The potential of amoeba-based processes for natural product syntheses

Kufs JE, Reimer C, Stallforth P, Hillmann F, Regestein L. The potential of amoeba-based processes for natural product syntheses. Current Opinion in Biotechnology. 2022;77: 102766.The identification of novel platform organisms for the production and discovery of small molecules is of high interest for the pharmaceutical industry. In particular, the structural complexity of most natural products with therapeutic potential restricts an industrial production since chemical syntheses often require complex multistep routes. The amoeba Dictyostelium discoideum can be easily cultivated in bioreactors due to its planktonic growth behavior and contains numerous polyketide and terpene synthase genes with only a few compounds being already elucidated. Hence, the amoeba both bears a wealth of hidden natural products and allows for the development of new bioprocesses for existing pharmaceuticals. In this mini review, we present D. discoideum as a novel platform for the production of complex secondary metabolites and discuss its suitability for industrial processes. We also provide initial insights into future bioprocesses, both involving bacterial coculture setups and for the production of plant-based pharmaceuticals

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

Reimer C, Kufs JE, Rautschek J, Regestein L, Valiante V, Hillmann F. Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides. Nature Biotechnology. 2022;40(5):751-758.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

Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid

Kufs JE, Reimer C, Steyer E, Valiante V, Hillmann F, Regestein L. Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid. Microbial Cell Factories. 2022;21(1): 217.**Background** The availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoebaDictyostelium discoideumrepresents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineeredD. discoideumstrain. **Results** In this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (εmax) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerableεmaxof 3.9 W/kg forD. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 µg/L, a productivity of 0.04 µg/L/h and a yield of 0.56 µg/g glucose. **Conclusion** We developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level forD. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass

Yellow polyketide pigment suppresses premature hatching in social amoeba

Günther M, Reimer C, Herbst R, et al. Yellow polyketide pigment suppresses premature hatching in social amoeba. Proceedings of the National Academy of Sciences. 2022;119(43).Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoebaDictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor

Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites

Efeito da radiação gama em soluções de aflatoxinas

Os fungos filamentosos, seres ubíquos na natureza, são muitas vezes parasitas de produtos alimentares, nomeadamente produtos agrícolas. A sua presença, embora encarada como natural, poderá não ser inócua, uma vez que alguns fungos são capazes de produzir compostos tóxicos, nomeadamente micotoxinas (e.g., aflatoxinas). As aflatoxinas, metabolito secundário produzido por Aspergillus flavus e Aspergillus parasiticus, são altamente tóxicas, mutagénicas e carcinogénicas. Por forma a garantir a segurança alimentar no que se refere à presença destes metabolitos em alimentos várias alternativas têm vindo a ser testadas. A irradiação de alimentos (e.g, radiação gama) é uma dessas alternativas [1]; no entanto, a identificação dos produtos de degradação das micotoxinas pela utilização desta tecnologia, assim como a avaliação da sua toxicidade, está por realizar. Com o trabalho realizado pretendeu-se verificar qual o efeito da radiação gama em soluções de aflatoxinas (B1, B2, G1 e G2). Para tal, soluções de aflatoxinas (B1, B2, G1 e G2) foram submetidas à radiação gama nas seguintes doses: 0, 0,5; 1,0; 3,0; 6,0 kGy. Após irradiação, a quantificação de aflatoxinas e a deteção de produtos de degradação foi efetuada por cromatografia líquida, com deteção por fluorescência e derivatização pós-coluna. Os resultados obtidos mostraram que existe uma diminuição da concentração de aflatoxinas nas soluções irradiadas, embora a sensibilidade de cada toxina à irradiação seja diferente. Para a dose mais elevada de radiação testada (6,0 kGy) a diminuição foi superior a 80 % para G1, G2 e B1. Além disto, em todas as amostras irradiadas, verifica-se a formação de compostos de degradação de aflatoxinas. A concentração destes compostos aumenta até à dose de 1,0 kGy, diminuindo nas restantes doses. O estudo da estrutura e da toxicidade destes compostos tem que ser levados a cabo de modo a ponderar a eficácia desta metodologia no controlo de micotoxinas em alimentos

DC subset-specific induction of T cell responses upon antigen uptake via Fc gamma receptors in vivo

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fc gamma receptors (Fc gamma Rs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory Fc gamma R in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating Fc gamma RIV is able to induce superior CD4(+) and CD8(+) T cell responses. Of note, this effect was independent of Fc gamma R intrinsic activating signaling pathways. Moreover, despite the expression of Fc gamma RIV on both conventional splenic DC subsets, the induction of CD8(+) T cell responses was largely dependent on CD11c(+) CD8(+) DCs, whereas CD11c(+) CD8-DCs were critical for priming CD4(+) T cell responses

DC subset-specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4(+) and CD8(+) T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8(+) T cell responses was largely dependent on CD11c(+)CD8(+) DCs, whereas CD11c(+)CD8(-) DCs were critical for priming CD4(+) T cell responses

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes

10.1038/NMICROBIOL.2016.166NATURE MICROBIOLOGY11

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.SCOPUS: ar.jinfo:eu-repo/semantics/publishe