Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Unraveling the multiplicity of geranylgeranyl reductases in Archaea: potential roles in saturation of terpenoids.

The enzymology of the key steps in the archaeal phospholipid biosynthetic pathway has been elucidated in recent years. In contrast, the complete biosynthetic pathways for proposed membrane regulators consisting of polyterpenes, such as carotenoids, respiratory quinones, and polyprenols remain unknown. Notably, the multiplicity of geranylgeranyl reductases (GGRs) in archaeal genomes has been correlated with the saturation of polyterpenes. Although GGRs, which are responsible for saturation of the isoprene chains of phospholipids, have been identified and studied in detail, there is little information regarding the structure and function of the paralogs. Here, we discuss the diversity of archaeal membrane-associated polyterpenes which is correlated with the genomic loci, structural and sequence-based analyses of GGR paralogs.

Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens.

Nonribosomal peptide synthetases (NRPS) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified ß-lactam antibiotics.

Identification of a conserved N-terminal domain in the first module of ACV synthetases.

The l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of ß-lactams. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l-α-aminoadipic acid, which is coupled to the α-amino group of l-cysteine through an unusual peptide bond, involving its δ-carboxyl group. Here we carried out an in-depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N-terminus which is structurally related to condensation domains, but smaller in size. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. The data indicate that the N-terminal domain is important for catalysis.

Single-molecule analysis of dynamics and interactions of the SecYEG translocon.

Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm2 ·s-1 . Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.

Biochemical characterization of the Nocardia lactamdurans ACV synthetase.

The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of ß-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position.

Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during ß-Lactam Production.

To produce high levels of ß-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 ß-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.IMPORTANCEPenicillium rubens is an important industrial producer of ß-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.

Two distinct anionic phospholipid-dependent events involved in SecA-mediated protein translocation.

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation.

Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin.

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.

The Canonical and Accessory Sec System of Gram-positive Bacteria.

The Sec system is present in all bacteria and responsible for the translocation of the majority of proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor protein, SecA, and the protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG.

Lipids Activate SecA for High Affinity Binding to the SecYEG Complex.

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, whereas acidic phospholipids allosterically activate SecA for ATP-dependent protein translocation.

Ciprofloxacin-Photoswitch Conjugates: A Facile Strategy for Photopharmacology.

Photopharmacology aims to locally treat diseases and study biological processes with photoresponsive drugs. Herein, easy access to photoswitchable drugs is crucial, which is supported by simple and robust drug modifications. We investigated the possibility of creating drugs that can undergo remote activation and deactivation with light, by conjugating molecular photoswitches to the exterior of an existing drug in a single chemical step. This facile strategy allows the convenient introduction of various photochromic systems into a drug molecule, rendering it photoresponsive. To demonstrate the feasibility of this approach, two photoswitch-modified ciprofloxacin antibiotics were synthesized. Remarkably, for one of them a 50-fold increase in activity compared to the original ciprofloxacin was observed. Their antimicrobial activity could be spatiotemporally controlled with light, which was exemplified by bacterial patterning studies.

Formation of the ether lipids archaetidylglycerol and archaetidylethanolamine in Escherichia coli.

In archaea, the membrane phospholipids consist of isoprenoid hydrocarbon chains that are ether-linked to a sn-glycerol1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these micro-organisms to extreme environments, but it also reflects an evolutionary marker that distinguishes archaea from bacteria and eukaryotes. CDP-archaeol is the central precursor for polar head group attachment. We examined various bacterial enzymes involved in the attachment of L-serine and glycerol as polar head groups for their promiscuity in recognizing CDP-archaeol as a substrate. Using a combination of mutated bacterial and archaeal enzymes, archaetidylethanolamine (AE) and archaetidylglycerol (AG) could be produced in vitro using nine purified enzymes while starting from simple building blocks. The ether lipid pathway constituted by a set of archaeal and bacterial enzymes was introduced into Escherichia coli, which resulted in the biosynthesis of AE and AG. This is a further step in the reprogramming of E. coli for ether lipid biosynthesis.

Characterization of the annular lipid shell of the Sec translocon.

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.

In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis.

The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers.

The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter.

Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

Biosynthesis of archaeal membrane ether lipids.

A vital function of the cell membrane in all living organism is to maintain the membrane permeability barrier and fluidity. The composition of the phospholipid bilayer is distinct in archaea when compared to bacteria and eukarya. In archaea, isoprenoid hydrocarbon side chains are linked via an ether bond to the sn-glycerol-1-phosphate backbone. In bacteria and eukarya on the other hand, fatty acid side chains are linked via an ester bond to the sn-glycerol-3-phosphate backbone. The polar head groups are globally shared in the three domains of life. The unique membrane lipids of archaea have been implicated not only in the survival and adaptation of the organisms to extreme environments but also to form the basis of the membrane composition of the last universal common ancestor (LUCA). In nature, a diverse range of archaeal lipids is found, the most common are the diether (or archaeol) and the tetraether (or caldarchaeol) lipids that form a monolayer. Variations in chain length, cyclization and other modifications lead to diversification of these lipids. The biosynthesis of these lipids is not yet well understood however progress in the last decade has led to a comprehensive understanding of the biosynthesis of archaeol. This review describes the current knowledge of the biosynthetic pathway of archaeal ether lipids; insights on the stability and robustness of archaeal lipid membranes; and evolutionary aspects of the lipid divide and the LUCA. It examines recent advances made in the field of pathway reconstruction in bacteria.

Identification of CDP-archaeol synthase, a missing link of ether lipid biosynthesis in Archaea.

Archaeal membrane lipid composition is distinct from Bacteria and Eukarya, consisting of isoprenoid chains etherified to the glycerol carbons. Biosynthesis of these lipids is poorly understood. Here we identify and characterize the archaeal membrane protein CDP-archaeol synthase (CarS) that catalyzes the transfer of the nucleotide to its specific archaeal lipid substrate, leading to the formation of a CDP-activated precursor (CDP-archaeol) to which polar head groups are attached. The discovery of CarS enabled reconstitution of the entire archaeal lipid biosynthesis pathway in vitro, starting from simple isoprenoid building blocks and using a set of five purified enzymes. The cell free synthetic strategy for archaeal lipids we describe opens opportunity for studies of archaeal lipid biochemistry. Additionally, insights into archaeal lipid biosynthesis reported here allow addressing the evolutionary hypothesis of the lipid divide between Archaea and Bacteria.

Novel key metabolites reveal further branching of the roquefortine/meleagrin biosynthetic pathway.

Metabolic profiling and structural elucidation of novel secondary metabolites obtained from derived deletion strains of the filamentous fungus Penicillium chrysogenum were used to reassign various previously ascribed synthetase genes of the roquefortine/meleagrin pathway to their corresponding products. Next to the structural characterization of roquefortine F and neoxaline, which are for the first time reported for P. chrysogenum, we identified the novel metabolite roquefortine L, including its degradation products, harboring remarkable chemical structures. Their biosynthesis is discussed, questioning the exclusive role of glandicoline A as key intermediate in the pathway. The results reveal that further enzymes of this pathway are rather unspecific and catalyze more than one reaction, leading to excessive branching in the pathway with meleagrin and neoxaline as end products of two branches.

Optical control of antibacterial activity.

Bacterial resistance is a major problem in the modern world, stemming in part from the build-up of antibiotics in the environment. Novel molecular approaches that enable an externally triggered increase in antibiotic activity with high spatiotemporal resolution and auto-inactivation are highly desirable. Here we report a responsive, broad-spectrum, antibacterial agent that can be temporally activated with light, whereupon it auto-inactivates on the scale of hours. The use of such a 'smart' antibiotic might prevent the build-up of active antimicrobial material in the environment. Reversible optical control over active drug concentration enables us to obtain pharmacodynamic information. Precisely localized control of activity is achieved, allowing the growth of bacteria to be confined to defined patterns, which has potential for the development of treatments that avoid interference with the endogenous microbial population in other parts of the organism.