Medicinal plants have been used by mankind since ancient times, and many bioactive plant secondary metabolites are applied nowadays both directly as drugs, and as raw materials for semi-synthetic ...modifications. However, the structural complexity often thwarts cost-efficient chemical synthesis, and the usually low content in the native plant necessitates the processing of large amounts of field-cultivated raw material. The biotechnological manufacturing of such compounds offers a number of advantages like predictable, stable, and year-round sustainable production, scalability, and easier extraction and purification. Plant cell and tissue culture represents one possible alternative to the extraction of phytochemicals from plant material. Although a broad commercialization of such processes has not yet occurred, ongoing research indicates that plant in vitro systems such as cell suspension cultures, organ cultures, and transgenic hairy roots hold a promising potential as sources for bioactive compounds. Progress in the areas of biosynthetic pathway elucidation and genetic manipulation has expanded the possibilities to utilize plant metabolic engineering and heterologous production in microorganisms. This review aims to summarize recent advances in the in vitro production of high-value plant secondary metabolites of medicinal importance.
Key points
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Bioactive plant secondary metabolites are important for current and future use in medicine
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In vitro production is a sustainable alternative to extraction from plants or costly chemical synthesis
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Current research addresses plant cell and tissue culture, metabolic engineering, and heterologous production
Graphical abstract
Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinomycetes from terrestrial sources have been studied and ...screened since the 1950s, yielding many important anti-infective and anti-cancer drugs. However, frequent re-discovery of the same compounds in terrestrial actinomycetes have made them less attractive for screening programs in the recent years. At the same time, actinomycetes isolated from the marine environment currently receive considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. This review highlights achievements and challenges in the isolation of marine actinomycetes, some examples of bioactive metabolites identified by conventional screening, and presents new developments in the field of genome mining and heterologous expression of biosynthetic gene clusters leading to the discovery of novel compounds.
Summary
For over seven decades, bacteria served as a valuable source of bioactive natural products some of which were eventually developed into drugs to treat infections, cancer and immune ...system‐related diseases. Traditionally, novel compounds produced by bacteria were discovered via conventional bioprospecting based on isolation of potential producers and screening their extracts in a variety of bioassays. Over time, most of the natural products identifiable by this approach were discovered, and the pipeline for new drugs based on bacterially produced metabolites started to run dry. This mini‐review highlights recent developments in bacterial bioprospecting for novel compounds that are based on several out‐of‐the‐box approaches, including the following: (i) targeting bacterial species previously unknown to produce any bioactive natural products, (ii) exploring non‐traditional environmental niches and methods for isolation of bacteria and (iii) various types of ‘genome mining’ aimed at unravelling genetic potential of bacteria to produce secondary metabolites. All these approaches have already yielded a number of novel bioactive compounds and, if used wisely, will soon revitalize drug discovery pipeline based on bacterial natural products.
This mini‐review highlights recent developments in bacterial bioprospecting for novel compounds based on targeting bacterial species previously unknown to produce any bioactive natural products, exploring non‐traditional environmental niches and methods for isolation of bacteria, and various types of ‘genome mining’ aimed at unraveling genetic potential of bacteria to produce secondary metabolites.
Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two ...vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the "capture" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Among the plant‐associated microorganisms, the so‐called endophytes continue to attract much attention because of their ability not only to protect host plants from biotic and abiotic stress factors, ...but also the potential to produce bioactive secondary metabolites. The latter property can elicit growth‐promoting effects on plants, as well as boost the production of plant‐specific secondary metabolites with valuable pharmacological properties. In addition, endophyte‐derived secondary metabolites may be a rich source for the discovery of drugs to treat various diseases, including infections and cancer. However, the full potential of endophytes to produce bioactive secondary metabolites is often not revealed upon conventional cultivation in the laboratory. New advances in genomics and metabolic engineering offer exciting opportunities for the exploration and exploitation of endophytes' biosynthetic potential. This review focuses on bacterial endophytes of medicinal plants, some of their secondary metabolites and recent advances in deciphering their biosynthesis. The latter may assist in genetic engineering efforts aimed at the discovery of novel bioactive compounds with the potential to be developed into drugs.
Bacteria dwelling within tissues of medicinal plants, the so‐called endophytes, represent a unique source of secondary metabolites with diverse biological activities. Modern approaches such as “genome mining” and metabolic engineering provide new opportunities for revealing the biosynthetic potential of bacterial endophytes and assisting in identification of novel compounds which may fuel drug discovery pipelines.
Marine actinobacteria are drawing more and more attention as a promising source of new natural products. Here we report isolation, genome sequencing and metabolic profiling of new strain Streptomyces ...sp. MP131-18 isolated from marine sediment sample collected in the Trondheim Fjord, Norway. The 16S rRNA and multilocus phylogenetic analysis showed that MP131-18 belongs to the genus Streptomyces. The genome of MP131-18 isolate was sequenced, and 36 gene clusters involved in the biosynthesis of 18 different types of secondary metabolites were predicted using antiSMASH analysis. The combined genomics-metabolics profiling of the strain led to the identification of several new biologically active compounds. As a result, the family of bisindole pyrroles spiroindimicins was extended with two new members, spiroindimicins E and F. Furthermore, prediction of the biosynthetic pathway for unusual α-pyrone lagunapyrone isolated from MP131-18 resulted in foresight and identification of two new compounds of this family - lagunapyrones D and E. The diversity of identified and predicted compounds from Streptomyces sp. MP131-18 demonstrates that marine-derived actinomycetes are not only a promising source of new natural products, but also represent a valuable pool of genes for combinatorial biosynthesis of secondary metabolites.
Abstract
DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, ...the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these ‘mutators’ was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10–5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.
Ethanol shock significantly affects expression of over 1200 genes in Streptomyces venezuelae NRRL B-65,442, including those involved in secondary metabolite biosynthesis and a cryptic gene pepX, ...which encodes a 19-amino acid peptide with an unknown function. To establish a possible correlation between the PepX peptide and secondary metabolism in S. venezuelae, its gene was deleted, followed by analyses of the transcriptome and secondary metabolome of the mutant. Although the secondary metabolome of the pepX mutant was not strongly affected, pepX deletion, similar to ethanol shock, mostly resulted in downregulated expression of secondary metabolite biosynthesis gene clusters (BGCs). At the same time, there was a reverse correlation between the expression of certain extracytoplasmic function sigma factors (ECFs) and several BGCs. Individual deletions of three selected ECF-coding genes conserved in Streptomyces that were upregulated upon both pepX deletion and ethanol shock, had a profound positive effect on the expression of BGCs, which also correlated with the overproduction of specific secondary metabolites. Deletion of one such ECF-coding gene in a marine sponge-derived Streptomyces sp. also significantly altered the secondary metabolite profile, suggesting an important role of this ECF in the regulation of secondary metabolism. These findings pave the way for the activation or upregulation of BGCs in Streptomyces bacteria harboring genes for ECFs homologous to those identified in this study, hereby assisting in the discovery of novel bioactive secondary metabolites.
Bacterial endophytes dwelling in medicinal plants represent an as yet underexplored source of bioactive natural products with the potential to be developed into drugs against various human diseases. ...For the first time, several Streptomyces spp. were isolated from the rare and endangered traditional medicinal plant Leontopodium nivale ssp. alpinum , also known as Edelweiss. In the search for novel natural products, nine endophytic Streptomyces spp. from Edelweiss were investigated via genome sequencing and analysis, followed by fermentation in different media and investigation of secondary metabolomes. A total of 214 secondary metabolite biosynthetic gene clusters (BGCs), of which 35 are presumably unique, were identified by the bioinformatics tool antiSMASH in the genomes of these isolates. LC-MS analyses of the secondary metabolomes of these isolates revealed their potential to produce both known and presumably novel secondary metabolites, whereby most of the identified molecules could be linked to their cognate BGCs. This work sets the stage for further investigation of endophytic streptomycetes from Edelweiss aimed at the discovery and characterization of novel bioactive natural products.
Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C₁₀H₂₂) to that of tetracontane (C₄₀H₈₂) as a sole carbon source. Two genes ...encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30°C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C₃₂H₆₆) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains.
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