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In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).
Tsetse flies are the transmitting vector of trypanosomes causing human sleeping sickness and animal trypanosomiasis in sub-saharan Africa. 3-alkylphenols are used as attractants in tsetse fly traps to reduce the spread of the disease. Here we present an inexpensive production method for 3-ethylphenol (3-EP) and 3-propylphenol (3-PP) by microbial fermentation of sugars. Heterologous expression in the yeast Saccharomyces cerevisiae of phosphopantetheinyltransferase-activated 6-methylsalicylic acid (6-MSA) synthase (MSAS) and 6-MSA decarboxylase converted acetyl-CoA as a priming unit via 6-MSA into 3-methylphenol (3-MP). We exploited the substrate promiscuity of MSAS to utilize propionyl-CoA and butyryl-CoA as alternative priming units and the substrate promiscuity of 6-MSA decarboxylase to produce 3-EP and 3-PP in yeast fermentations. Increasing the formation of propionyl-CoA by expression of a bacterial propionyl-CoA synthetase, feeding of propionate and blocking propionyl-CoA degradation led to the production of up to 12.5 mg/L 3-EP. Introduction of a heterologous ‘reverse ß-oxidation’ pathway provided enough butyryl-CoA for the production of 3-PP, reaching titers of up to 2.6 mg/L. As the concentrations of 3-alkylphenols are close to the range of the concentrations deployed in tsetse fly traps, the yeast broths might become promising and inexpensive sources for attractants, producible on site by rural communities in Africa.
De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT—malonyl‐/acetyltransferase) and the condensation (KS—β‐ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate‐binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.
Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS) are responsible for the production of a large spectrum of bioactive polyketides (PK), which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the "assembly-line synthetic concept" of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS) are and will be valuable objects for further developing this field.
Fatty acids (FAs) are considered strategically important platform compounds that can be accessed by sustainable microbial approaches. Here we report the reprogramming of chain-length control of Saccharomyces cerevisiae fatty acid synthase (FAS). Aiming for short-chain FAs (SCFAs) producing baker’s yeast, we perform a highly rational and minimally invasive protein engineering approach that leaves the molecular mechanisms of FASs unchanged. Finally, we identify five mutations that can turn baker’s yeast into a SCFA producing system. Without any further pathway engineering, we achieve yields in extracellular concentrations of SCFAs, mainly hexanoic acid (C6-FA) and octanoic acid (C8-FA), of 464 mg l−1 in total. Furthermore, we succeed in the specific production of C6- or C8-FA in extracellular concentrations of 72 and 245 mg l−1, respectively. The presented technology is applicable far beyond baker’s yeast, and can be plugged into essentially all currently available FA overproducing microorganisms.
Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.
Results: The previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.
Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
Most fungal fatty acid synthases assemble from two multidomain subunits, α and β, into a heterododecameric FAS complex. It has been recently shown that the complex assembly occurs in a cotranslational manner and is initiated by an interaction between the termini of α and β subunits. This initial engagement of subunits may be the rate-limiting phase of the assembly and subject to cellular regulation. Therefore, we hypothesized that bypassing this step by genetically fusing the subunits could be beneficial for biotechnological production of fatty acids. To test the concept, we expressed fused FAS subunits engineered for production of octanoic acid in Saccharomyces cerevisiae. Collectively, our data indicate that FAS activity is a limiting factor of fatty acid production and that FAS fusion proteins show a superior performance compared to their split counterparts. This strategy is likely a generalizable approach to optimize the production of fatty acids and derived compounds in microbial chassis organisms.
Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum.
The authors regret that there is an error present in the units displayed in the sentence “The dissociation constant of docking domains or modules connected by docking domains was found to be KD 70–130 mM (ref. 35) and KD 1–2 mM (ref. 59), respectively.” within Section 3.1. Module–module exchanges. The corrected version of this sentence is as follows:
The dissociation constant of docking domains or modules connected by docking domains was found to be KD 70–130 μM (ref. 35) and KD 1–2 mM (ref. 59), respectively.
The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.
Covering: mid 1990s to 2018
Over the last two decades, diverse approaches have been explored to generate new polyketides by engineering polyketide synthases (PKSs). Although it has been proven possible to produce new compounds by designed PKSs, engineering strategies failed to make polyketides available via widely applicable rules and protocols. Still, organic synthetic routes have to be employed whenever new polyketides are needed for applications in medicine, agriculture, and industry. In light of the rising demand for commodity products from feedstock and for fast and cheap access to pharmaceutical compounds, the need for harnessing PKSs to produce such molecules is more urgent than ever before. In this review, we focus on a multitude of approaches to engineer modular PKSs by swapping and replacing PKS modules and domains, which we analyze in the light of recent structural and biochemical data. We conclude with an outlook on possible strategies on how to increase success rates of PKS engineering in future.
Lieblingsbild
(2017)
The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the β-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.
Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1 Å resolution map of yeast FAS from 15 000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.
Flavins are employed to transform physical input into biological output signals. In this function, flavins catalyze a variety of light-induced reactions and redox processes. However, nature also provides flavoproteins with the ability to uncouple the mediation of signals. Such proteins are the riboflavin-binding proteins (RfBPs) with their function to store riboflavin for fast delivery of FMN and FAD. Here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin is an RfBP, and we reveal that riboflavin storage is not restricted to eukaryotes. However, the function of the prokaryotic RfBP dodecin seems to be adapted to the requirement of a monocellular organism. While in eukaryotes RfBPs are involved in trafficking riboflavin, and dodecin is responsible for the flavin homeostasis of the cell. Although only 68 amino acids in length, dodecin is of high functional versatility in neutralizing riboflavin to protect the cellular environment from uncontrolled flavin reactivity. Besides the predominant ultrafast quenching of excited states, dodecin prevents light-induced riboflavin reactivity by the selective degradation of riboflavin to lumichrome. Coordinated with the high affinity for lumichrome, the directed degradation reaction is neutral to the cellular environment and provides an alternative pathway for suppressing uncontrolled riboflavin reactivity. Intriguingly, the different structural and functional properties of a homologous bacterial dodecin suggest that dodecin has different roles in different kingdoms of life.
Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins.
Modularity is an aspect of a decomposable system with a coordinating authority that acts as a glue which holds the loosely held components. These multi-component entities (“modules”) facilitate rewiring into different designs allowing for change. Such modular character is a fundamental property of many biological entities, especially the family of megasynthases such as polyketide synthases (PKSs). The ability of these PKSs to produce diverse product spectra is strongly coupled to their broad architectural modularity. Decoding the molecular basis of modularity, i.e. identifying the folds and domains that comprise the modules as well as understanding constrains of the assembly of modules, is of utmost importance for harnessing megasynthases for the synthesis of designer compounds. In this study, we exploit the close semblance between PKSs and animal FAS to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by structural and sequence information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel engineered modules resemble all four common module types of PKSs and demonstrate that this approach can be a powerful tool to create higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.
Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.
Single-particle electron cryo-microscopy (cryoEM) has undergone a “resolution revolution” that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 Å map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.