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The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg(2+)-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg(2+) enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.Cell Research advance online publication 29 September 2017; doi:10.1038/cr.2017.122.

One of the key aspects that defines a cell as a living entity is its ability to self-reproduce. In this process, membrane biogenesis is an essential element. Here, we developed an in vitro phospholipid biosynthesis pathway based on a cascade of eight enzymes, starting from simple fatty acid building blocks and glycerol 3-phosphate. The reconstituted system yields multiple phospholipid species that vary in acyl-chain and polar head group compositions. Due to the high fidelity and versatility, complete conversion of the fatty acid substrates into multiple phospholipid species is achieved simultaneously, leading to membrane expansion as a first step towards a synthetic minimal cell.

Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N-terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d-glucose and 4% d-xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d-xylose over d-glucose with high d-xylose transport rates. This mutant supported efficient sugar fermentation of both d-glucose and d-xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937-1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

To produce high levels of β-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for β-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production. IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

A critical aspect of a synthetic minimal cell is expansion of the surrounding boundary layer. This layer should consist of phospholipids (mimics) as these molecules assemble into a bilayer, creating a functional barrier with specific phospholipid species that are essential for membrane related processes. As a first step towards synthetic cells, an in vitro phospholipid biosynthesis pathway has been constructed that utilizes fatty acids as precursors to produce a wide variety of phospholipid species, thereby driving membrane growth. This now needs to be developed further into a sustainable expanding system, meanwhile keeping simplicity in mind. The non-enzymatic synthesis of phospholipid-like molecules forms a realistic alternative for natural enzymatic-based pathways, that nowadays can even support functional membrane proteins. Eventually, coupling to in vitro transcription/translation is required, for which efficient mechanisms of insertion and folding of the involved membrane proteins need to be developed. Such an integrated system will form a suitable foundation of a synthetic minimal cell that eventually can be coupled to other cellular processes such as division.

Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. Conclusion: Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.

Background: Filamentous fungi are important producers of secondary metabolites, low molecular weight molecules that often have bioactive properties. Calbistrin A is a secondary metabolite with an interesting structure that was recently found to have bioactivity against leukemia cells. It consists of two polyketides linked by an ester bond: a bicyclic decalin containing polyketide with structural similarities to lovastatin, and a linear 12 carbon dioic acid structure. Calbistrin A is known to be produced by several uniseriate black Aspergilli, Aspergillus versicolor-related species, and Penicillia. Penicillium decumbens produces calbistrin A and B as well as several putative intermediates of the calbistrin pathway, such as decumbenone A-B and versiol. Results: A comparative genomics study focused on the polyketide synthase (PKS) sets found in three full genome sequence calbistrin producing fungal species, P. decumbens, A. aculeatus and A. versicolor, resulted in the identification of a novel, putative 13-membered calbistrin producing gene cluster (calA to calM). Implementation of the CRISPR/Cas9 technology in P. decumbens allowed the targeted deletion of genes encoding a polyketide synthase (calA), a major facilitator pump (calB) and a binuclear zinc cluster transcription factor (calC). Detailed metabolic profiling, using UHPLC-MS, of the ∆calA (PKS) and ∆calC (TF) strains confirmed the suspected involvement in calbistrin productions as neither strains produced calbistrin nor any of the putative intermediates in the pathway. Similarly analysis of the excreted metabolites in the ∆calB (MFC-pump) strain showed that the encoded pump was required for efficient export of calbistrin A and B. Conclusion: Here we report the discovery of a gene cluster (calA-M) involved in the biosynthesis of the polyketide calbistrin in P. decumbens. Targeted gene deletions proved the involvement of CalA (polyketide synthase) in the biosynthesis of calbistrin, CalB (major facilitator pump) for the export of calbistrin A and B and CalC for the transcriptional regulation of the cal-cluster. This study lays the foundation for further characterization of the calbistrin biosynthetic pathway in multiple species and the development of an efficient calbistrin producing cell factory.

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed predominantly by the Sec translocase. This system consists of the membrane-embedded protein-conducting channel SecYEG, the motor ATPase SecA, and the heterotrimeric SecDFyajC membrane protein complex. Previous studies suggest that anionic lipids are essential for SecA activity and that the N-terminus of SecA is capable of penetrating the lipid bilayer. The role of lipid binding, however, has remained elusive. By employing differently sized nanodiscs reconstituted with single SecYEG complexes and comprising varying amounts of lipids, we establish that SecA gains access to the SecYEG complex via a lipid-bound intermediate state, whilst acidic phospholipids allosterically activate SecA for ATP-dependent protein translocation.

CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

Filamentous fungi such as Aspergillus and Penicillium are widely used as hosts for the industrial products such as proteins and secondary metabolites. Although filamentous fungi are versatile in recognizing transcriptional and translational elements present in genes from other filamentous fungal species, only few promoters have been applied and compared in performance so far in Penicillium chrysogenum. Therefore, a set of homologous and heterologous promoters were tested in a reporter system to obtain a set of potential different strengths. Through in vivo homologous recombination in Saccharomyces cerevisiae, twelve Aspergillus niger and P. chrysogenum promoter-reporter pathways were constructed that drive the expression of Green fluorescent protein while concurrent expression of the Red fluorescent protein was used as an internal standard and placed under control of the PcPAF promoter. The pathways were integrated into the genome of P. chrysogenum and tested using the BioLector system for fermentation. Reporter gene expression was monitored during growth and classified according to promoter strength and expression profile. A set of novel promoters was obtained that can be used to tune the expression of target genes in future strain engineering programs.

The Pc21 g14570 gene of Penicillium chrysogenum encodes an ortholog of a class 2 histone deacetylase termed HdaA which may play a role in epigenetic regulation of secondary metabolism. Deletion of the hdaA gene induces a significant pleiotropic effect on the expression of a set of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS)‐encoding genes. The deletion mutant exhibits a decreased conidial pigmentation that is related to a reduced expression of the PKS gene Pc21 g16000 (pks17) responsible for the production of the pigment precursor naphtha‐γ‐pyrone. Moreover, the hdaA deletion caused decreased levels of the yellow pigment chrysogine that is associated with the downregulation of the NRPS‐encoding gene Pc21 g12630 and associated biosynthetic gene cluster. In contrast, transcriptional activation of the sorbicillinoids biosynthetic gene cluster occurred concomitantly with the overproduction of associated compounds . A new compound was detected in the deletion strain that was observed only under conditions of sorbicillinoids production, suggesting crosstalk between biosynthetic gene clusters. Our present results show that an epigenomic approach can be successfully applied for the activation of secondary metabolism in industrial strains of P. chrysogenum.

Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences . This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacteriumEscherichia coliBy boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viableE. colistrain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and thatE. colihas the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20-30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell's phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.

One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium

Background l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Results Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2, had been deleted. Sugar transport assays indicated that this fungal transporter, designated as PcAraT, is a high-affinity (Km = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10−3 and 1.8 g L−1, respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of PcAraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L−1 l-arabinose and 20 g L−1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Conclusion Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make PcAraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates. Electronic supplementary material The online version of this article (10.1186/s13068-018-1047-6) contains supplementary material, which is available to authorized users.

Chrysogine is a yellow pigment produced by Penicillium chrysogenum and other filamentous fungi. Although the pigment was first isolated in 1973, its biosynthetic pathway has so far not been resolved. Here, we show that deletion of the highly expressed nonribosomal peptide synthetase (NRPS) gene Pc21g12630 ( chyA ) resulted in a decrease in the production of chrysogine and 13 related compounds in the culture broth of P. chrysogenum . Each of the genes of the chyA -containing gene cluster was individually deleted, and corresponding mutants were examined by metabolic profiling in order to elucidate their function. The data suggest that the NRPS ChyA mediates the condensation of anthranilic acid and alanine into the intermediate 2-(2-aminopropanamido)benzoic acid, which was verified by feeding experiments of a ΔchyA strain with the chemically synthesized product. The remainder of the pathway is highly branched, yielding at least 13 chrysogine-related compounds. IMPORTANCE Penicillium chrysogenum is used in industry for the production of β-lactams, but also produces several other secondary metabolites. The yellow pigment chrysogine is one of the most abundant metabolites in the culture broth, next to β-lactams. Here, we have characterized the biosynthetic gene cluster involved in chrysogine production and elucidated a complex and highly branched biosynthetic pathway, assigning each of the chrysogine cluster genes to biosynthetic steps and metabolic intermediates. The work further unlocks the metabolic potential of filamentous fungi and the complexity of secondary metabolite pathways.

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems

Summary Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems., Graphical Abstract, Highlights • CryoET and ssNMR give complementary information about proteins in native membranes • One sample can be prepared for both methods without the use of detergents • Hybrid method shows differences between purified and native preparations of YidC • Sample preparation reduces costs and time and suggests new strategy for assignment, Membrane proteins are often treated with detergents, which can affect structure and activity. Baker et al. apply a hybrid method to bacterial membrane proteins in native membranes without detergent, using solid-state NMR spectroscopy and electron cryotomography. They find that the structure and function of YidC differ with and without detergent.

Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantaruml-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h−1 on l-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on l-arabinose in the presence of d-glucose and d-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2N376T or Gal2N376I substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2T89I substitution, which was subsequently also detected in the independently evolved strain IMS0010. In 14C-sugar-transport assays, Gal2N376S, Gal2N376T and Gal2N376I substitutions showed a much lower glucose sensitivity of l-arabinose transport and a much higher Km for d-glucose transport than wild-type Gal2. Introduction of the Gal2N376I substitution in a non-evolved strain enabled growth on l-arabinose in the presence of d-glucose. Gal2N376T, T89I and Gal2T89I variants showed a lower Km for l-arabinose and a higher Km for d-glucose than wild-type Gal2, while reverting Gal2N376T, T89I to Gal2N376 in an evolved strain negatively affected anaerobic growth on l-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex ‘transporter landscapes’, consisting of sugar transporters with complementary kinetic and regulatory properties., This research describes the construction, laboratory evolution and characterization of l-arabinose fermenting Saccharomyces cerevisiae strains that are able to consume specifically l-arabinose in the presence of d-glucose and d-xylose.

About 30% of proteins synthesized in bacteria perform their functions outside of the cytoplasm and have to be inserted into or translocated across the cytoplasmic membrane. The primary system for protein translocation is the Secretory (Sec) pathway. Its essential components include the membrane-embedded protein-conducting channel SecYEG, the motor ATPase SecA, and the YidC insertase, and a number of accessory integral and peripheral membrane proteins, which facilitate targeting and translocation. Structural and in vitro functional studies on the Sec pathway have been carried out either in detergents or in model membranes, such as lipid monolayers, supported lipid bilayers, and (proteo-)liposomes. However, detergents may alter structural and functional properties of studied proteins, while the sample heterogeneity and protein aggregation occurring in large-scale model membranes often interfere with experimental analysis. Here, we review a recent progress in isolating Sec components within lipid-based particles, nanodiscs, for biophysical, biochemical, and structural analysis. Nanodiscs have been successfully applied to investigate oligomeric states of individual Sec components, to monitor structural dynamics of proteins and their assembly into functional complexes, and to reconstitute translocation and membrane insertion reactions. Cryo-electron microscopy of nanodisc-reconstituted SecYEG and YidC in complex with ribosomes visualized intermediates on membrane protein insertion and demonstrated structural dynamics of insertases. Nanodisc-based experiments have highlighted the importance of the physiologically relevant molecular environment for functionality of membrane-embedded components, but also for membrane-associated targeting machinery, and suggested nanodiscs as a powerful platform for further studies, including high-resolution structural analysis.

The bacterial Sec translocase in its minimal form consists of a membrane-embedded protein-conducting pore SecYEG that interacts with the motor protein SecA to mediate the translocation of secretory proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids.

M.H.M. was supported by a Rubicon fellowship of the Netherlands Organization for Scientific Research (NWO;Rubicon 825.13.001). The work of R.K. was supported by the European Union’s Seventh Framework Programme(Joint Call OCEAN.2011–2: Marine microbial diversity—new insights into marine ecosystems functioning and its biotechnological potential) under the grant agreement no.287589 (Micro B3). M.C. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC)studentship (BB/J014478/1). The GSC is supported by funding from the Natural Environment Research Council(UK), the National Institute for Energy Ethics and Society(NIEeS; UK), the Gordon and Betty Moore Foundation,the National Science Foundation (NSF; US) and the US Department of Energy. The Manchester Synthetic Biology Research Centre, SYNBIOCHEM, is supported by BBSRC/Engineering and Physical Sciences Research Council(EPSRC) grant BB/M017702/1, Medema, M.H., Kottmann, R., Yilmaz, P., Cummings, M., Biggins, J.B., Blin, K., De Bruijn, I., Chooi, Y.H., Claesen, J., Coates, R.C., Cruz-Morales, P., Duddela, S., Düsterhus, S., Edwards, D.J., Fewer, D.P., Garg, N., Geiger, C., Gomez-Escribano, J.P., Greule, A., Hadjithomas, M., Haines, A.S., Helfrich, E.J.N., Hillwig, M.L., Ishida, K., Jones, A.C., Jones, C.S., Jungmann, K., Kegler, C., Kim, H.U., Kötter, P., Krug, D., Masschelein, J., Melnik, A.V., Mantovani, S.M., Monroe, E.A., Moore, M., Moss, N., Nützmann, H.-W., Pan, G., Pati, A., Petras, D., Reen, F.J., Rosconi, F., Rui, Z., Tian, Z., Tobias, N.J., Tsunematsu, Y., Wiemann, P., Wyckoff, E., Yan, X., Yim, G., Yu, F., Xie, Y., Aigle, B., Apel, A.K., Balibar, C.J., Balskus, E.P., Barona-Gómez, F., Bechthold, A., Bode, H.B., Borriss, R., Brady, S.F., Brakhage, A.A., Caffrey, P., Cheng, Y.Q., Clardy, J., Cox, R.J., De Mot, R., Donadio, S., Donia, M.S., Van Der Donk, W.A., Dorrestein, P.C., Doyle, S., Driessen, A.J.M., Ehling-Schulz, M., Entian, K.-D., Fischbach, M.A., Gerwick, L., Gerwick, W.H., Gross, H., Gust, B., Hertweck, C., Höfte, M., Jensen, S.E., Ju, J., Katz, L., Kaysser, L., Klassen, J.L., Keller, N.P., Kormanec, J., Kuipers, O.P., Kuzuyama, T., Kyrpides, N.C., Kwon, H.-J., Lautru, S., Lavigne, R., Lee, C.Y., Linquan, B., Liu, X., Liu, W., Luzhetskyy, A., Mahmud, T., Mast, Y., Méndez, C., Metsä-Ketelä, M., Micklefield, J., Mitchell, D.A., Moore, B.S., Moreira, L.M., Müller, R., Neilan, B.A., Nett, M., Nielsen, J., O'Gara, F., Oikawa, H., Osbourn, A., Osburne, M.S., Ostash, B., Payne, S.M., Pernodet, J.-L., Petricek, M., Piel, J., Ploux, O., Raaijmakers, J.M., Salas, J.A., Schmitt, E.K., Scott, B., Seipke, R.F., Shen, B., Sherman, D.H., Sivonen, K., Smanski, M.J., Sosio, M., Stegmann, E., Süssmuth, R.D., Tahlan, K., Thomas, C.M., Tang, Y., Truman, A.W., Viaud, M., Walton, J.D., Walsh, C.T., Weber, T., Van Wezel, G.P., Wilkinson, B., Willey, J.M., Wohlleben, W., Wright, G.D., Ziemert, N., Zhang, C., Zotchev, S.B., Breitling, R., Takano, E., Glöckner, F.O.