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Journal of Biological Engineering May 2024Cladosporium phlei is a phytopathogenic fungus that produces a pigment called phleichrome. This fungal perylenequinone plays an important role in the production of a...
BACKGROUND
Cladosporium phlei is a phytopathogenic fungus that produces a pigment called phleichrome. This fungal perylenequinone plays an important role in the production of a photosensitizer that is a necessary component of photodynamic therapy. We applied synthetic biology to produce phleichrome using Saccharomyces cerevisiae.
RESULTS
The gene Cppks1, which encodes a non-reducing polyketide synthase (NR-PKS) responsible for the biosynthesis of phleichrome in C. phlei, was cloned into a yeast episomal vector and used to transform S. cerevisiae. In addition, a gene encoding a phosphopantetheinyl transferase (PPTase) of Aspergillus nidulans was cloned into a yeast integrative vector and also introduced into S. cerevisiae for the enzymatic activation of the protein product of Cppks1. Co-transformed yeasts were screened on a leucine/uracil-deficient selective medium and the presence of both integrative as well as episomal recombinant plasmids in the yeast were confirmed by colony PCR. The episomal vector for Cppks1 expression was so dramatically unstable during cultivation that most cells lost their episomal vector rapidly in nonselective media. This loss was also observed to a less degree in selective media. This data strongly suggests that the presence of the Cppks1 gene exerts a significant detrimental effect on the growth of transformed yeast cells and that selection pressure is required to maintain the Cppks1-expressing vector. The co-transformants on the selective medium showed the distinctive changes in pigmentation after a period of prolonged cultivation at 20 °C and 25 °C, but not at 30 °C. Furthermore, thin layer chromatography (TLC) revealed the presence of a spot corresponding with the purified phleichrome in the extract from the cells of the co-transformants. Liquid chromatography (LC/MS/MS) verified that the newly expressed pigment was indeed phleichrome.
CONCLUSION
Our results indicate that metabolic engineering by multiple gene expression is possible and capable of producing fungal pigment phleichrome in S. cerevisiae. This result adds to our understanding of the characteristics of fungal PKS genes, which exhibit complex structures and diverse biological activities.
PubMed: 38741106
DOI: 10.1186/s13036-024-00429-0 -
ACS Omega May 2024is a fungal pathogen capable of producing two mycotoxins of concern, ochratoxin A (OTA) and citrinin (CIT). The production profile of these two mycotoxins is not well...
is a fungal pathogen capable of producing two mycotoxins of concern, ochratoxin A (OTA) and citrinin (CIT). The production profile of these two mycotoxins is not well understood but could help mitigate co-contamination in the food supply. As such, the production of OTA and CIT from DAOMC 242724 was investigated under different growing conditions in liquid culture. We found that among the different liquid media chosen, liquid YES (yeast extract sucrose) medium induced the highest production of both OTA and CIT, when DAOMC 242724 was cultured in stationary mode. Shake culture significantly reduced the amounts of OTA and CIT produced. Among all culture conditions tested, far greater amounts of CIT were produced compared to OTA. Consequently, upon transcriptomic data analysis, a statistically significant increase in the expression of CIT biosynthetic genes was easier to detect than the expression of OTA biosynthetic genes. Our study also revealed that the putative biosynthetic gene clusters of OTA and CIT in DAOMC 242724 are likely distinct from each other. It appears that despite sharing a highly similar structure, the isocoumarin rings of OTA and CIT are each assembled by a specialized polyketide synthase enzyme. Our data identified a putative nonreducing polyketide synthase responsible for assembling the carbo-skeleton of CIT. In contrast, a highly reducing polyketide synthase appears to be involved in the biosynthesis of OTA.
PubMed: 38737015
DOI: 10.1021/acsomega.4c00874 -
Applied Microbiology and Biotechnology May 2024Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary...
Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.
Topics: Chaetomium; Multigene Family; Secondary Metabolism; Histone Deacetylases; Polyketides; Gene Deletion; Gene Expression Regulation, Fungal; Polyketide Synthases; Biosynthetic Pathways; Epigenesis, Genetic
PubMed: 38734756
DOI: 10.1007/s00253-024-13173-8 -
Molecules (Basel, Switzerland) Apr 2024Chalkophomycin is a novel chalkophore with antibiotic activities isolated from sp. CB00271, while its potential in studying cellular copper homeostasis makes it an...
Chalkophomycin is a novel chalkophore with antibiotic activities isolated from sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of -hydroxylpyrrole, 2-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the biosynthetic gene cluster from . sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 -type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.
Topics: Multigene Family; Streptomyces; Polyketide Synthases; Peptide Synthases; Bacterial Proteins
PubMed: 38731473
DOI: 10.3390/molecules29091982 -
Nature May 2024QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex...
QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.
Topics: Adjuvants, Immunologic; Biosynthetic Pathways; Drug Design; Enzymes; Metabolic Engineering; Plants; Saccharomyces cerevisiae; Saponins; Structure-Activity Relationship
PubMed: 38720067
DOI: 10.1038/s41586-024-07345-9 -
Applied Microbiology and Biotechnology May 2024Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic...
Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.
Topics: Antarctic Regions; Multigene Family; Phylogeny; Soil Microbiology; Geologic Sediments; Secondary Metabolism; Actinobacteria; Genome, Bacterial; Biotechnology; Biosynthetic Pathways; Peptide Synthases; Polyketide Synthases
PubMed: 38717668
DOI: 10.1007/s00253-024-13154-x -
BMC Biology May 2024Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan...
BACKGROUND
Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited.
RESULTS
In this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and L-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria.
CONCLUSIONS
Our results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms.
Topics: Ciliophora; Genomics; Genome, Protozoan; Transcriptome
PubMed: 38715037
DOI: 10.1186/s12915-024-01904-2 -
Microbiology Spectrum Jun 2024The cyanosiphophage Mic1 specifically infects the bloom-forming FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded...
UNLABELLED
The cyanosiphophage Mic1 specifically infects the bloom-forming FACHB 1339 from Lake Chaohu, China. Previous genomic analysis showed that its 92,627 bp double-stranded DNA genome consists of 98 putative open reading frames, 63% of which are of unknown function. Here, we investigated the transcriptome dynamics of Mic1 and its host using RNA sequencing. In the early, middle, and late phases of the 10 h lytic cycle, the Mic1 genes are sequentially expressed and could be further temporally grouped into two distinct clusters in each phase. Notably, six early genes, including that encodes a TnpB-like transposase, immediately reach the highest transcriptional level in half an hour, representing a pioneer cluster that rapidly regulates and redirects host metabolism toward the phage. An in-depth analysis of the host transcriptomic profile in response to Mic1 infection revealed significant upregulation of a polyketide synthase pathway and a type III-B CRISPR system, accompanied by moderate downregulation of the photosynthesis and key metabolism pathways. The constant increase of phage transcripts and relatively low replacement rate over the host transcripts indicated that Mic1 utilizes a unique strategy to gradually take over a small portion of host metabolism pathways after infection. In addition, genomic analysis of a less-infective Mic1 and a Mic1-resistant host strain further confirmed their dynamic interplay and coevolution via the frequent horizontal gene transfer. These findings provide insights into the mutual benefit and symbiosis of the highly polymorphic cyanobacteria and cyanophages.
IMPORTANCE
The highly polymorphic is one of the predominant bloom-forming cyanobacteria in eutrophic freshwater bodies and is infected by diverse and abundant cyanophages. The presence of a large number of defense systems in genome suggests a dynamic interplay and coevolution with the cyanophages. In this study, we investigated the temporal gene expression pattern of Mic1 after infection and the corresponding transcriptional responses of its host. Moreover, the identification of a less-infective Mic1 and a Mic1-resistant host strain provided the evolved genes in the phage-host coevolution during the multiple-generation cultivation in the laboratory. Our findings enrich the knowledge on the interplay and coevolution of and its cyanophages and lay the foundation for the future application of cyanophage as a potential eco-friendly and bio-safe agent in controlling the succession of harmful cyanobacterial blooms.
Topics: Microcystis; Bacteriophages; China; Transcriptome; Lakes; Genome, Viral; Evolution, Molecular
PubMed: 38695606
DOI: 10.1128/spectrum.00298-24 -
Acta Biochimica Et Biophysica Sinica Jun 2024Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of... (Review)
Review
Biological control of pests and pathogens has attracted much attention due to its green, safe and effective characteristics. However, it faces the dilemma of insignificant effects in large-scale applications. Therefore, an in-depth exploration of the metabolic potential of biocontrol fungi based on big omics data is crucial for a comprehensive and systematic understanding of the specific modes of action operated by various biocontrol fungi. This article analyzes the preferences for extracellular carbon and nitrogen source degradation, secondary metabolites (nonribosomal peptides, polyketide synthases) and their product characteristics and the conversion relationship between extracellular primary metabolism and intracellular secondary metabolism for eight different filamentous fungi with characteristics appropriate for the biological control of bacterial pathogens and phytopathogenic nematodes. Further clarification is provided that , encoding a large number of hydrolase enzymes capable of degrading pathogen protection barrier, can be directly applied in the field as a predatory biocontrol fungus, whereas , as an antibiosis-active biocontrol control fungus, can form dominant strains on preferred substrates and produce a large number of secondary metabolites to achieve antibacterial effects. By clarifying the levels of biological control achievable by different biocontrol fungi, we provide a theoretical foundation for their application to cropping habitats.
Topics: Fungi; Secondary Metabolism; Carbon; Biological Control Agents; Pest Control, Biological; Nitrogen; Animals; Metabolomics
PubMed: 38686460
DOI: 10.3724/abbs.2024056 -
Microbial Biotechnology May 2024Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal...
Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 μg/mL - 4.36-fold higher than WT value (48.9 μg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 μg/mL. Overexpression of fatty acid β-oxidation pathway genes in the strain further increased B1a titer to 452.8 μg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 μg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.
Topics: Polyketide Synthases; Metabolic Engineering; Acyl Coenzyme A; Streptomyces; Metabolic Networks and Pathways; Ivermectin; Bacterial Proteins
PubMed: 38683675
DOI: 10.1111/1751-7915.14470