ABSTRACT
Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.
ABSTRACT
Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.
ABSTRACT
Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.
Subject(s)
Maltose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Maltose/metabolism , Microbial Viability , Gene Deletion , Sorbitol/metabolism , Sorbitol/pharmacology , Xylose/metabolism , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Glucose/metabolismABSTRACT
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.
Subject(s)
Adenosine Triphosphate , Arginine , Adenosine Triphosphate/metabolism , Arginine/metabolism , Artificial Cells/metabolism , Glycerophosphates/metabolism , Glycerol Kinase/metabolism , Glycerol Kinase/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Lipids/biosynthesis , Phospholipids/metabolism , Metabolic Networks and PathwaysABSTRACT
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.
Subject(s)
Archaea , Terpenes , Terpenes/metabolism , Archaea/genetics , Archaea/metabolism , Phospholipids/metabolism , Oxidoreductases/genetics , Oxidoreductases/chemistry , Oxidoreductases/metabolismABSTRACT
Naringenin is a natural product with several reported bioactivities and is the key intermediate for the entire class of plant flavonoids. The translation of flavonoids into modern medicine as pure compounds is often hampered by their low abundance in nature and their difficult chemical synthesis. Here, we investigated the possibility to use the filamentous fungus Penicillium rubens as a host for flavonoid production. P. rubens is a well-characterized, highly engineered, traditional "workhorse" for the production of ß-lactam antibiotics. We integrated two plant genes encoding enzymes in the naringenin biosynthesis pathway into the genome of the secondary metabolite-deficient P. rubens 4xKO strain. After optimization of the fermentation conditions, we obtained an excellent molar yield of naringenin from fed p-coumaric acid (88%) with a titer of 0.88 mM. Along with product accumulation over 36 h, however, we also observed rapid degradation of naringenin. Based on high-resolution mass spectrometry analysis, we propose a naringenin degradation pathway in P. rubens 4xKO, which is distinct from other flavonoid-converting pathways reported in fungi. Our work demonstrates that P. rubens is a promising host for recombinant flavonoid production, and it represents an interesting starting point for further investigation into the utilization of plant biomass by filamentous fungi.
Subject(s)
Flavanones , Penicillium , Flavanones/chemistry , Flavonoids/chemistry , Penicillium/genetics , Penicillium/metabolism , Fungi/metabolismABSTRACT
Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.
Subject(s)
Anti-Infective Agents , Peptidomimetics , Peptidomimetics/pharmacology , Peptidomimetics/metabolism , Antimicrobial Cationic Peptides/chemistry , Anti-Infective Agents/chemistry , Staphylococcus aureus , Molecular Dynamics Simulation , Cell Membrane/metabolism , Anti-Bacterial Agents/chemistry , Microbial Sensitivity TestsABSTRACT
Rapid and effective consumption of D-xylose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. Hence, heterologous D-xylose metabolic pathways have been introduced into S. cerevisiae. An effective solution is based on a xylose isomerase in combination with the overexpression of the xylulose kinase (Xks1) and all genes of the non-oxidative branch of the pentose phosphate pathway. Although this strain is capable of consuming D-xylose, growth inhibition occurs at higher D-xylose concentrations, even abolishing growth completely at 8% D-xylose. The decreased growth rates are accompanied by significantly decreased ATP levels. A key ATP-utilizing step in D-xylose metabolism is the phosphorylation of D-xylulose by Xks1. Replacement of the constitutive promoter of XKS1 by the galactose tunable promoter Pgal10 allowed the controlled expression of this gene over a broad range. By decreasing the expression levels of XKS1, growth at high D-xylose concentrations could be restored concomitantly with increased ATP levels and high rates of xylose metabolism. These data show that in fermentations with high D-xylose concentrations, too high levels of Xks1 cause a major drain on the cellular ATP levels thereby reducing the growth rate, ultimately causing substrate accelerated death. Hence, expression levels of XKS1 in S. cerevisiae needs to be tailored for the specific growth conditions and robust D-xylose metabolism.
ABSTRACT
Cellular membranes are essential for compartmentalization, maintenance of permeability, and fluidity in all three domains of life. Archaea belong to the third domain of life and have a distinct phospholipid composition. Membrane lipids of archaea are ether-linked molecules, specifically bilayer-forming dialkyl glycerol diethers (DGDs) and monolayer-forming glycerol dialkyl glycerol tetraethers (GDGTs). The antifungal allylamine terbinafine has been proposed as an inhibitor of GDGT biosynthesis in archaea based on radiolabel incorporation studies. The exact target(s) and mechanism of action of terbinafine in archaea remain elusive. Sulfolobus acidocaldarius is a strictly aerobic crenarchaeon thriving in a thermoacidophilic environment, and its membrane is dominated by GDGTs. Here, we comprehensively analyzed the lipidome and transcriptome of S. acidocaldarius in the presence of terbinafine. Depletion of GDGTs and the accompanying accumulation of DGDs upon treatment with terbinafine were growth phase-dependent. Additionally, a major shift in the saturation of caldariellaquinones was observed, which resulted in the accumulation of unsaturated molecules. Transcriptomic data indicated that terbinafine has a multitude of effects, including significant differential expression of genes in the respiratory complex, motility, cell envelope, fatty acid metabolism, and GDGT cyclization. Combined, these findings suggest that the response of S. acidocaldarius to terbinafine inhibition involves respiratory stress and the differential expression of genes involved in isoprenoid biosynthesis and saturation.
Subject(s)
Allylamine , Sulfolobus acidocaldarius , Terbinafine/pharmacology , Terbinafine/metabolism , Sulfolobus acidocaldarius/genetics , Sulfolobus acidocaldarius/metabolism , Allylamine/metabolism , Glycerol/metabolism , Membrane Lipids/metabolism , Archaea/geneticsABSTRACT
Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.
Subject(s)
RNA, Long Noncoding , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , RNA, Long Noncoding/genetics , Ethanol/pharmacology , Ethanol/metabolismABSTRACT
Archaeal glycerophospholipids are the main constituents of the cytoplasmic membrane in the archaeal domain of life and fundamentally differ in chemical composition compared to bacterial phospholipids. They consist of isoprenyl chains ether-bonded to glycerol-1-phosphate. In contrast, bacterial glycerophospholipids are composed of fatty acyl chains ester-bonded to glycerol-3-phosphate. This largely domain-distinguishing feature has been termed the "lipid-divide". The chemical composition of archaeal membranes contributes to the ability of archaea to survive and thrive in extreme environments. However, ether-bonded glycerophospholipids are not only limited to extremophiles and found also in mesophilic archaea. Resolving the structural basis of glycerophospholipid biosynthesis is a key objective to provide insights in the early evolution of membrane formation and to deepen our understanding of the molecular basis of extremophilicity. Many of the glycerophospholipid enzymes are either integral membrane proteins or membrane-associated, and hence are intrinsically difficult to study structurally. However, in recent years, the crystal structures of several key enzymes have been solved, while unresolved enzymatic steps in the archaeal glycerophospholipid biosynthetic pathway have been clarified providing further insights in the lipid-divide and the evolution of early life.
Subject(s)
Archaea , Membrane Lipids , Archaea/metabolism , Bacteria/metabolism , Ethers/chemistry , Ethers/metabolism , Glycerol/metabolism , Glycerophospholipids/metabolism , Membrane Lipids/metabolism , Phosphates/metabolismABSTRACT
Filamentous fungi are highly productive cell factories, many of which are industrial producers of enzymes, organic acids, and secondary metabolites. The increasing number of sequenced fungal genomes revealed a vast and unexplored biosynthetic potential in the form of transcriptionally silent secondary metabolite biosynthetic gene clusters (BGCs). Various strategies have been carried out to explore and mine this untapped source of bioactive molecules, and with the advent of synthetic biology, novel applications, and tools have been developed for filamentous fungi. Here we summarize approaches aiming for the expression of endogenous or exogenous natural product BGCs, including synthetic transcription factors, assembly of artificial transcription units, gene cluster refactoring, fungal shuttle vectors, and platform strains.
ABSTRACT
Acetic acid is a growth inhibitor generated during alcoholic fermentation and pretreatment of lignocellulosic biomass, a major feedstock to produce bioethanol. An understanding of the acetic acid tolerance mechanisms is pivotal for the industrial production of bioethanol. One of the mechanisms for acetic acid tolerance is transporter-mediated secretion where individual transporters have been implicated. Here, we deleted the transporters Aqr1, Tpo2, and Tpo3, in various combinations, to investigate their combined role in acetic acid tolerance. Single transporter deletions did not impact the tolerance at mild acetic acid stress (20 mM), but at severe stress (50 mM) growth was decreased or impaired. Tpo2 plays a crucial role in acetic acid tolerance, while the AQR1 deletion has a least effect on growth and acetate efflux. Deletion of both Tpo2 and Tpo3 enhanced the severe growth defects at 20 mM acetic acid concomitantly with a reduced rate of acetate secretion, while TPO2 and/or TPO3 overexpression in ∆tpo2∆tpo3∆ restored the tolerance. In the deletion strains, the acetate derived from sugar metabolism accumulated intracellularly, while gene transcription analysis suggests that under these conditions, ethanol metabolism is activated while acetic acid production is reduced. The data demonstrate that Tpo2 and Tpo3 together fulfill an important role in acetate efflux and the acetic acid response.
ABSTRACT
The evolutionary conserved YidC is a unique dual-function membrane protein that adopts insertase and chaperone conformations. The N-terminal helix of Escherichia coli YidC functions as an uncleaved signal sequence and is important for membrane insertion and interaction with the Sec translocon. Here, we report the first crystal structure of Thermotoga maritima YidC (TmYidC) including the N-terminal amphipathic helix (N-AH) (PDB ID: 6Y86). Molecular dynamics simulations show that N-AH lies on the periplasmic side of the membrane bilayer forming an angle of about 15° with the membrane surface. Our functional studies suggest a role of N-AH for the species-specific interaction with the Sec translocon. The reconstitution data and the superimposition of TmYidC with known YidC structures suggest an active insertase conformation for YidC. Molecular dynamics (MD) simulations of TmYidC provide evidence that N-AH acts as a membrane recognition helix for the YidC insertase and highlight the flexibility of the C1 region underlining its ability to switch between insertase and chaperone conformations. A structure-based model is proposed to rationalize how YidC performs the insertase and chaperone functions by re-positioning of N-AH and the other structural elements.
Subject(s)
Bacterial Proteins/chemistry , Cell Membrane/metabolism , Membrane Transport Proteins/chemistry , Molecular Dynamics Simulation , Thermotoga maritima/metabolism , Bacterial Proteins/metabolism , Crystallography, X-Ray , Membrane Transport Proteins/metabolism , Protein ConformationABSTRACT
Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.
Subject(s)
Fungi/genetics , Synthetic Biology/methods , CRISPR-Cas Systems/genetics , Cloning, Molecular/methods , Genetic Engineering/methods , Genetic Vectors/genetics , Plasmids/genetics , Promoter Regions, Genetic/geneticsABSTRACT
Simple, robust and versatile LC-MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC.
Subject(s)
1-Butanol/chemistry , Lipids/chemistry , Chromatography, High Pressure Liquid , Spectrometry, Mass, Electrospray IonizationABSTRACT
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.