Complete Genome Sequences of Five Burkholderia Strains with Biocontrol Activity against Various Lettuce Pathogens

Numerous bacterial strains from the Burkholderia cepacia complex display biocontrol activity. Here, we report the complete genome sequences of five Burkholderia strains isolated from soil. Biosynthetic gene clusters responsible for the production of antimicrobial compounds were found in the genome of these strains, which display biocontrol activity against various lettuce pathogens.

Horizontal Transfers Lead to the Birth of Momilactone Biosynthetic Gene Clusters in Grass

Momilactone A, an important plant labdane-related diterpenoid, functions as a phytoalexin against pathogens and an allelochemical against neighboring plants. The genes involved in biosynthesis of momilactone A are found in clusters, i.e., MABGCs (Momilactone A biosynthetic gene clusters), in the rice and barnyardgrass genomes. How MABGCs originate and evolve is still not clear. Here, we integrated results from comprehensive phylogeny and comparative genomic analyses of the core genes of MABGC-like clusters and MABGCs in 40 monocot plant genomes, providing convincing evidence for the birth and evolution of MABGCs in grass species. The MABGCs found in the PACMAD clade of the core grass lineage (including Panicoideae and Chloridoideae) originated from a MABGC-like cluster in Triticeae (BOP clade) via horizontal gene transfer (HGT) and followed by recruitment of MAS and CYP76L1 genes. The MABGCs in Oryzoideae originated from PACMAD through another HGT event and lost CYP76L1 afterwards. The Oryza MABGC and another Oryza diterpenoid cluster c2BGC are two distinct clusters, with the latter being originated from gene duplication and relocation within Oryzoideae. Further comparison of the expression patterns of the MABGC genes between rice and barnyardgrass in response to pathogen infection and allelopathy provides novel insights into the functional innovation of MABGCs in plants. Our results demonstrate HGT-mediated origination of MABGCs in grass and shed lights into the evolutionary innovation and optimization of plant biosynthetic pathways.

FunOrder 2.0 – a fully automated method for the identification of co-evolved genes

Coevolution is an important biological process that shapes interacting species or even proteins – may it be physically interacting proteins or consecutive enzymes in a metabolic pathway. The detection of co-evolved proteins will contribute to a better understanding of biological systems. Previously, we developed a semi-automated method, termed FunOrder, for the detection of co-evolved genes from an input gene or protein set. We demonstrated the usability and applicability of FunOrder by identifying essential genes in biosynthetic gene clusters from different ascomycetes. A major drawback of this original method was the need for a manual assessment, which may create a user bias and prevents a high-throughput application. Here we present a fully automated version of this method termed FunOrder 2.0. To fully automatize the method, we used several mathematical indices to determine the optimal number of clusters in the FunOrder output, and a subsequent k-means clustering based on the first three principal components of a principal component analysis of the FunOrder output. Further, we replaced the BLAST with the DIAMOND tool, which enhanced speed and allows the future integration of larger proteome databases. The introduced changes slightly decreased the sensitivity of this method, which is outweighed by enhanced overall speed and specificity. Additionally, the changes lay the foundation for future high-throughput applications of FunOrder 2.0 in different phyla to solve different biological problems.

Draft Genome Sequence of Streptomyces albidoflavus 09MW18-IS, Cultivated from the Atlantic Ocean off the Coast of Virginia

We report a draft genome sequence for Streptomyces albidoflavus strain 09MW18-IS, isolated from the Atlantic slope off the coast of Virginia. The whole-genome sequence will provide novel insights into biosynthetic gene clusters and ecological adaptation in an oligotrophic environment.

A Glossary for Chemical Approaches towards Unlocking the Trove of Metabolic Treasures in Actinomycetes

Actinobacterial natural products showed a critical basis for the discovery of new antibiotics as well as other lead secondary metabolites. Varied environmental and physiological signals touch the antibiotic machinery that faced a serious decline in the last decades. The reason was exposed by genomic sequencing data, which revealed that Actinomycetes harbor a large portion of silent biosynthetic gene clusters in their genomes that encrypt for secondary metabolites. These gene clusters are linked with a great reservoir of yet unknown molecules, and arranging them is considered a major challenge for biotechnology approaches. In the present paper, we discuss the recent strategies that have been taken to augment the yield of secondary metabolites via awakening these cryptic genes in Actinomycetes with emphasis on chemical signaling molecules used to induce the antibiotics biosynthesis. The rationale, types, applications and mechanisms are discussed in detail, to reveal the productive path for the unearthing of new metabolites, covering the literature until the end of 2020.

Diverse Protein Architectures and α-N-Methylation Patterns Define Split Borosin RiPP Biosynthetic Gene Clusters

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylations installed on the peptide backbone that impart unique properties like proteolytic stability to these natural products. The borosin RiPP family was initially reported only in fungi until our recent discovery and characterization of a Type IV split borosin system in the metal-respiring bacterium Shewanella oneidensis. Here, we used hidden Markov models and sequence similarity networks to identify over 1,600 putative pathways that show split borosin biosynthetic gene clusters are widespread in bacteria. Noteworthy differences in precursor and α-N-methyltransferase open reading frame sizes, architectures, and core peptide properties allow further subdivision of the borosin family into six additional discrete structural types, of which five have been validated in this study.

Unique physiological and genetic features of ofloxacin-resistant Streptomyces mutants

New antimicrobial agents are urgently needed to combat the emergence and spread of multidrug-resistant bacteria. Activating the cryptic biosynthetic gene clusters for actinomycete secondary metabolites can provide essential clues for research into new antimicrobial agents. An effective method for this purpose is based on drug resistance selection. This report describes interesting results for drug resistance selection using antibiotics that target DNA replication can effectively potentiate secondary metabolite production by actinomycetes. Ofloxacin-resistant mutants were isolated from five different streptomycetes. Ofloxacin is an antibiotic that binds to DNA complexes and type II topoisomerase, causing double-stranded breaks in bacterial chromosomes. Physiological and genetic characterization of the mutants revealed that the development of ofloxacin resistance in streptomycetes leads to the emergence of various types of secondary metabolite-overproducing strains. In Streptomyces coelicolor A3(2), ofloxacin-resistant mutants that overproduced actinorhodin, undecylprodigiosin, or carotenoid were identified. Also, an ofloxacin-resistant mutant that overproduces methylenomycin A, whose biosynthetic gene cluster is located on the endogenous plasmid, SCP1, was isolated. These observations indicate that ofloxacin resistance might activate biosynthetic genes on both chromosomes and on endogenous plasmids. We also identified the mutations that are probably involved in the phenotype of ofloxacin resistance and secondary metabolite overproduction in S. coelicolor A3(2). Furthermore, we observed an interesting phenomenon in which several ofloxacin-resistant mutants overproduced antibiotics in the presence of ofloxacin. Based on these results, we present the unique physiological and genetic characteristics of ofloxacin-resistant Streptomyces mutants and discuss the importance and potential development of the new findings. IMPORTANCE The abuse or overuse of antibacterial agents for therapy and animal husbandry has caused an increased population of antimicrobial-resistant bacteria in the environment. Consequently, there are now fewer effective antimicrobials available. Due to the depleted antibiotic pipeline, pandemic outbreaks caused by antimicrobial-resistant bacteria are deeply concerned, and the development of new antibiotics is now an urgent issue. Promising sources of antimicrobial agents include cryptic biosynthetic gene clusters for secondary metabolites in streptomycetes and rare actinomycetes. This study’s significance is an unprecedented activation method to accelerate drug discovery research on a global scale. The technique developed in this study could allow for simultaneous drug discovery in different countries, maximizing the world’s microbial resources.

Streptomyces as Microbial Chassis for Heterologous Protein Expression

Heterologous production of recombinant proteins is gaining increasing interest in biotechnology with respect to productivity, scalability, and wide applicability. The members of genus Streptomyces have been proposed as remarkable hosts for heterologous production due to their versatile nature of expressing various secondary metabolite biosynthetic gene clusters and secretory enzymes. However, there are several issues that limit their use, including low yield, difficulty in genetic manipulation, and their complex cellular features. In this review, we summarize rational engineering approaches to optimizing the heterologous production of secondary metabolites and recombinant proteins in Streptomyces species in terms of genetic tool development and chassis construction. Further perspectives on the development of optimal Streptomyces chassis by the design-build-test-learn cycle in systems are suggested, which may increase the availability of secondary metabolites and recombinant proteins.

Genome and Metabolome MS-Based Mining of a Marine Strain of Aspergillus affinis

Aspergillus section Circumdati encompasses several species that express both beneficial (e.g., biochemical transformation of steroids and alkaloids, enzymes and metabolites) and harmful compounds (e.g., production of ochratoxin A (OTA)). Given their relevance, it is important to analyze the genetic and metabolic diversity of the species of this section. We sequenced the genome of Aspergillus affinis CMG 70, isolated from sea water, and compared it with the genomes of species from section Circumdati, including A. affinis’s strain type. The A. affinis genome was characterized considering secondary metabolites biosynthetic gene clusters (BGCs), carbohydrate-active enzymes (CAZymes), and transporters. To uncover the biosynthetic potential of A. affinis CMG 70, an untargeted metabolomics (LC-MS/MS) approach was used. Cultivating the fungus in the presence and absence of sea salt showed that A. affinis CMG 70 metabolite profiles are salt dependent. Analyses of the methanolic crude extract revealed the presence of both unknown and well-known Aspergillus compounds, such as ochratoxin A, anti-viral (e.g., 3,5-Di-tert-butyl-4-hydroxybenzoic acid and epigallocatechin), anti-bacterial (e.g., 3-Hydroxybenzyl alcohol, L-pyroglutamic acid, lecanoric acid), antifungal (e.g., L-pyroglutamic acid, 9,12,13-Trihydroxyoctadec-10-enoic acid, hydroxyferulic acid), and chemotherapeutic (e.g., daunomycinone, mitoxantrone) related metabolites. Comparative analysis of 17 genomes from 16 Aspergillus species revealed abundant CAZymes (568 per species), secondary metabolite BGCs (73 per species), and transporters (1359 per species). Some BGCs are highly conserved in this section (e.g., pyranonigrin E and UNII-YC2Q1O94PT (ACR toxin I)), while others are incomplete or completely lost among species (e.g., bikaverin and chaetoglobosins were found exclusively in series Sclerotiorum, while asperlactone seemed completely lost). The results of this study, including genome analysis and metabolome characterization, emphasize the molecular diversity of A. affinis CMG 70, as well as of other species in the section Circumdati.