understanding of the characteristics of polyketide biosynthesis and can be used in the development of technology for tacrolimus production.

Conservation of griseofulvin genes in the gsf gene cluster among fungal genomes.

The polyketide griseofulvin is a natural antifungal compound and research in griseofulvin has been key in establishing our current understanding of polyketide biosynthesis. Nevertheless, the griseofulvin gsf biosynthetic gene cluster (BGC) remains poorly understood in most fungal species, including Penicillium griseofulvum where griseofulvin was first isolated. To elucidate essential genes involved in griseofulvin biosynthesis, we performed third-generation sequencing to obtain the genome of P. griseofulvum strain D-756. Furthermore, we gathered publicly available genome of 11 other fungal species in which gsf gene cluster was identified. In a comparative genome analysis, we annotated and compared the gsf BGC of all 12 fungal genomes. Our findings show no gene rearrangements at the gsf BGC. Furthermore, seven gsf genes are conserved by most genomes surveyed whereas the remaining six were poorly conserved. This study provides new insights into differences between gsf BGC and suggests that seven gsf genes are essential in griseofulvin production.

Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis.

The medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]‐spiroketal pharmacophore that was recently shown to be assembled by flavin‐dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]‐spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon‐carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals.

Catalytic Control of Spiroketal Formation in Rubromycin Polyketide Biosynthesis

AbstractThe medically important bacterial aromatic polyketide natural products typically feature a planar, polycyclic core structure. An exception is found for the rubromycins, whose backbones are disrupted by a bisbenzannulated [5,6]‐spiroketal pharmacophore that was recently shown to be assembled by flavin‐dependent enzymes. In particular, a flavoprotein monooxygenase proved critical for the drastic oxidative rearrangement of a pentangular precursor and the installment of an intermediate [6,6]‐spiroketal moiety. Here we provide structural and mechanistic insights into the control of catalysis by this spiroketal synthase, which fulfills several important functions as reductase, monooxygenase, and presumably oxidase. The enzyme hereby tightly controls the redox state of the substrate to counteract shunt product formation, while also steering the cleavage of three carbon‐carbon bonds. Our work illustrates an exceptional strategy for the biosynthesis of stable chroman spiroketals.

Visualizing transiently associated catalytic domains in assembly-line biosynthesis using cryo-electron microscopy

While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein–protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure–function relationships. Here, we outline examples in which transient protein–protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein–protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein–protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.