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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.
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)
