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Last year ActinoBase, a Wiki-style initiative supported by the UK Microbiology Society, published a review highlighting the research of particular interest to the actinomycete community. Here, we present the second ActinoBase review showcasing selected reports published in 2020 and early 2021, integrating perspectives in the actinomycete field. Actinomycetes are well-known for their unsurpassed ability to produce specialised metabolites, of which many are used as therapeutic agents with antibacterial, antifungal, or immunosuppressive activities. Much research is carried out to understand the purpose of these metabolites in the environment, either within communities or in host interactions. Moreover, many efforts have been placed in developing computational tools to handle big data, simplify experimental design, and find new biosynthetic gene cluster prioritisation strategies. Alongside, synthetic biology has provided advances in tools to elucidate the biosynthesis of these metabolites. Additionally, there are still mysteries to be uncovered in understanding the fundamentals of filamentous actinomycetes' developmental cycle and regulation of their metabolism. This review focuses on research using integrative methodologies and approaches to understand the bigger picture of actinomycete biology, covering four research areas: i) technology and methodology; ii) specialised metabolites; iii) development and regulation; and iv) ecology and host interactions.

Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. It is produced from daidzein by fastidious and oxygen-susceptible intestinal bacteria, which hinders their use at an industrial scale. Therefore, expressing the equol production machinery into easily-cultivable hosts would expedite the heterologous production of this compound. In this work, four genes (racemase, tdr, ddr and dzr) coding for key enzymes involved in equol production in Adlercreutzia equolifaciens DSM19450T were synthesized and cloned in a pUC-derived vector (pUC57-equol) that was introduced in Escherichia coli. Recombinant clones of E. coli produced equol in cultures supplemented with daidzein (equol precursor) and dihydrodaidzein (intermediate compound). To check whether equol genes were expressed in Gram-positive bacteria, the pUC57-equol construct was cloned into the low-copy-number vector pIL252, and the new construct (pIL252-pUC57-equol) introduced into model strains of Lacticaseibacillus casei and Lactococcus lactis. L. casei clones carrying pIL252-pUC57-equol produced a small amount of equol from dihydrodaidzein but not from daidzein, while L. lactis recombinant clones produced no equol from either of the substrates. This is the first time that A. equolifaciens equol genes have been cloned and expressed in heterologous hosts. E. coli clones harboring pUC57-equol could be used for biotechnological production of equol., Heterologous equol production from synthetic genes based on those of Adlercreutzia equolifaciens.

Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology., Transporter protein expression enables the optimization of microbial cell factories for the bioproduction of organic acids.

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools., In this review, we discuss our current understanding of phage-encoded inhibitors that block prokaryotic CRISPR-Cas systems, their mechanisms, structures and applications.

Microbial production of chemical compounds often requires highly engineered microbial cell factories. During the last years, CRISPR-Cas nucleases have been repurposed as powerful tools for genome editing. Here, we briefly review the most frequently used CRISPR-Cas tools and describe some of their applications. We describe the progress made with respect to CRISPR-based multiplex genome editing of industrial bacteria and eukaryotic microorganisms. We also review the state of the art in terms of gene expression regulation using CRISPRi and CRISPRa. Finally, we summarize the pillars for efficient multiplexed genome editing and present our view on future developments and applications of CRISPR-Cas tools for multiplex genome editing., Current status of multiplex genome editing in bacteria and eukaryotic microorganisms using CRISPR-Cas tools.

A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 × 1018 m3 and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 1012 cells per millilitre, exceeding eukaryotic densities of around 106 cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project ‘INMARE’., The success of using marine resources for the development of industrial products and processes depends on the manner they are screened, produced and tested on a larger scale.

This mini-review provides a perspective of traditional, emerging and future applications of lactic acid bacteria (LAB) and how genome editing tools can be used to overcome current challenges in all these applications. It also describes available tools and how these can be further developed, and takes current legislation into account. Genome editing tools are necessary for the construction of strains for new applications and products, but can also play a crucial role in traditional ones, such as food and probiotics, as a research tool for gaining mechanistic insights and discovering new properties. Traditionally, recombinant DNA techniques for LAB have strongly focused on being food-grade, but they lack speed and the number of genetically tractable strains is still rather limited. Further tool development will enable rapid construction of multiple mutants or mutant libraries on a genomic level in a wide variety of LAB strains. We also propose an iterative Design–Build–Test–Learn workflow cycle for LAB cell factory development based on systems biology, with ‘cell factory’ expanding beyond its traditional meaning of production strains and making use of genome editing tools to advance LAB understanding, applications and strain development., Traditional, emerging and future applications of lactic acid bacteria can all benefit from genome editing and a proposed Design–Build–Test–Learn workflow cycle for advancement of strain development.

Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1–2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genes cas1 and cas2 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation in Synechocystis sp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array., Conserved nucleotide motifs in the CRISPR leader sequence control spacer acquisition levels of CRISPR-Cas systems

Fungi are renowned producers of natural compounds, also known as secondary metabolites (SMs) that display a wide array of biological activities. Typically, the genes that are involved in the biosynthesis of SMs are located in close proximity to each other in so-called secondary metabolite clusters. Many plant-pathogenic fungi secrete SMs during infection in order to promote disease establishment, for instance as cytocoxic compounds. Verticillium dahliae is a notorious plant pathogen that can infect over 200 host plants worldwide. However, the SM repertoire of this vascular pathogen remains mostly uncharted. To unravel the potential of V. dahliae to produce SMs, we performed in silico predictions and in-depth analyses of its secondary metabolite clusters. Using distinctive traits of gene clusters and the conserved signatures of core genes 25 potential SM gene clusters were identified. Subsequently, phylogenetic and comparative genomics analyses were performed, revealing that two putative siderophores, ferricrocin and TAFC, DHN-melanin and fujikurin may belong to the SM repertoire of V. dahliae., In total, 25 potential SMCs were identified in the genome of the plant pathogenic fungus Verticillium dahliae, including loci that can be implicated in DHN-melanin, ferricrocin, triacetyl fusarinine and fujikurin production.

Scaling up industrial microbial processes for commercial production is a high-stakes endeavor, requiring time and investment often exceeding that for laboratory microbe and process development. Omissions, oversights and errors can be costly, even fatal to the program. Approached properly, scale-up can be executed successfully. Three guiding principles are provided as a basis: begin with the end in mind; be diligent in the details; prepare for the unexpected. A detailed roadmap builds on these principles. There is a special emphasis on the fermentation step, which is usually the costliest and also impacts downstream processing. Examples of common scale-up mistakes and the recommended approaches are given. It is advised that engineering resources skilled in integrated process development and scale-up be engaged from the very beginning of microbe and process development to guide ongoing R&D, thus ensuring a smooth and profitable path to the large-scale commercial end., Scaling up industrial microbial processes is a costly, high-stakes endeavor that can be executed successfully if approached properly.

Their biochemical versatility and biotechnological importance make actinomycete bacteria attractive targets for ambitious genetic engineering using the toolkit of synthetic biology. But their complex biology also poses unique challenges. This mini review discusses some of the recent advances in synthetic biology approaches from an actinomycete perspective and presents examples of their application to the rational improvement of industrially relevant strains., Recent advances in synthetic biology driven engineering of the actinomycetes for the production of valuable drug compounds.