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Abstract Background Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches. Results We established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation. The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains. Conclusions We propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies.

Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Some microbial toxins also target the producer species itself, necessitating a means of self-protection. The M2 double-stranded RNA (dsRNA) killer virus in Saccharomyces cerevisiae contains a single open reading frame (ORF) encoding both the secreted pore-forming toxin K2 as well as a cognate immunity factor. Here, we show that expression of a 49-amino acid N-terminal peptide from the K2 precursor is both necessary and sufficient for immunity. This immunity peptide simultaneously functions as a signal peptide for toxin secretion and protects the cell against the cytotoxic K2 α subunit. The K2 toxin and immunity factor can be functionally separated into two ORFs, yielding a modular toxin-immunity system. This case further shows how a (signal) peptide can carry the potential for providing cellular protection against an antimicrobial toxin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Interactions between bacteria and microalgae are important for the functioning of aquatic ecosystems, yet interactions based on the biodiversity of these two taxonomic domains have been scarcely studied. Specifically, it is unclear whether a positive biodiversity-productivity relationship in phytoplankton is largely facilitated by niche partitioning among the phytoplankton organisms themselves or whether associated bacterial communities play an additional role in modifying these diversity effects. Moreover, the effects of intraspecific diversity in phytoplankton communities on bacterial community diversity have not been tested. To address these points, we factorially manipulated both species and intraspecific richness of three diatoms to test the effects of diatom species/strain diversity on biomass production and bacterial diversity in algae-bacteria communities. The results show that diatom intraspecific diversity has significant positive effects on culture biomass and the diversity of the associated free-living bacterial community (0.2-3 μm size fraction), which are comparable in magnitude to species diversity effects. However, there were little to no effects of diatom diversity on host-associated bacterial diversity (>3 μm size fraction), or of bacterial diversity on biomass production. These results suggest a decoupling of bacterial diversity from the diatom diversity-productivity relationship and provide early insights regarding the relations between diversity across domains in aquatic ecosystems., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The ability to precisely edit genomes by deleting or adding genetic information enables the study of biological functions and the building of efficient cell factories. In many unconventional yeasts, such as those promising new hosts for cell factory design but also human pathogenic yeasts and food spoilers, this progress has been limited by the fact that most yeasts favour non-homologous end joining (NHEJ) over homologous recombination (HR) as a DNA repair mechanism, impairing genetic access to these hosts. In mammalian cells, small molecules that either inhibit proteins involved in NHEJ, enhance protein function in HR, or arrest the cell cycle in HR-dominant phases are regarded as promising agents for the simple and transient increase of HR-mediated genome editing without the need for a priori host engineering. Only a few of these chemicals have been applied to the engineering of yeast, although the targeted proteins are mostly conserved, making chemical agents a yet-underexplored area for enhancing yeast engineering. Here, we consolidate knowledge of the available small molecules that have been used to improve HR efficiency in mammalian cells and the few ones that have been used in yeast. We include available high-throughput-compatible NHEJ/HR quantification assays that could be used to screen for and isolate yeast-specific inhibitors., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Information is the cornerstone of research, from experimental (meta)data and computational processes to complex inventories of reagents and equipment. These 10 simple rules discuss best practices for leveraging laboratory information management systems to transform this large information load into useful scientific findings., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests:J.P., S.P., and K.M. have a financial interest in GenoFAB, Inc., M.K. is an employee of LanzaTech. N.J.H. has a financial interest in TeselaGen Biotechnology, Inc. and Ansa Biotechnologies, Inc. GenoFAB Inc. and TeselaGen Biotechnology, Inc. provide research information management systems. These companies may benefit or be perceived as benefiting from this publication., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate─a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the Saccharomyces cerevisiae -based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK ( C. glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.

Synthetic biologists have made great progress over the past decade in developing methods for modular assembly of genetic sequences and in engineering biological systems with a wide variety of functions in various contexts and organisms. However, current paradigms in the field entangle sequence and functionality in a manner that makes abstraction difficult, reduces engineering flexibility and impairs predictability and design reuse. Functional Synthetic Biology aims to overcome these impediments by focusing the design of biological systems on function, rather than on sequence. This reorientation will decouple the engineering of biological devices from the specifics of how those devices are put to use, requiring both conceptual and organizational change, as well as supporting software tooling. Realizing this vision of Functional Synthetic Biology will allow more flexibility in how devices are used, more opportunity for reuse of devices and data, improvements in predictability and reductions in technical risk and cost., Competing Interests: No competing interest is declared., (© The Author(s) 2023. Published by Oxford University Press.)

Interactions between marine diatoms and bacteria have been studied for decades. However, the visualization of physical interactions between these diatoms and their colonizers is still limited. To enhance our understanding of these specific interactions, a new Thalassiosira rotula isolate from the North Sea (strain 8673) was characterized by scanning electron microscopy and confocal laser scanning microscopy (CLSM) after staining with fluorescently labeled lectins targeting specific glycoconjugates. To investigate defined interactions of this strain with bacteria the new strain was made axenic and co-cultivated with a natural bacterial community and in two- or three-partner consortia with different bacteria of the Roseobacter group, Gammaproteobacteria and Bacteroidetes. The CLSM analysis of the consortia identified six out of 78 different lectins as very suitable to characterize glycoconjugates of T. rotula. The resulting images show that fucose-containing threads were the dominant glycoconjugates secreted by the T. rotula cells but chitin and to a lesser extent other glycoconjugates were also identified. Bacteria attached predominantly to the fucose glycoconjugates. The colonizing bacteria showed various attachment patterns such as adhering to the diatom threads in aggregates only or attaching to both the surfaces and the threads of the diatom. Interestingly the colonization patterns of single bacteria differed strikingly from those of bacterial co-cultures, indicating that interactions between two bacterial species impacted the colonization of the diatom. Our observations help to better understand physical interactions and specific colonization patterns of distinct bacterial mono- and co-cultures with an abundant diatom of costal seas., (© 2022 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

Cyclodipeptides are diverse chemical scaffolds that show a broad range of bioactivities relevant for medicine, agriculture, chemical catalysis, and material sciences. Cyclodipeptides can be synthesized enzymatically through two unrelated enzyme families, non-ribosomal peptide synthetases (NRPS) and cyclodipeptide synthases (CDPSs). The chemical diversity of cyclodipeptides is derived from the two amino acid side chains and the modification of those side-chains by cyclodipeptide tailoring enzymes. While a large spectrum of chemical diversity is already known today, additional chemical space - and as such potential new bioactivities - could be accessed by exploring yet undiscovered NRPS and CDPS gene clusters as well as via engineering. Further, to exploit cyclodipeptides for applications, the low yield of natural biosynthesis needs to be overcome. In this review we summarize current knowledge on NRPS and CDPS-based cyclodipeptide biosynthesis, engineering approaches to further diversity the natural chemical diversity as well as strategies for high-yield production of cyclodipeptides, including a discussion of how advancements in synthetic biology and metabolic engineering can accelerate the translational potential of cyclodipeptides., Competing Interests: The authors have no financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work., (© 2022 The Author(s).)

Synthetic biology (SynBio) is a rapidly growing scientific discipline. In the Netherlands, various universities and companies are tackling a variety of opportunities and challenges within this field. In this perspective article, we review the current synthetic biology landscape in the Netherlands across academia, industry, politics, and society. Especially within Dutch academia there is an active, though only partially connected, research community involved in various domains of SynBio. Mostly supported by governmental funding, academic research is focusing on top-down synthetic biology, involving the engineering of, for example, bacteria and yeast for bioproduction, as well as bottom-up and cell-free synthetic biology aiming to understand life and build synthetic cells. There is also a large number of talented and motivated students interested in the field, exemplified by the participation and success of Dutch teams in the international iGEM synthetic biology competition. Commercial synthetic biology activities are taking place in various large industrial companies, as well as in start-ups and spin-offs, mostly divided over several 'SynBio hubs' in the Netherlands. However, the investment, regulatory and public-perception landscape is not yet optimal to stimulate entrepreneurial activities in SynBio. The Dutch and global society can further benefit from the large promise of SynBio through better integration of people active in the Dutch SynBio field, frequent political and public dialogue, and more attention towards regulatory issues. The recently founded Dutch synthetic biology association SynBioNL aims to contribute to realizing a positive impact on society by stimulating advances of the field in the Netherlands and beyond., Competing Interests: The authors have no conflicts of interest to disclose., (© 2022 The Authors.)

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells., (© The Author(s) 2022. Published by Oxford University Press.)

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ∼350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Building multicellular microbial consortia that communicate with each other and perform programmed functionalities is the next milestone for synthetic biology. Achieving cell-cell communication within these communities requires programming of the transduction of an extracellular signal into a customized intracellular response. G-protein-coupled receptors (GPCRs) are attractive candidates for engineering signal transduction as they can sense extracellular events with high sensitivity and specificity and transduce them into complex intracellular programs. We recently developed a scalable cell-cell communication language based on fungal mating GPCRs and their secreted peptide ligands. This language allows the assembly of engineered yeast strains into multicellular communication networks and allows them to be made interdependent by peptide signaling. In peptide signaling, one cell secretes a peptide that supports the growth of another cell at nanomolar concentrations, a scalable approach for engineering interdependence. Here we address the challenge of correlating the doubling time of Saccharomyces cerevisiae cells with an increasing external peptide concentration by linking GPCR activation to the expression of an essential gene. The required fine-tuning of downstream signaling is achieved via the transcriptional titration of a set of orthogonal GPCR-activated transcription factors, a series of corresponding promoters with different output dynamics, and the use of chemically recoded peptide ligands with varying activation potentials. As such, our work establishes three control points that allow the tuning of the basal and maximal activation of the GPCR response, fold change activation, and response sensitivity. The presented results enable the implementation of peptide-dependent and peptide-tunable growth but could also facilitate the design and calibration of more complex GPCR-controlled synthetic functionality in the future.

Background: Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system., Results: We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations., Conclusion: Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.

Antimicrobial residues in milk have been discussed as a possible selector for Enterobacteriaceae that produce extended-spectrum β-lactamases (ESBL) in dairy herds. Such residues are found in waste milk after antibiotic treatment of mastitis, but antibiotic dry cow therapy might also lead to antibiotic residues in colostrum and in milk during early lactation. While it is known that feeding of waste milk selects ESBL bacteria in calves, this was not investigated for colostrum yet, which is supposed to contain much lower antibiotic concentrations than waste milk. In this observational prospective case study on 2 farms, we hypothesized that blanket dry cow treatment with β-lactams would have more selective (here: increasing) effects on ESBL concentrations than selective (here: individually chosen) antibiotic dry cow therapy. Thus, we compared concentrations of ESBL-producing Enterobacteriaceae in feces of calves (n = 50) at 2 dairy farms with different management of antibiotic dry cow therapy. Considerably higher concentrations of ESBL-producing Escherichia coli were observed in blanket antibiotic dry cow therapy on d 3 of the calf's life (7.6 vs. 5.3 log cfu/g of calf feces). Both farms used narrow-spectrum penicillin combined with aminoglycosides for drying off, and the majority of ESBL isolates (93%) were co-resistant to aminoglycosides. No waste milk was fed to calves and no calf was treated with β-lactam antibiotics or aminoglycosides during the first 3 d of life, thus differences were most likely associated with different frequency of antibiotic dry cow therapy on farms (19 of 25 mother cows on farm A, 9 of 25 on farm B). Even though the presumable selection effect of antibiotics used for drying off decreased within the next 3 wk, this result further emphasizes the need for the reduction and prudent use of antibiotic dry cow therapy on farms., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'J.D.B. is a founder and Director of the following: Neochromosome, Inc., the Center of Excellence for Engineering Biology, and CDI Labs, Inc. and serves on the Scientific Advisory Board of the following: Modern Meadow, Inc., Recombinetics, Inc., and Sample6, Inc.'. This has now been corrected in both the PDF and HTML versions of the Article.

Engineering multicellularity is one of the next breakthroughs for Synthetic Biology. A key bottleneck to building multicellular systems is the lack of a scalable signaling language with a large number of interfaces that can be used simultaneously. Here, we present a modular, scalable, intercellular signaling language in yeast based on fungal mating peptide/G-protein-coupled receptor (GPCR) pairs harnessed from nature. First, through genome-mining, we assemble 32 functional peptide-GPCR signaling interfaces with a range of dose-response characteristics. Next, we demonstrate that these interfaces can be combined into two-cell communication links, which serve as assembly units for higher-order communication topologies. Finally, we show 56 functional, two-cell links, which we use to assemble three- to six-member communication topologies and a three-member interdependent community. Importantly, our peptide-GPCR language is scalable and tunable by genetic encoding, requires minimal component engineering, and should be massively scalable by further application of our genome mining pipeline or directed evolution.

Deciphering the ecological traits of abundant marine bacteria is a major challenge in marine microbial ecology. In the current study, we linked compositional and functional predictions to elucidate such traits for abundant bacterioplankton lineages in the North Sea. For this purpose, we investigated entire and active bacterioplankton composition along a transect ranging from the German Bight to the northern North Sea by pyrotag sequencing of bacterial 16S rRNA genes and transcripts. Functional profiles were inferred from 16S rRNA data using Tax4Fun. Bacterioplankton communities were dominated by well-known marine lineages including clusters/genera that are affiliated with the Roseobacter group and the Flavobacteria . Variations in community composition and function were significantly explained by measured environmental and microbial properties. Turnover of dissolved free amino acids (DFAA) showed the strongest correlation to community composition and function. We applied multinomial models, which enabled us to identify bacterial lineages involved in DFAA turnover. For instance, the genus Planktomarina was more abundant at higher DFAA turnover rates, suggesting its vital role in amino acid degradation. Functional predictions further indicated that Planktomarina is involved in leucine and isoleucine degradation. Overall, our results provide novel insights into the biogeochemical significance of abundant bacterioplankton lineages in the North Sea.

Background: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool., Methods: We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins., Results: We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability., Conclusions: Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins.

Heterotrophic bacterioplankton communities play an important role in organic matter processing in the oceans worldwide. In order to investigate the significance of distinct phylogenetic bacterial groups it is not only important to assess their quantitative abundance but also their growth dynamics in relation to the entire bacterioplankton. Therefore bacterial abundance, biomass production and the composition of the entire and cell-proliferating bacterioplankton community were assessed in North Sea surface waters between the German Bight and 58°N in early summer by applying catalyzed reporter deposition (CARD-FISH) and bromodeoxyuridine fluorescence in situ hybridization (BrdU-FISH). Bacteroidetes and the Roseobacter group dominated the cell-proliferating fraction with 10-55 and 8-31% of total BrdU-positive cells, respectively. While Bacteroidetes also showed high abundances in the total bacterial fraction, roseobacters constituted only 1-9% of all cells. Despite abundances of up to 55% of total bacterial cells, the SAR11 clade constituted <6% of BrdU-positive cells. Gammaproteobacteria accounted for 2-16% of the total and 2-13% of the cell-proliferating cells. Within the two most active groups, BrdU-positive cells made up 28% of Bacteroidetes as an overall mean and 36% of roseobacters. Estimated mean growth rates of Bacteroidetes and the Roseobacter group were 1.2 and 1.5 day -1 , respectively, and much higher than bulk growth rates of the bacterioplankton whereas those of the SAR11 clade and Gammaproteobacteria were 0.04 and 0.21 day -1 , respectively, and much lower than bulk growth rates. Only numbers of total and cell-proliferating roseobacters but not those of Bacteroidetes and the other groups were significantly correlated to chlorophyll fluorescence and bacterioplankton biomass production. The Roseobacter group, besides Bacteroidetes , appeared to be a major player in processing phytoplankton derived organic matter despite its low partitioning in the total bacterioplankton community.

The availability of simple, specific, and inexpensive on-site detection methods is of key importance for deployment of pathogen surveillance networks. We developed a nontechnical and highly specific colorimetric assay for detection of pathogen-derived peptides based on Saccharomyces cerevisiae -a genetically tractable model organism and household product. Integrating G protein-coupled receptors with a visible, reagent-free lycopene readout, we demonstrate differential detection of major human, plant, and food fungal pathogens with nanomolar sensitivity. We further optimized a one-step rapid dipstick prototype that can be used in complex samples, including blood, urine, and soil. This modular biosensor can be economically produced at large scale, is not reliant on cold-chain storage, can be detected without additional equipment, and is thus a compelling platform scalable to global surveillance of pathogens.

Enzymatic reaction networks are unique in that one can operate a large number of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biological processes such as glycolysis. For this, we adapt the characterization of the biochemical system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply standard system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chemical production purposes.

The identification and functional characterization of microbial communities remains a prevailing topic in microbial oceanography as information on environmentally relevant pelagic prokaryotes is still limited. The Roseobacter group, an abundant lineage of marine Alphaproteobacteria, can constitute large proportions of the bacterioplankton. Roseobacters also occur associated with eukaryotic organisms and possess streamlined as well as larger genomes from 2.2 to >5 Mpb. Here, we show that one pelagic cluster of this group, CHAB-I-5, occurs globally from tropical to polar regions and accounts for up to 22% of the active North Sea bacterioplankton in the summer. The first sequenced genome of a CHAB-I-5 organism comprises 3.6 Mbp and exhibits features of an oligotrophic lifestyle. In a metatranscriptome of North Sea surface waters, 98% of the encoded genes were present, and genes encoding various ABC transporters, glutamate synthase and CO oxidation were particularly upregulated. Phylogenetic gene content analyses of 41 genomes of the Roseobacter group revealed a unique cluster of pelagic organisms distinct from other lineages of this group, highlighting the adaptation to life in nutrient-depleted environments.

The Roseobacter group is one of the predominant lineages in the marine environment. While most investigations focus on pelagic roseobacters, the distribution and metabolic potential of benthic representatives is less understood. In this study, the diversity of the Roseobacter group was characterized in sediment and water samples along the German/Scandinavian North Sea coast by 16S rRNA gene analysis and cultivation-based methods. Molecular analysis indicated an increasing diversity between communities of the Roseobacter group from the sea surface to the seafloor and revealed distinct compositions of free-living and attached fractions. Culture media containing dimethyl sulfide (DMS), dimethyl sulfonium propionate (DMSP) or dimethyl sulfoxide (DMSO) stimulated growth of roseobacters showing highest most probable numbers (MPN) in DMSO-containing dilutions of surface sediments (2.1 × 10(7) roseobacters cm(-3)). Twenty roseobacters (12 from sediments) were isolated from DMSP- and DMS-containing cultures. Sequences of the isolates represented 0.04% of all Bacteria and 4.7% of all roseobacters in the pyrosequencing dataset from sediments. Growth experiments with the isolate Shimia sp. SK013 indicated that benthic roseobacters are able to switch between aerobic and anaerobic utilization of organic sulfur compounds. This response to changing redox conditions might be an adaptation to specific environmental conditions on particles and in sediments., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Gram-negative alphaproteobacterium Octadecabacter temperatus SB1 (DSM 26878) belongs to the marine Roseobacter clade. The genome of this strain is the smallest closed genome of the Roseobacter clade. O. temperatus SB1 is the first described nonpolar mesophilic isolate of the genus Octadecabacter and the type strain of the species., (Copyright © 2015 Voget et al.)

A heterotrophic, Gram-negative, aerobic bacterium, designated strain SB1T, was isolated from surface water of the southern North Sea. Comparison of 16S rRNA gene sequences revealed that strain SB1T is affiliated to the genus Octadecabacter within the marine Roseobacter clade (family Rhodobacteraceae), with Octadecabacter antarcticus as the closest described species (98.2 % sequence similarity to the type strain). DNA-DNA hybridization indicated that SB1T represents a distinct species within this genus. On marine agar, strain SB1T formed beige, circular and convex colonies. Cells were irregular, motile rods. Growth occurred between 4 and 25 °C and was optimal at 20 °C, and at pH 7-9 (optimum pH 7.5-8.5) and NaCl concentrations between 1 and 6 % (optimum 2-4 %). The DNA G+C content of SB1T was 54.7 mol%. The fatty acids (>1 %) comprised 10 : 0 3-OH, 12 : 1 3-OH, 16 : 1ω7c, 16 : 0, 18 : 2ω7,12, 18 : 1ω7c, 18 : 0 and 11-methyl 18 : 1ω7c. The sole respiratory lipoquinone was ubiquinone Q-10 and the polar lipid pattern indicated the presence of the phospholipids phosphatidylglycerol and phosphatidylcholine, as well as unidentified aminolipid AL1, phospholipids PL1 and PL3 and lipids L1, L2 and L4. On the basis of phylogenetic and phenotypic differences, strain SB1T represents a novel species in the genus Octadecabacter, for which we propose the name Octadecabacter temperatus sp. nov. The type strain is SB1T ( = DSM 26878T = LMG 27946T). Furthermore, our results suggest the reclassification of Octadecabacter jejudonensis as the type species of a new genus, Pseudooctadecabacter gen. nov., as Pseudooctadecabacter jejudonensis comb. nov. (type strain SSK2-1T = KCTC 32535T = CECT 8397T). Finally, emended descriptions of the genus Octadecabacter and its species Octadecabacter antarcticus and Octadecabacter arcticus are also provided.

The RCA (Roseobacter clade affiliated) cluster, with an internal 16S rRNA gene sequence similarity of >98%, is the largest cluster of the marine Roseobacter clade and most abundant in temperate to (sub)polar oceans, constituting up to 35% of total bacterioplankton. The genome analysis of the first described species of the RCA cluster, Planktomarina temperata RCA23, revealed that this phylogenetic lineage is deeply branching within the Roseobacter clade. It shares not >65.7% of homologous genes with any other organism of this clade. The genome is the smallest of all closed genomes of the Roseobacter clade, exhibits various features of genome streamlining and encompasses genes for aerobic anoxygenic photosynthesis (AAP) and CO oxidation. In order to assess the biogeochemical significance of the RCA cluster we investigated a phytoplankton spring bloom in the North Sea. This cluster constituted 5.1% of the total, but 10-31% (mean 18.5%) of the active bacterioplankton. A metatranscriptomic analysis showed that the genome of P. temperata RCA23 was transcribed to 94% in the bloom with some variations during day and night. The genome of P. temperata RCA23 was also retrieved to 84% from metagenomic data sets from a Norwegian fjord and to 82% from stations of the Global Ocean Sampling expedition in the northwestern Atlantic. In this region, up to 6.5% of the total reads mapped on the genome of P. temperata RCA23. This abundant taxon appears to be a major player in ocean biogeochemistry.

Despite their importance for ecosystem functioning, little is known about the composition of active marine bacterioplankton communities. Hence, this study was focused on assessing the diversity of these communities in the southern North Sea and examining the impact of a phytoplankton spring bloom on the ambient bacterioplankton community. Community composition in and outside the bloom was assessed in 14 samples by pyrosequencing-based analysis of 16S rRNA gene amplicons generated from environmental RNA. The data set comprised of 211 769 16S rRNA gene sequences. Proteobacteria were the predominant phylogenetic group with Alphaproteobacteria and Gammaproteobacteria as the most abundant classes. Actinobacteria and Bacteroidetes were identified in minor abundances. Active bacterial communities were dominated by few lineages such as the Roseobacter RCA cluster and the SAR92 clade. Community structures of three selected samples were also assessed by direct sequencing of cDNA generated from rRNA-depleted environmental RNA. Generated data sets comprised of 988 202 sequences. Taxonomic assignment of the reads confirmed the predominance of Proteobacteria. The examined phytoplankton spring bloom affected the bacterioplankton community structures significantly. Bacterial richness was reduced in the bloom area, and the abundance of certain bacterial groups was affected by bloom presence. The SAR92 clade and the Roseobacter RCA cluster were significantly more abundant and active in the bloom. Functions affected by the bloom include photosynthesis, protein metabolism, and DNA metabolism., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

In vitro biocatalytic systems have moved far beyond established uses in food, diagnostic, and chemical applications. As new strategies to construct and manage multiple enzymes in ever more complex systems are developed, novel applications emerge. In the field of chemistry, complex protein networks are applied to enable the production of fine chemicals, such as dihydroxyacetone phosphate, and even bulk chemicals, such as biofuels, from cheap sugars. Cell-free protein synthesis is applied to expanding protein and nucleic acid biochemistry and enabling novel assay formats, while programmable DNA-circuits can be exploited to engineer sensitive detection methods. Novel developments in chemical analytics such as real-time mass spectrometry to follow the metabolism online, directed physical assembly of network members facilitating substrate channeling, and encapsulation forming biofunctional subunits enable a better control and potential for optimization., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single-ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The orchestration of a multitude of enzyme catalysts allows cells to carry out complex and thermodynamically unfavorable chemical conversions. In an effort to recruit these advantages for in vitro biotransformations, we have assembled a 10-step catalytic system-a system of biotransformations (SBT)-for the synthesis of unnatural monosaccharides based on the versatile building block dihydroxyacetone phosphate (DHAP). To facilitate the assembly of such a network, we have insulated a production pathway from Escherichia coli's central carbon metabolism. This pathway consists of the endogenous glycolysis without triose-phosphate isomerase to enable accumulation of DHAP and was completed with lactate dehydrogenase to regenerate NAD(+). It could be readily extended for the synthesis of unnatural sugar molecules, such as the unnatural monosaccharide phosphate 5,6,7-trideoxy-D-threo-heptulose-1-phosphate from DHAP and butanal. Insulation required in particular inactivation of the amn gene encoding the AMP nucleosidase, which otherwise led to glucose-independent DHAP production from adenosine phosphates. The work demonstrates that a sufficiently insulated in vitro multi-step enzymatic system can be readily assembled from central carbon metabolism pathways., (2010 Wiley Periodicals, Inc.)